HANDBOOK OF
Seafood and Seafood Products Analysis
HANDBOOK OF
Seafood and Seafood Products Analysis Edited by
LEO M.L. NOLLET FIDEL TOLDRÁ
Boca Raton London New York
CRC Press is an imprint of the Taylor & Francis Group, an informa business
CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-4633-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of seafood and seafood products analysis / editors, Leo M.L. Nollet, Fidel Toldrá. p. cm. Includes bibliographical references and index. ISBN 978-1-4200-4633-5 (hardcover : alk. paper) 1. Seafood--Analysis--Handbooks, manuals, etc. I. Nollet, Leo M. L., 1948- II. Toldrá, Fidel. III. Title. TX385.H36 2010 641.3’92--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com
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Contents Preface ..................................................................................................................................ix Editors ..................................................................................................................................xi Contributors ...................................................................................................................... xiii
PART I: CHEMISTRY AND BIOCHEMISTRY 1 Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts.....................................................................................3 JÖRG OEHLENSCHLÄGER
2 Peptides and Proteins .................................................................................................11 TURID RUSTAD
3 Proteomics ..................................................................................................................21 HÓLMFRÍÐUR SVEINSDÓTTIR, ÁGÚSTA GUÐMUNDSDÓTTIR, AND ODDUR VILHELMSSON
4 Seafood Genomics ......................................................................................................43 ASTRID BÖHNE, DELPHINE GALIANA-ARNOUX, CHRISTINA SCHULTHEIS, FRÉDÉRIC BRUNET, AND JEAN-NICOLAS VOLFF
5 Nucleotides and Nucleosides ......................................................................................57 M. CONCEPCIÓN ARISTOY, LETICIA MORA, ALEIDA S. HERNÁNDEZ-CÁZARES, AND FIDEL TOLDRÁ
6 Lipid Compounds.......................................................................................................69 SANTIAGO P. AUBOURG
7 Lipid Oxidation ..........................................................................................................87 TURID RUSTAD
8 Volatile Aroma Compounds in Fish ...........................................................................97 GUÐRÚN ÓLAFSDÓTTIR AND RÓSA JÓNSDÓTTIR
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PART II: PROCESSING CONTROL 9 Basic Composition: Rapid Methodologies ...............................................................121 HEIDI NILSEN, KARSTEN HEIA, AND MARGRETHE ESAIASSEN
10 Microstructure .........................................................................................................139 ISABEL HERNANDO, EMPAR LLORCA, ANA PUIG, AND MARÍA-ANGELES LLUCH
11 Chemical Sensors .....................................................................................................153 CORRADO DI NATALE
12 Physical Sensors and Techniques .............................................................................169 RUTH DE LOS REYES CÁNOVAS, PEDRO JOSÉ FITO SUÑER, ANA ANDRÉS GRAU, AND PEDRO FITO-MAUPOEY
13 Methods for Freshness Quality and Deterioration...................................................189 YESIM OZOGUL
14 Analytical Methods to Differentiate Farmed from Wild Seafood ............................215 ICIAR MARTÍNEZ, INGER BEATE STANDAL, MARIT AURSAND, YUMIKO YAMASHITA, AND MICHIAKI YAMASHITA
15 Smoke Flavoring Technology in Seafood .................................................................233 VINCENT VARLET, THIERRY SEROT, AND CAROLE PROST
PART III: NUTRITIONAL QUALITY 16 Composition and Calories ........................................................................................257 EVA FALCH, INGRID OVERREIN, CHRISTEL SOLBERG, AND RASA SLIZYTE
17 Essential Amino Acids ..............................................................................................287 M. CONCEPCIÓN ARISTOY AND FIDEL TOLDRÁ
18 Antioxidants .............................................................................................................309 NICK KALOGEROPOULOS AND ANTONIA CHIOU
19 Vitamins ...................................................................................................................327 YOUNG-NAM KIM
20 Minerals and Trace Elements ...................................................................................351 JÖRG OEHLENSCHLÄGER
21 Analysis of n-3 and n-6 Fatty Acids ..........................................................................377 VITTORIO M. MORETTI AND FABIO CAPRINO
PART IV: SENSORY QUALITY 22 Quality Assessment of Fish and Fishery Products by Color Measurement ..............395 REINHARD SCHUBRING
23 Instrumental Texture ...............................................................................................425 ISABEL SÁNCHEZ-ALONSO, MARTA BARROSO, AND MERCEDES CARECHE
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24 Aroma .......................................................................................................................439 JOHN STEPHEN ELMORE
25 Quality Index Methods ............................................................................................463 GRETHE HYLDIG, EMILÍA MARTINSDÓTTIR, KOLBRÚN SVEINSDÓTTIR, RIAN SCHELVIS, AND ALLAN BREMNER
26 Sensory Descriptors ..................................................................................................481 GRETHE HYLDIG
27 Sensory Aspects of Heat-Treated Seafood.................................................................499 GRETHE HYLDIG
PART V: SAFETY 28 Assessment of Seafood Spoilage and the Microorganisms Involved.........................515 ROBERT E. LEVIN
29 Detection of Fish Spoilage........................................................................................537 GEORGE-JOHN E. NYCHAS AND E.H. DROSINOS
30 Detection of the Principal Foodborne Pathogens in Seafoods and Seafood-Related Environments ................................................................................557 DAVID RODRÍGUEZ-LÁZARO AND MARTA HERNANDEZ
31 Parasites....................................................................................................................579 JUAN ANTONIO BALBUENA AND JUAN ANTONIO RAGA
32 Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases .................603 CARLOS PEREIRA DOPAZO AND ISABEL BANDÍN
33 Marine Toxins ..........................................................................................................649 CARA EMPEY CAMPORA AND YOSHITSUGI HOKAMA
34 Detection of Adulterations: Addition of Foreign Proteins .......................................675 VÉRONIQUE VERREZ-BAGNIS
35 Detection of Adulterations: Identification of Seafood Species .................................687 ANTONIO PUYET AND JOSÉ M. BAUTISTA
36 Veterinary Drugs ......................................................................................................713 ANTON KAUFMANN
37 Differentiation of Fresh and Frozen–Thawed Fish ...................................................735 MUSLEH UDDIN
38 Spectrochemical Methods for the Determination of Metals in Seafood .....................................................................................................................751 JOSEPH SNEDDON AND CHAD A. THIBODEAUX
39 Food Irradiation and Its Detection ..........................................................................773 YIU CHUNG WONG, DELLA WAI MEI SIN, AND WAI YIN YAO
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40 Analysis of Dioxins in Seafood and Seafood Products .............................................797 LUISA RAMOS BORDAJANDI, BELÉN GÓMARA, AND MARÍA JOSÉ GONZÁLEZ
41 Environmental Contaminants: Persistent Organic Pollutants .................................817 MONIA PERUGINI
42 Biogenic Amines in Seafood Products......................................................................833 CLAUDIA RUIZ-CAPILLAS AND FRANCISCO JIMÉNEZ-COLMENERO
43 Residues of Food Contact Materials .........................................................................851 EMMA L. BRADLEY AND LAURENCE CASTLE
44 Detection of GM Ingredients in Fish Feed ...............................................................871 KATHY MESSENS, NICOLAS GRYSON, KRIS AUDENAERT, AND MIA EECKHOUT
Index .................................................................................................................................889
Preface There are several seafood and seafood products, which represent some of the most important foods in almost all types of societies, including those in developed and developing countries. The intensive production of fish and shellfish has raised some concerns related to the nutritional and sensory qualities of cultured fish in comparison to their wild-catch counterparts. In addition, there are several processing and preservation technologies, from traditional drying or curing to high-pressure processing, and different methods of storage. This increase of variability in products attending the consumers’ demands necessitates the use of adequate analytical methodologies as presented in this book. These analyses will be focused on the chemistry and biochemistry of postmortem seafood; the technological, nutritional, and sensory qualities; as well as the safety aspects related to processing and preservation. This book contains 44 chapters. Part I—Chemistry and Biochemistry (Chapters 1 through 8)—focuses on the analysis of the main chemical and biochemical compounds of seafood. Chapter 1 provides a general introduction to the topics covered in this book. Part II—Processing Control (Chapters 9 through 15)—describes the analysis of technological quality and the use of some nondestructive techniques. Various methods to differentiate between farmed and wild seafood, to check freshness, and to evaluate smoke flavoring are discussed in these chapters. Part III—Nutritional Quality (Chapters 16 through 21)—deals with the analysis of nutrients in muscle foods such as essential amino acids, omega fatty acids, antioxidants, vitamins, minerals, and trace elements. Part IV—Sensory Quality (Chapters 22 through 27)—covers the sensory quality and the main analytical tools to determine the color texture, the flavor and off-flavor, etc. Sensory descriptors and sensory aspects of heat-treated seafood are also discussed. Finally, Part V—Safety (Chapters 28 through 44)—is concerned with safety, especially related to analytical tools, for the detection of pathogens, parasites, viruses, marine toxins, antibiotics, adulterations, and chemical toxic compounds from the environment generated during processing, or intentionally added, that can be found in either cultured or wild-catch seafood. The last chapter also deals with the analysis of genetically modified ingredients in fish feed. This book provides an overview of the analytical tools available for the analysis of seafood, either cultured fish or their wild-catch counterparts, and its derived products. It also provides an extensive description of techniques and methodologies for quality assurance, and describes analytical methodologies for safety control. In summary, this handbook deals with the main types of analytical techniques available worldwide, and the methodologies for the analysis of seafood and seafood products. ix
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We would like to thank all the contributors for their excellent work. Their hard work and dedication have resulted in this comprehensive and prized handbook. We wish them all the very best in their academic and/or scientific careers. Leo M.L. Nollet Fidel Toldrá
Editors Dr. Leo M.L. Nollet is the editor and associate editor of several books. He edited for Marcel Dekker, New York—now CRC Press of Taylor & Francis Group—the first and second editions of Food Analysis by HPLC and the Handbook of Food Analysis. The Handbook of Food Analysis is a three-volume book. He also edited the third edition of the Handbook of Water Analysis, Chromatographic Analysis of the Environment (CRC Press) and the second edition of the Handbook of Water Analysis (CRC Press) in 2007. He coedited two books with F. Toldrá that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). He also coedited Radionuclide Concentrations in Foods and the Environment with M. Pöschl in 2006 (CRC Press). Nollet has coedited several books with Y.H. Hui and other colleagues: the Handbook of Food Product Manufacturing (Wiley, 2007); the Handbook of Food Science, Technology and Engineering (CRC Press, 2005); and Food Biochemistry and Food Processing (Blackwell Publishing, 2005). Finally, he also edited the Handbook of Meat, Poultry and Seafood Quality (Blackwell Publishing, 2007). He has worked on the following five books on analysis methodologies with F. Toldrá for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods He has worked with Professor H. Rathore on the Handbook of Pesticides: Methods of Pesticides Residues Analysis, which was published by CRC Press in 2009. Dr. Fidel Toldrá is a research professor in the Department of Food Science at the Instituto de Agroquímica y Tecnología de Alimentos (CSIC) and serves as the European editor of Trends in Food Science & Technology, the editor-in-chief of Current Nutrition & Food Science, and as a member of the Flavorings and Enzymes Panel at the European Food Safety Authority. In recent years, he has served as an editor or associate editor of several books. He was the editor of Research Advances in the Quality of Meat and Meat Products (Research Signpost, 2002) and the associate editor of the Handbook of Food and Beverage Fermentation Technology and the Handbook of Food Science, xi
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Technology and Engineering published in 2004 and 2006, respectively, by CRC Press. He coedited two books with L. Nollet that were published in 2006: Advanced Technologies for Meat Processing (CRC Press) and Advances in Food Diagnostics (Blackwell Publishing). Both he and Nollet are also associate editors of the Handbook of Food Product Manufacturing published by John Wiley & Sons in 2007. Professor Toldrá has edited Safety of Meat and Processed Meat (Springer, 2009) and has also authored Dry-Cured Meat Products (Food & Nutrition Press—now Wiley-Blackwell, 2002). He has worked on the following five books on analysis methodologies with L. Nollet for foods of animal origin, all to be published by CRC Press: Handbook of Muscle Foods Analysis Handbook of Processed Meats and Poultry Analysis Handbook of Seafood and Seafood Products Analysis Handbook of Dairy Foods Analysis Handbook of Analysis of Edible Animal By-Products Handbook of Analysis of Active Compounds in Functional Foods Toldrá was awarded the 2002 International Prize for Meat Science and Technology by the International Meat Secretariat. He was elected as a fellow of the International Academy of Food Science & Technology in 2008 and as a fellow of the Institute of Food Technologists in 2009.
Contributors M. Concepción Aristoy Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Santiago P. Aubourg Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Científicas Vigo, Spain Kris Audenaert Department of Plant Production Faculty of Biosciences and Landscape Architecture University College Ghent Ghent, Belgium Marit Aursand SINTEF Fisheries and Aquaculture Trondheim, Norway Juan Antonio Balbuena Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain
Isabel Bandín Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain Marta Barroso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain José M. Bautista Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Astrid Böhne Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Luisa Ramos Bordajandi Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain
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Emma L. Bradley Food and Environment Research Agency York, United Kingdom Allan Bremner Allan Bremner and Associates Mount Coolum, Queensland, Australia Frédéric Brunet Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Cara Empey Campora Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Fabio Caprino Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Mercedes Careche Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain Laurence Castle Food and Environment Research Agency York, United Kingdom
Corrado Di Natale Department of Electronic Engineering University of Rome Tor Vergata Rome, Italy Carlos Pereira Dopazo Departamento de Microbiología y Parasitología Instituto de Acuicultura Universidad de Santiago de Compostela Santiago de Compostela, Spain E.H. Drosinos Laboratory of Food Quality Control and Hygiene Department of Food Science & Technology Agricultural University of Athens Athens, Greece Mia Eeckhout Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium John Stephen Elmore Department of Food Biosciences University of Reading Reading, United Kingdom Margrethe Esaiassen Nofima Marked Tromsø, Norway
Antonia Chiou Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece
Eva Falch Mills DA Trondheim, Norway
Ruth De los Reyes Cánovas Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain
Pedro Fito-Maupoey Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain
Contributors
Delphine Galiana-Arnoux Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Belén Gómara Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain María José González Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute Consejo Superior de Investigaciones Científicas Madrid, Spain Ana Andrés Grau Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain Nicolas Gryson Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Ágústa Guðmundsdóttir Department of Food Science and Nutrition School of Health Sciences Science Institute University of Iceland Reykjavik, Iceland Karsten Heia Nofima Marine Tromsø, Norway
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Marta Hernandez Molecular Biology and Microbiology Laboratory Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Aleida S. Hernández-Cázares Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain Isabel Hernando Department of Food Technology Universidad Polite′cnica de Valencia Valencia, Spain Yoshitsugi Hokama Department of Pathology John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Grethe Hyldig Aquatic Process and Product Technology National Institute of Aquatic Resources (DTU Aqua) Technical University of Denmark Kongens Lyngby, Denmark Francisco Jiménez-Colmenero Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Rósa Jónsdóttir Matís Icelandic Food Research Reykjavik, Iceland Nick Kalogeropoulos Department of Science of Dietetics-Nutrition Harokopio University Athens, Greece
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Anton Kaufmann Kantonales Labor Zurich Zurich, Switzerland Young-Nam Kim Department of Nutrition and Health Sciences Duksung Women’s University Seoul, South Korea Robert E. Levin Department of Food Science University of Massachusetts Amherst, Massachusetts Empar Llorca Departamento de Tecnología de Alimentos Universidad Politécnica de Valencia Valencia, Spain María-Angeles Lluch Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain Iciar Martínez Instituto de Investigaciones Marinas (CSIC) Consejo Superior de Investigaciones Científicas Vigo, Spain Emilía Martinsdóttir Matís Iceland Food Research Reykjavík, Iceland Kathy Messens Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Ghent University Association Ghent, Belgium Leticia Mora Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain
Vittorio M. Moretti Dipartimento de Scienze e Technologie Veterinari per la Sicurezza Alimentare Università degli Studi di Milano Milan, Italy Heidi Nilsen Nofima Marine Tromsø, Norway George-John E. Nychas Laboratory of Microbiology and Biotechnology of Foods Department of Food Science and Technology Agricultural University of Athens Athens, Greece Jörg Oehlenschläger Max Rubner-Institute Federal Research Centre for Nutrition and Food Hamburg, Germany Guðrún Ólafsdóttir Syni Laboratory Services and University of Iceland Reykjavik, Iceland Ingrid Overrein SINTEF Fisheries and Aquaculture and Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway Yesim Ozogul Department of Seafood Processing Technology Faculty of Fisheries Cukurova University Adana, Turkey Monia Perugini Department of Food Science University of Teramo Teramo, Italy
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Carole Prost Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France
Isabel Sánchez-Alonso Instituto del Frío Consejo Superior de Investigaciones Científicas Madrid, Spain
Ana Puig Department of Food Technology Universidad Politécnica de Valencia Valencia, Spain
Rian Schelvis Wageningen IMARES Institute for Marine Resources & Ecosytem Studies IJmuiden, the Netherlands
Antonio Puyet Faculty of Veterinary Sciences Department of Biochemistry and Molecular Biology IV Universidad Complutense de Madrid Ciudad Universitaria Madrid, Spain Juan Antonio Raga Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Valencia, Spain David Rodríguez-Lázaro Food Safety and Technology Research Group Instituto Tecnologico Agrario de Castilla y León Valladolid, Spain Claudia Ruiz-Capillas Department of Meat and Fish Science and Technology Instituto del Frío Consejo Superior de Investigaciones Científicas Ciudad Universitaria Madrid, Spain Turid Rustad Department of Biotechnology Norwegian University of Science and Technology Trondheim, Norway
Reinhard Schubring Department of Safety and Quality of Milk and Fish Products Federal Research Institute for Nutrition and Food Max Rubner-Institut Hamburg, Germany Christina Schultheis Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France Thierry Serot Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Della Wai Mei Sin Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China Rasa Slizyte SINTEF Fisheries and Aquaculture Trondheim, Norway Joseph Sneddon Department of Chemistry McNeese State University Lake Charles, Louisiana
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Christel Solberg Faculty of Biosciences and Aquaculture Bodø University College Bodø, Norway Inger Beate Standal SINTEF Fisheries and Aquaculture Trondheim, Norway
Vincent Varlet Food Aroma Quality Group LBAI—ENITIAA Rue de la Géraudière Nantes, France Véronique Verrez-Bagnis Ifremer Nantes, France
Pedro José Fito Suñer Institute of Food Engineering for Development Polytechnic University of Valencia Valencia, Spain
Oddur Vilhelmsson Department of Science University of Akureyri Akureyri, Iceland
Hólmfríður Sveinsdóttir Division of Biotechnology and Biomolecules Matís Iceland Food Research SauđárkrÓkur, Iceland
Jean-Nicolas Volff Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon University of Lyon Lyon, France
Kolbrún Sveinsdóttir Matís Iceland Food Research Reykjavik, Iceland
Yiu Chung Wong Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China
Chad A. Thibodeaux Department of Chemistry McNeese State University Lake Charles, Louisiana
Michiaki Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama, Japan
Fidel Toldrá Instituto de Agroquímica y Tecnología de Alimentos Consejo Superior de Investigaciones Científicas Burjassot, Valencia, Spain
Yumiko Yamashita Food Biotechnology Section National Research Institute of Fisheries Science Yokohama, Japan
Musleh Uddin Corporate Quality Assurance Albion Fisheries Ltd. Vancouver, British Columbia, Canada
Wai Yin Yao Analytical and Advisory Services Division Government Laboratory Hong Kong, People’s Republic of China
CHEMISTRY AND BIOCHEMISTRY
I
Chapter 1
Introduction—Importance of Analysis in Seafood and Seafood Products, Variability and Basic Concepts Jörg Oehlenschläger Contents 1.1 World Catch and Harvest .................................................................................................. 3 1.2 Variability of Aquatic Animals ........................................................................................... 5 1.3 Special Problems with Aquatic Animals ............................................................................. 5 1.4 Benefits and Risks .............................................................................................................. 6 1.5 Sampling ............................................................................................................................ 6 1.6 Analytical Methodologies................................................................................................... 7 1.7 Analytical Problems ........................................................................................................... 8 1.8 Trends and Outlook ........................................................................................................... 9 References ..................................................................................................................................10
1.1
World Catch and Harvest
Seafood has by far the greatest variety of all animal-based foods. Whereas the species consumed as warm-blooded mammals (beef, pork, lamb, goat, and donkey) or poultry (hen, turkey, geese, and duck) are represented by very few species, fishes and other aquatic animals show an abundant 3
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Handbook of Seafood and Seafood Products Analysis
number of species and variability. The fish group alone is represented by 25,000–35,000 species. However, only a little proportion of this large number of about 5% is present in the world’s oceans in amounts huge enough to allow an economical use (catch and following processing). Further, only some of these 5% have the desired sensory properties and give a good or satisfying fillet yield that catching and processing them can be justified. Another difference compared with land-living animals is the fact that the quality (size, state of maturity, nutritional status, infestation with parasites, burden of pollutants, etc.) of aquatic animals when captured by fishing techniques is—with few exceptions—completely unknown. Although land-based animals are today tailor made according to industry’s and consumer’s wishes in weight, body composition, appearance, and sensory properties, in the case of captured seafood we have to accept what we find in the trawl despite modern advanced technology of sonar and echo sounders. Further, fish and other seafood are highly perishable products when stored without chilling. They deteriorate at ambient temperature in a few days, and only correct storage of wet fish in melting ice or of certain products at chilled temperatures can prolong the shelf life up to weeks or months. The total world seafood supply for 2007 amounted to 143 million tons. The world’s aquaculture provided 52 million tons (36%), and the captured fish, 91 million tons (64%) of the total supply. Although the amount of captured fish is almost constant at a level around 90 million tons/ year since 1990 after a continuous growth for more than 40 years, aquaculture is dramatically growing (1960: 2 million tons, 1970: 4 million tons, 1980: 7 million tons, 1990: 16 million tons, 2000: 40 million tons, including plants) [1]. The stagnation of captured fish is mainly due to fully exploited or partially overfished stocks. The most important primary product producing countries of marine and inland (freshwater) fisheries in 2005 were China (17.1 million tons), Peru (9.4 million tons), the United States (4.9 million tons), Indonesia (4.4 million tons), Chile (4.3 million tons), Japan (4.1 million tons), India (3.5 million tons), Russia (3.2 million tons), Thailand (2.6 million tons), and Norway (2.4 million tons). The top 10 species being caught in huge amounts in 2005 were Anchoveta (10.2 million tons), Alaska Pollock (2.8 million tons), Atlantic herring (2.3 million tons), Skipjack tuna (2.3 million tons), Blue whiting (2.1 million tons), Chub mackerel (2.0 million tons), Chilean jack mackerel (1.7 million tons), Japanese anchovy (1.6 million tons), Largehead hairtail (1.4 million tons), and Yellowfin tuna (1.3 million tons). Most fish was caught in the Pacific Ocean (Northeast and Southeast) followed by Northeast Atlantic Ocean. The major aquaculture (excluding plants) producers (>1 million tons) in 2005 were China (32.4 million tons, whereof the major part are cyprinids like carp), India (2.8 million tons), Vietnam (1.4 million tons, mostly Pangasius species), Indonesia (1.1 million tons), and Thailand (1.1 million tons). By major groupings, fish is the top group in aquaculture at 47.4% by quantity. Aquatic plants that are popular in Southeast Asia are second in quantity at 23.4%, whereas crustaceans are fourth by quantity at 6.2% but second by value at 20.4%. Mollusks (bivalves and cephalopods) are the third most important group both by quantity and by value at 22.3% and 14.2%, respectively. About 75% of the world’s total seafood supply is used for human consumption, 25% is converted into fishmeal and other nonfood products, 40% is consumed as wet fish without any further technological processing or preservation, about 20% is converted into deep frozen products, 8% is transformed into cured products, and another 8% into canned products.
Introduction ◾ 5
1.2 Variability of Aquatic Animals The variability of aquatic animals can be described and explained in many different ways. Based on taxonomic criteria, we have different groups such as bony and cartilaginous fishes, crustaceans, and mollusks, which are very different from each other in appearance, composition, and nutritive properties. When concentrating on fish as the major group contributing to the world’s fish supply, we arrange them in order according to their shape into round fish, flat fishes, eellike fishes, and so forth, or according to their occurrence in the ocean’s water column into pelagic fish, bottom fish, demersal fish, and ground fish. We can also group them according to their fat content into three groups: lean fish species (<1% fat), medium fatty fish species (>1% to <10% fat), and fatty fish species (>10% fat). However, these are all very rough classifications. In addition, the main difficulty in the analysis of fish and other seafood is that there is not only a big variation between groups of species and species but also within a given species. Not only weight and length are varying with age but also other factors such as proximate composition, mineral, and trace element content, which are subject to variations based on state of maturity, fishing area, season, pollution of water, and so on. This means that each fish can be different and unique, and before analyzing fish, a careful consideration has to be made if the variation is important and if it is worth or essential knowing (leading to analysis of individuals) or if a more general impression about the target component is sufficient (pooled samples). A drastic example illustrating the variability in fish is the Atlantic mackerel. The prespawning fish can have a fat content in fillet up to 35%, and the spawned fish can exhibit fillet fat contents of down to 5%. Mackerel is a typical pelagic swarm fish occurring in big schools. When captured during the spawning season, in one haul specimen of 5% fat and 35% fat are present. This can lead to extreme problems not only in processing but also in analysis, since parallel with fat content, other parameters such as organic pollutant concentrations vary. Also within the fish body, a certain degree of variability is found. Components like water, fat, and protein are not even distributed in the edible part and also trace element concentrations vary from head to tail or back to belly. With all these variations in the raw seafood material before the analysis of any components, decisions must be made where the results should be used and how detailed an analysis must be.
1.3 Special Problems with Aquatic Animals The main problem with aquatic animals is the fact that from the moment that they are caught or harvested, a change in properties starts, which continues until a state of spoilage is reached. After catch and harvest, not only spoilage and freshness parameters are changing due to metabolic (autolytic) and microbiological processes but also the microbial flora is changing. Besides this more general aspect, some groups offer special problems to which a lot of attention has to be given: aquatic animals may contain parasites (e.g., nematodes, cestodes) that can be harmful to humans when they enter live and intact into the human body. Predatory fish species such as sharks, which are at the end of the marine food web, can accumulate mercury during their long life span to quantities that exceed legal limits. Toxins from dinoflagellates can accumulate in bivalve mollusks, leading to several diseases such as diarrhetic shellfi sh poisoning (DSP), paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), and
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amnesic shellfish poisoning (ASP), and in fish, leading to ciguatera or maitotoxin poisoning. In the digestive glands of mollusks (hepatopancreas) such as cephalopods and mussels, cadmium is accumulated to amounts that exceed any legal limits by far. When not eviscerated immediately after catch, cadmium from hepatopancreas penetrates into the edible part (mantle) during storage, leading to elevated cadmium concentrations also in this body compartment. In products that have not undergone thermal treatment and that are offered to the consumer as ready to eat (e.g., cold smoked products, gravad products, sushi, and sashimi), there is an inherent microbial risk. Fish and other aquatic animals from areas that are polluted (rivers, inshore waters, estuaries, seas with no or limited water exchange with world oceans such as Baltic Sea, Mediterranean Sea, Caspian Sea, or Black Sea) can carry a high burden of environmental pollutants, especially in their organs responsible for detoxification such as liver and kidney. Aquatic animals from some areas of the world can carry viruses and microorganisms (e.g., Vibrio sp.) that are harmful to human health and must be destroyed or removed before marketing of the products.
1.4 Benefits and Risks Seafood is a rich source for a great number of nutritive and important components. The high amount of long-chain polyunsaturated fatty acids of the n-3 series such as eicosapentanoic acid (20:5) and docosahexanoic acid (22:6); the vitamins A, D, E, and B12; the well-balanced content of essential amino acids; the high amount of taurine; the presence of antioxidants such as tocopherols; the exceptional concentrations of essential elements such as selenium and iodine; and the good digestibility of fish protein due to low amounts of connective tissue are some examples of the many benefits seafood offers when consumed. On the other hand, we have the risk of viruses and microorganisms; we are confronted with toxins in mussels and fish; we have sometimes a parasitical problem; we may find high amounts of inorganic toxic elements and organic pollutants (POP, persistent organic pollutants), and residues of pharmaceuticals and hormones used in aquaculture can be detected and more. All of these parameters and substances have to be carefully analyzed and quantified to allow a risk benefit analysis, which can give reliable advice and guidance for wise and responsible seafood consumption. Unfortunately, only few quantitative analytical data have entered these assessments, with the consequence that recommendations are mostly restricted to a few factors being appropriately analyzed but not based on all factors. Considering the great variability of seafood described here, a tremendous amount of analytic work in seafood has to be done.
1.5
Sampling
Sampling, which means here the selection of an appropriate number and part of aquatic animals under well-defined conditions, is very often underestimated. Most errors and most erroneous results arising from analytical methods are based on poor or even wrong sampling plans and practices. Before starting the sampling procedure, a sampling plan has to be developed describing the numbers of samples to be taken, the body compartments to be dissected, and the measures to be taken to avoid any contamination as well as the storage and transport conditions of the samples after sample preparation.
Introduction ◾ 7
The number of individuals should be big when a small specimen has to be analyzed, smaller when medium-sized animals are the target, and only a few samples are taken from big individuals. In small specimens that are consumed totally, the whole body may be sampled and analyzed (mussels, sprat, snails); in medium-sized specimen, always the whole edible part (fillet, tail muscle) must be taken due to intrinsic variations in fillet parts and after homogenization subsamples can be taken; and in big fish (tuna, shark), it is advisable to concentrate on a muscle part that is simple to identify and can be dissected without destroying the fish completely (examples are muscle below gill cover, head end, or tail end of fillet). While sampling is done, precaution must be taken not to contaminate the sample by instruments used during manipulation (scissors, knives) or by protective clothes or gloves. When sampling is done onboard a vessel, a careful selection of individuals that have not been mechanically damaged by the catching technique, other species or mud, sand, and so forth, is necessary. When sampling for later microbiological analyses, it has to be made under strict hygienic conditions to avoid any microbial contamination. After sampling is completed successfully, it is recommended to store all samples (also solutions) in deep frozen conditions (<−18°C, preferably at −30°C) until analysis.
1.6 Analytical Methodologies The improvement and further development of analytical methods in the field of seafood research in Europe were initiated and brought forward by a number of research projects and concerted actions (CA) financed by the European Union within the research and technological development (RTD) framework programs 3 to 6. These projects brought the scientists together in conferences, workshops, and practical work-ins and allowed on-site measurements, calibrations, and comparative analyses with different instruments. The first concerted action in this area was “Evaluation of fish freshness” from 1995 to 1997, and the second concerted action was “Fish quality labeling and monitoring” (FQLM) from 1998 to 2000. To be also mentioned are the research project “Multisensor techniques for monitoring the quality of fish” (MUSTEC) from 1999 to 2002 and the research project SEQUID “A new method for measurement of the quality of seafood” from 2001 to 2003. The main results of these projects have been published in books [2–4,14] that form a very rich source of information about seafood analysis. In addition, two books shall be mentioned that have been published earlier but still contain a significant amount of basic knowledge about analytical methods for seafood quality determination [5,6]. The analytical methods used for seafood analyses can be divided into objective methods and sensory methods. The objective methods are chemical/biochemical methods, physical methods, and microbiological methods. The chemical/biochemical methods are mostly traditional methods that were developed earlier than the physical (instrumental) methods and have been mostly applied as methods for freshness/spoilage determinations. Methods that are still in use are among others k-value, which is based on ATP breakdown products, analysis of trimethyl amine, dimethyl amine, ammonia, trimethyl amine oxide, and total volatile basic nitrogen (TVB-N); determination of thiobarbituric acid and formaldehyde; and analysis of biogenic amines as histamine or cadaverine. More chemical methods have been developed for differentiation between fresh and frozen/thawed products (see Chapter 48) and for species identification and authenticity (see Chapters 37 and 38). Another method that was developed recently is the two-dimensional gel electrophoresis (2DE).
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Physical methods comprise microscopy, pH measurement, texture and texture profile analysis (e.g., Kramer test, Warner–Bratzler test, puncture, tensile, and compression tests, viscoelastic methods such as stress relaxation, creep, and oscillatory measurements); analysis of electrical resistance or conductivity by Torrymeter, Intellectron Fischtester VI, RT Freshness grader, and time domain spectroscopy (TDR) [7]. Further, we have color measurement, image analysis, differential scanning calorimetry (DSC), near-infrared spectroscopy (NIR), UV and visible light spectroscopy, nuclear magnetic resonance (magnetic resonance imaging (MRI), low-field (LF) NMR, and high-resolution NMR (HR-NMR)), which are noninvasive and nondestructive techniques for the sample. Other methods that are rapid, not harmful for the operator, and nonpolluting for the environment are, among others, electronic noses and electronic tongues [14]. Frequently used microbiological methods are total viable count (TVC), determination of specific spoilage organisms (SSO), polymerase chain reaction (PCR), oligonucleotide probes, antibody techniques, and bacterial sensors. Sensory methods are often considered to be subjective methods. However, a well-trained sensory panel in which the human senses are used as measuring instruments has been shown to give reliable, reproducible, and objective results. The sensory methods can also be divided into two principal methodologies: methods based on outer inspection of the sample (without cooking) and methods based on assessing the cooked sample. Outer inspection is done by the European Union quality-grading scheme and by the quality index method (QIM), whereas the Torry sensory scheme and the flavor profile analysis are performed on cooked samples. Two more systematic methods that involve some analytical methods are the hazard analysis critical control point (HACCP) and traceability.
1.7
Analytical Problems
Despite the great progress that has been made, there are still many problems left in the analysis of seafood and seafood-based products. The main problem is that there is no single method existing that can give sufficient information about the quality (freshness) of seafood, with the exception of sensory methods. Sensory methods, however, are time consuming, need trained personal, and are, therefore, expensive. Instrumental methods are fast, comparatively cheap, and can be used after a short training period by nonscientific educated personnel. When using instrumental methods nowadays, always a combination of several methods is necessary to give sufficient information equal to sensory assessment [8]. An instrumental method that is fast, cheap, can be used by untrained personal, and can be applied on many species and also on processed seafood products, with the same degree and quality of information obtained by sensory assessment, is still missing. Although many instrumental analytical methods have been developed and have been intensively tested and proven in research to be working sufficiently and reliably on seafood and seafood products, many of them have not graduated from research to seafood industrial application. The reasons are the relatively complex and difficult handling of the instruments and the need of being applied and maintained by educated personnel, which is usually not present in seafood industry. The European seafood sector where the majority of enterprises are small and medium sized (SMEs) hesitate to apply new instrumentation and prefer to rely on the methods they know, are experienced in, and have used since many years [9]. It is, therefore, of utmost importance to introduce the newly developed analytical methods into the industry for better product and raw material analysis and quality assurance.
Introduction ◾ 9
For some analytical methods, more research is needed to make them simpler to apply and to increase the speed of analysis. This holds for all methods for species differentiation, for almost all microbiological methods, and for many methods of trace element and residue analysis. In the area of sensory methods, it is necessary that the schemes for the QIM as the quality method of the future are extended to all species on the market (about 100). New methods are also urgently needed for the reduction of microbial risk, bacterial pathogens, and virus contamination in seafood. In this field, however, remarkably developments have occurred very recently [10–12]. There are many seafood products on our markets that have not been characterized by analytical methods at all. Many exotic fish, crustacean, and mollusk species from tropical and subtropical countries enter our markets in large quantities or as single fish specimen and are not thoroughly investigated for their microbiological status including viruses, their spoilage characteristics and shelf life, the presence of parasites, toxins such as ciguatera, inorganic and organic residues, allergens, sensory characteristics, food additives, pharmaceuticals, and contents of all the beneficial components. Th is is a large area where a significant amount of analytical input is needed.
1.8 Trends and Outlook In the future, most chemical and biochemical analytical methods that use a huge amount of chemicals and manpower will be substituted by instrumental methods that are more reliable, more cost efficient, and more environmental friendly. Some methods that are well known such as k-value or TVB-N will disappear. Analytical instruments that are simple to use, robust, and have a wide range of applicability will be built. This next generation of instruments will then also find its way into the fish industry and fish inspection. Although the lipids in seafood are analyzed very intensively, the protein and peptides are analyzed to a much lesser extent. Recent findings show that seafood contains important functional proteins and peptides [13]. More research and development of analytical methodology will be initiated by these new findings. The QIM will be further developed, and QIM schemes will also be developed for exotic species on our markets and for processed products. QIM will be digitalized and will work in combination with image analysis and electronic nose without sensory experts involved. PCR-based methods will soon take the place of the traditional microbiological methods and will enable the checking of microbiologic status of samples in minutes or hours. This will shorten delays in seafood trade. Almost all analytical methods for seafood analysis will be developed further to avoid time and chemicals and to minimize sample preparation and digestion steps. The method of the future will analyze a well-homogenized sample without any other sample preparatory steps except homogenizing. However, all progress in analytical methods and instrumentation needs an analyst who is responsible and follows the guidelines and advice for analytical quality assurance. Without a well-documented and traceable analytical quality assurance (reference materials, proficiency tests, own standards, justification of methods used, sampling strategy) showing that the results obtained are accurate and correct, journals will in the near future no longer accept manuscripts in this field.
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References 1. Anon., FAO Fisheries Department. State of world aquaculture 2006. FAO Fisheries Technical Paper. No. 500. Rome, FAO. 2006. 134p. 2. Olafsdottir, G., Luten, J., Dalgaard, P., Careche, M., Verrez-Bagnis, V., Martinsdottir, E., and Heia, K. (Eds.), Methods to Determine the Freshness of Fish in Research and Industry, International Institute of Refrigeration, Paris, 1998, 396p. 3. Luten, J.B., Oehlenschläger, J., and Olafsdottir, G. (Eds.), Quality of Fish from Catch to Consumer—Labelling, Monitoring and Traceability, Wageningen Academic Publishers, Wageningen, 2003, 456p. 4. Luten, J.B., Jacobsen C., Bekaert, K., Sæbø, A., and Oehlenschläger, J. (Eds.), Seafood Research from Fish to Dish, Wageningen Academic Publishers, Wageningen, 2006, 567p. 5. Botta, J.R., Evaluation of Seafood Freshness Quality, VCH, New York, 1995, 180p. 6. Connell, J.J., Control of Fish Quality (3rd edn.), Fishing News Books, Farnham, Surrey, 1990, 227p. 7. Kent, M., Knöchel, R., Barr, U.-K., Tejada, M., Nunes, L., and Oehlenschläger, J. (Eds.), SEQUID: A New Method for Measurement of the Quality of Seafood, Shaker Verlag GmbH, Aachen, 2005, 216p. 8. Olafsdottir, G. et al., Multisensor for fish quality determination, Trends Food Sci. Technol., 15, 86, 2004. 9. Jørgensen, B.M. et al., A study of the attitudes of the European fish sector towards quality monitoring and labeling, in Quality of Fish from Catch to Consumer—Labelling, Monitoring and Traceability, Luten, J.B., Oehlenschläger, J., and Olafsdottir, G. (Eds.), Wageningen Academic Publishers, Wageningen, 2003, 57p. 10. Bosch, A. et al., Detecting virus contamination in seafood, in Improving Seafood Products for the Consumer, Børresen, T. (Ed.), Woodhead Publishing Limited, Cambridge, U.K., 2008, 194p. 11. Pommepuy, M. et al., Reducing microbial risk associated with shellfish in European countries, in Improving Seafood Products for the Consumer, Børresen, T. (Ed.), Woodhead Publishing Limited, Cambridge, U.K., 2008, 212p. 12. Lee, R.J. et al., Bacterial pathogens in seafood, in Improving Seafood Products for the Consumer, Børresen, T. (Ed.), Woodhead Publishing Limited, Cambridge, U.K., 2008, 247p. 13. Thorkelsson, G. et al., Mild processing techniques and development of functional marine protein and peptide ingredients, in Improving Seafood Products for the Consumer, Børresen, T. (Ed.), Woodhead Publishing Limited, Cambridge, U.K., 2008, 363p. 14. Rehbein, H., Oehlenschläger, J. (Eds.), Fishery Products—Quality, Safety and Authenticity, WileyBlackwell, 2009, 477p.
Chapter 2
Peptides and Proteins Turid Rustad Contents 2.1 Introduction ......................................................................................................................11 2.2 Total Content of Proteins ................................................................................................. 12 2.3 Protein Solubility Classes ..................................................................................................13 2.4 Analysis of Soluble Proteins ...............................................................................................14 2.5 Immunoassays ...................................................................................................................16 2.6 Electrophoresis-Based Methods .........................................................................................16 2.7 Peptide Characterization ...................................................................................................16 2.8 Protein Modifications........................................................................................................17 References ..................................................................................................................................18
2.1
Introduction
Protein analysis is highly important for the food industry, including the fish industry. Both the content and the properties of the proteins are important for the value and the quality of the products [1]. Both for quality control and food labeling it is therefore important to have methods to determine not only the total content of proteins in a raw material or a product, but it is also important to have methods to determine the type and the origin of the proteins present. For product and process development it is important to have methods to determine the properties of the proteins and how these change during processing and storage, and how these properties are influenced by food additives and other components. Fish provides about 14% of the world’s need for animal proteins and 4%–5% of the total protein requirement [2]. Both the amino acid composition and the digestibility of fish proteins are excellent. Fish are regarded as an excellent source of high-quality protein, particularly the 11
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essential amino acids lysine and methionine. In addition to the high nutritional value, fish proteins also have good functional properties such as water-holding capacity, gelling, emulsification, and textural properties. For products such as fish mince and surimi, the water-holding capacity and the gelling properties which determine the textural attributes of the products are important quality parameters [3]. Retaining the functional properties through preservation and processing operations is therefore of great importance.
2.2
Total Content of Proteins
The total content of proteins is usually determined by the Kjeldahl or the Dumas method. It is also possible to determine the nitrogen content using elemental analysis (C/N analyzers) [4]. It is important that the methods to analyze food proteins are robust [1]. This means that it should be possible to use the method on different types of foods, both different types of raw materials and processed foods, and that other components in the food such as lipids and pigments should not interfere with the analysis. The method should also require minimal sample pretreatment to decrease analytical error and reduce costs. The Kjeldahl method was first published in 1883 but has been extensively modified since then. The method includes sample digestion, neutralization, distillation, and trapping of ammonia and titration steps. The advantage of this method is that it gives accurate results for all types of samples. This method is used as a reference method by many national and international organizations. The disadvantage is that the method requires use of hazardous and toxic chemicals. The Dumas method is quicker and cheaper and easier to perform and is therefore now considered on equal terms with the Kjeldahl method [1]. The Kjeldahl method determines the nitrogen content as ammonia, and the amount of protein is calculated by multiplication with a Kjeldahl factor. This factor is the amount of nitrogen that contains 1 g of nitrogen. For animal proteins the value 6.25 is usually used, assuming a nitrogen content of 16% in the proteins. Many proteins have protein contents that deviate from this, for instance collagen has a nitrogen content of 18%, which gives a factor of 5.56 [1]. The factor can be calculated from the amino acid composition of the proteins. Tables of conversion factors are given in several papers such as [1,5]. For fish, values from 5.43 to 5.82 are given. Mariotti and coworkers discuss conversion factors in their critical review and conclude that even if a factor of 6.25, which has been used for more than 75 years, has been shown to be too high for animal proteins, it is difficult to start using other and more correct factors [5]. When the protein content is calculated based on determination of the nitrogen content, nitrogen from other nitrogen-containing compounds such as free amino acids, nucleotides, amines, and urea will also contribute to the calculated protein content. Originally only sulfuric acid was used for digestion of the samples, but other chemicals such as potassium sulfate and mercury oxide are also used. During digestion the nitrogen in the sample is converted to ammonium sulfate. The ammonium sulfate is converted into ammonia, which is distilled and trapped in boric acid, and the amount of nitrogen is determined by titration [1]. It is also possible to determine the amount of ammonia by different colorimetric methods [1]. The Kjeldahl method has been automated and several instruments for automated analysis are available. The Kjel-Foss® instrument mechanizes the entire micro-Kjeldahl procedure while the Kjel-Tec® instrument uses a digestion block together with an apparatus for automated distillation and titration. The Technicon AutoAnalyzer uses continuous flow analysis [1].
Peptides and Proteins ◾ 13
The Dumas method was first published in 1831 and the first instruments used were not user friendly. Today accurate combustion nitrogen analyzers are used. The sample is put in a furnace (950°C–1050°C), purged free of atmospheric gas, and filled with pure oxygen. After cooling of the gas mixture, the CO2, SO2, O2, and water are removed; the NO2 is reduced to N2 and measured with a thermal conductivity meter [1]. The combustion method has been calibrated with the Kjeldahl method and this has led to approval of the method by Association of Official Analytical Chemists (AOAC), American Oil Chemists’ Society (AOCS), and several other organizations [1]. Quantitative amino acid analysis is one of the most reliable methods for quantification of food proteins. This procedure involves hydrolyzation of the food sample using concentrated hydrochloric acid, determination of the amino acid profile, and calculation of the amount of different amino acids. The principle of quantitative amino acids is described in Owusu-Apenten [1]. It is also described in Chapter 16. Near infrared spectroscopy can also be used to determine protein content. The advantages of this method are that it is rapid, nondestructive, and can be used online. It is easy to perform, but the instrumentation is expensive and the method requires calibration. It has been successfully used to determine protein and water content of salmon fillets [6] as well as of surimi [7]. It can also be used to determine the properties of food proteins [8] and has been used to detect adulteration of beef with animal and plant proteins as well as classify tenderness of beef in two categories.
2.3
Protein Solubility Classes
Fish muscle proteins can be divided into three groups, sarcoplasmic, myofibrillar, and connective proteins, based on differences in solubility [9,10]. The sarcoplasmic proteins consist mainly of enzymes and can be extracted using water or buffers with low ionic strength such as for instance 50 mM phosphate buffer. The myofibrillar proteins, also called the salt-soluble proteins can be extracted in buffers with an ionic strength of >0.3. The connective tissue proteins are often called the insoluble proteins and can be extracted using alkali or acid. The methods for extraction are not standardized so the amount of proteins extracted will vary with the method used. However, changes in solubility can be used to measure changes in protein structure caused by denaturation during storage and processing. Fish muscle proteins are more sensitive and less stable than proteins from mammals. A few examples of methods to extract proteins from fish muscle are given here. Hultmann and Rustad [11] used a modification of the method by Anderson and Ravesi [12] and Licciardello and coworkers [13]. Four grams of muscle was homogenized for 20 s in 80 mL 50 mM phosphate buffer, pH 7. After centrifugation, the supernatant was decanted and the volume made up to 100 mL—this was the water-soluble fraction. The precipitate was homogenized in 80 mL phosphate buffer with 0.5 M KCl and centrifuged as above. The volume of the supernatant was made up to 100 mL. This was the salt-soluble fraction. Martinez-Alvarez and Gomez-Guillen [14] used a modification on the method of Stefansson and Hultin [15]. The soluble protein was extracted in distilled water (low ionic strength), and in 0.86 M NaCl solution (high ionic strength). Two grams of minced muscle was homogenized at low temperature for 1 min in 50 mL of distilled water. The homogenates of these solutions were stirred constantly for 30 min at 2°C, then centrifuged (6000 g) for 30 min at 3°C. Kelleher and Hultin compared the use of NaCl, KCl, and LiCl for extraction of protein from fish muscle and concluded that LiCl was a better extractant of fish muscle proteins over a wider range of conditions than NaCl or KCl [16].
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Solubility of collagen can be determined by extraction in alkali or acid as described by Eckhoff and coworkers [17], which is a modification of the method described by Sato et al. [18]. Samples were homogenized in 0.1 M NaOH and centrifuged. The extraction in NaOH was repeated five times and the supernatants were pooled for the analysis of alkali-soluble collagen content. The precipitate was stirred with 0.5 M acetic acid for 2 days at room temperature and centrifuged as above. This was the acid-soluble collagen. After extraction, the concentration of the soluble proteins can be analyzed with a wide variety of methods. Since all proteins absorb UV/visible light to varying degrees, one of the simplest methods is to determine absorbance in the far UV range. The protein concentration can then be calculated from the Lambert–Beer law: A = ε cl where A is the absorption at a given wavelength c is the molar protein concentration l is the path length for the light (cm) e is the molar absorption or extinction coefficient (M−1 cm−1) The molar absorptivity can be determined by dry weight estimation of a purified protein, by absorbance at 205 nm or from knowledge of amino acid composition [19]. Measurement of UV absorption at 280 nm is a simple and popular method to determine protein concentration. However mg quantities of protein are generally required. Absorption at 280 nm is mainly due to tryptophan and tyrosine residues with smaller contributions from phenylalanine and the sulfur-containing amino acids, the method therefore has protein-to-protein variations. In addition, the presence of nonprotein UV-absorbing groups such as nucleic acids and nucleotides which absorb strongly at 260 nm further complicate matters. Light scattering because of large particles or aggregates can also lead to errors. Methods exist to correct for the influence of light scattering and nucleic acids/ nucleotides [19].
2.4 Analysis of Soluble Proteins There are many indirect colorimetric methods to determine protein content, and a few of them will be treated here. The Biuret method is based on the formation of complexes between copper salts and peptide bonds under alkaline conditions. The purple complex is relatively stable and has an absorption maximum at 540–560 nm. A standard curve is needed, but the method is simple and inexpensive. The method is not very sensitive, measuring concentrations between 1 and 10 mg/mL. The sensitivity can be increased by measuring absorbance at 310 nm or by increasing the time for the Biuret reaction. However, some of these methods reduce the speed and simplicity of the method [19]. The Lowry method [20] is based on a Biuret-type reaction between protein and copper(II) ions under alkaline conditions, the complexes react with the Folin-phenol reagent a mixture of phosphotungstic acid and phosphomolybdic acid in phenol. The product becomes reduced to molybdenum/tungsten blue and can be measured at 750 nm. The reactions are highly pH dependent. Peterson have reviewed the Lowry method [21] and listed interfering substances, giving upper tolerable limits for a long range of these as well as some methods for coping with the effect of these substances. Reducing agents and sucrose as well as several common buffers interfere with the Lowry method. The review also discusses many of the modifications that
Peptides and Proteins ◾
15
have been suggested for the Lowry method. Finally he compares the Lowry method with other methods to determine protein concentration and concludes that the advantages of the Lowry method are simplicity, sensitivity, and precision, the disadvantages are interfering substances and time—compared to some of the dye-binding methods such as the Coomassie Blue methods. Use of bicinchonic acid (BCA) was introduced as an easier way to determine protein; it uses only one reagent instead of two as in the Lowry procedure [1]. Sensitivity is similar to the Lowry procedure, but detergents, buffer salts, and denaturing agents such as urea and guanidine hydrochloride cause less interference. However, for lipids, reducing agents, chelators such as EDTA, and acids and alkali cause interference. There are different dye-binding methods, and one of the most widely used is the Biorad method based on binding of Coomassie Brilliant Blue G (CBBG) [22]. This method is based on the color change taking place when CBBG binds to proteins under acidic conditions. Th is method is faster to perform than the Lowry procedure (5 min development compared to 30–45 min), and stable reagents and kits are available. The method is compatible with a wide range of buffers/substances. The Coomassie Brilliant Blue method is also used for visualizing proteins in electrophoretic gels. The ability of proteins to bind silver has also been used as a very sensitive method to visualize proteins in gel electrophoresis. The silver staining methods are 100 times more sensitive than the CBBG staining. Silver binding is also being used as a method to analyze concentration of soluble proteins [19]. All the methods discussed above are highly protein dependent and this should be kept in mind when applying these methods for analysis of the protein content. It would be best if the protein being analyzed could be used as the standard protein; however, this is often not possible or practical. The Lowry method determines both proteins, small peptides and free amino acids, while methods such as Biuret and Biorad only determine peptide chains above a certain length. However, as different amino acids and peptides give different colors in the Lowry method, the method is highly protein dependent (Table 2.1). The amount of collagen can be determined by analysis of the hydroxylysine content by the Neuman and Logan method as modified by Leach [23]. Hydroxylysine is an amino acid that is almost exclusively found in collagen. However, an accurate determination requires that the amount of hydroxylysine residues per 100 residues in the collagen is known. Th is figure varies for different collagen types such as collagen from fish skins from different fish species [24]. Table 2.1 Comparison of Useful Range for Methods to Determine Protein Concentration Method
Range (μg)
Kjeldahl
500–30,000
Biuret
1,000–10,000
Lowry
10–300
Biorad (Coomassie Brilliant Blue)
20–140
Biorad (Coomassie Brilliant Blue)—micro
1–20
Bicinchonic acid
1–50
Absorption at 280 nm
100–300
16 ◾ Handbook of Seafood and Seafood Products Analysis
2.5
Immunoassays
The amount of a specific protein in a mixture can be determined by enzyme-linked immunosorbent assays (ELISA). It is then necessary to have the antibody of the protein that one seeks to quantify. A polyclonal or monoclonal antibody against the protein of interest is then bound to a film through the Fc region of the antibody. Bovine serum albumin (BSA) is then added to block nonspecific binding sites. After washing, a second antibody is bound to the protein bound to the primary antibody. The amount of secondary antibody bound is proportional to the amount of the specific protein in the sample. The secondary antibody is usually linked to peroxidase or alkaline phosphatase. These enzymes can convert a colorless substrate to a colored product which can then be detected. The method is very sensitive but requires available antibodies.
2.6 Electrophoresis-Based Methods The molecular weight of proteins and peptides is often of interest and this can be determined by several different methods. In native gel filtration chromatography, the proteins are separated based on their size and shape (Stokes’ radii). For low- and medium-pressure chromatography, beads are made of open, cross-linked three-dimensional polymer networks such as agarose, dextrans, cellulose, polyacrylamide, and combinations of these. For high-pressure systems, macroporous silica, porous glass, or inorganic–organic composites are used as support media [1]. Small proteins can enter all the pores in the beads, while larger proteins can only enter the largest pores. As the protein solution moves down the column, smaller proteins will, on the average, spend more time inside the beads and the larger proteins will emerge from the column first. How a certain protein behaves in a gel filtration column can be described by the coefficient Kav which defines the proportion of pores that are accessible to that molecule. Kav = (Ve − V0)/(Vt − V0), where Ve is the elution volume of the molecule, V0 is the void volume of the column, and Vt is the total volume of the column. By using standard proteins of known molecular weight, a standard curve can be made allowing determination of the molecular weight distribution in a protein mixture. Molecular weight can also be determined by electrophoresis. One of the most commonly used methods is SDS-PAGE, using gels of polyacrylamide and denaturing the samples by boiling in a solution of sodium dodecyl sulfate (SDS). SDS binds to proteins in a weight ratio of 1:1.4, which gives one SDS molecule for every two amino acids. Since SDS is charged, this results in a charged complex where the charge is proportional to the molecular weight of the protein. Dithiothreitol (DTT) or mercaptoethanol is often added to reduce disulfide bonds. The most commonly used system is that of Laemmli [25]. The denatured proteins are applied to the gel and an electric current is applied, causing the negatively charged proteins to migrate across the gel toward the anode. The proteins will migrate based on their size; smaller proteins will travel farther down the gel, while larger ones travel a shorter distance. By using markers of known molecular weight, a standard curve can be made in the same way as for gel chromatography and the weight of the unknown proteins determined.
2.7 Peptide Characterization Studying the composition and properties of peptides in seafood is often of interest, for instance after enzymatic hydrolysis of proteins or during processing and storage of seafood. Many peptides are bioactive and have physiological properties, such as immunostimulating or antihypertensive
Peptides and Proteins ◾
17
properties. For characterization of mixtures of peptides, especially after enzymatic degradation/ hydrolysis, the term degree of hydrolysis describes the extent to which peptide bonds are broken by the enzymatic hydrolysis reaction. The measurement shows the number of specific peptide bonds broken in hydrolysis as a percent of the total number of peptide bonds present in the intact protein. Several methods to determine this value exist. One of these is the determination of free amino groups after reaction with trinitrobenzene-sulfonic acid (TNBS) [26]; this is spectrophotometric method determining the amount of the chromophore formed when TNBS reacts with primary amines. The reaction takes place under slightly alkaline conditions and is stopped by lowering the pH in the solution. Another widely used method is the determination of free amino groups after titration with formaldehyde [27]. Formaldehyde reacts with unprotonated primary amine groups resulting in loss of protons. The amount of liberated protons can be determined by titration. Studying the peptide fraction can give a lot of useful information as peptides may have several functions in the food. The peptides may also give valuable information about the quality of the food, such as provide information about the enzymes that are active during storage. For determination of the amount of peptides below a certain chain length, selective precipitation using ethanol, methanol, or trichloroacetic acid can be used [28]. The amount of peptides soluble in different concentrations of ethanol was found to be dependent on the chain length as well as on the hydrophobicity of the peptides. Precipitation of the proteins makes it possible to study peptides which are found in lower concentrations using different chromatographic methods such as LC–MS or electrophoretic methods. Mass spectroscopy can be used to determine the molecular mass of the peptides, and by using tandem mass spectroscopy detailed information of the structure of the peptides can be found. Bauchart and coworkers [29] studied the peptides in rainbow trout using precipitation with perchloric acid followed by electrophoresis and MS-analysis in order to study proteolytic degradation.
2.8
Protein Modifications
During storage and processing of marine raw materials, changes take place in the proteins and it is often of interest to quantify these changes. In addition to lipids and pigments, muscle proteins are also vulnerable to oxidative attack during processing and storage of muscle foods [30]. Oxidation can occur at both the protein backbone and on the amino acid side chains, and can result in major physical changes in protein structure ranging from fragmentation of the backbone to oxidation of the side chains. Oxidation of protein side chains can give rise to unfolding and conformational changes in protein and also to dimerization or aggregation [31]. Oxidative modification often leads to alterations in the functional, nutritional, and sensory properties of the muscle proteins, including gelation, emulsification, viscosity, solubility, and water-holding capacity. Several methods are used to determine protein oxidation, the most used are determination of formation of carbonyl groups [32,33] and reduction in SH-groups. The content of sulfhydryl groups can be determined using DTNB by the method of [34] with the modification of [35]. Formation of dityrosine is also used to determine the degree of protein oxidation. In addition oxidation can be measured as loss of functional properties such as loss of solubility, loss of water-holding capacity, gelling and emulsification properties, and formation of aggregates. However, these properties are not only dependent on the oxidation state of the proteins, and changes in these properties may be due to other factors. Changes in proteins during storage and processing will often result in changes in the functional properties of the proteins. One much used definition of functional properties is this: Those physical and chemical properties that influence the behavior of proteins in food systems during
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processing, storage, cooking, and consumption [36]. A description of the properties of the proteins important for functional properties was given by Damodaran [37]: The physicochemical properties that influence functional behavior of proteins in food include their size, shape, amino acid composition and sequence, net charge, distribution, hydrophobicity, hydrophilicity, structures (secondary, tertiary, and quaternary), molecular flexibility/rigidity in response to external environment (pH, temperature, salt concentration), or interaction with other food constituents. Nutritional, sensory, and biological values are sometimes included in the functional properties. Functional properties can be divided in several groups. It is usual to classify them according to mechanism of action into three main groups: (1) properties related with hydration (absorption of water/oil, solubility, thickening, wettability), (2) properties related with the protein structure and rheological characteristics (viscosity, elasticity, adhesiveness, aggregation, and gelation), and (3) properties related with the protein surface (emulsifying and foaming activities, formation of protein–lipid films, whippability). Methods to determine functional properties are often developed for a particular use in a specific food system resulting in a vast number of different methods. It is therefore difficult to compare results from different laboratories. The book edited by Hall [38] gives a good overview of methods to determine protein functionality.
References 1. Owusu-Apenten, R.K., Food Protein Analysis: Quantitative Eff ects on Processing. New York: Marcel Dekker. 2002, p. 463. 2. Venugopal, V., Methods for processing and utilization of low cost fishes: A critical appraisal. Journal of Food Science & Technology, 1995. 32: 1–12. 3. Venugopal, V. and F. Shahidi, Value added products from underutilised fish species. Journal of Food Science & Nutrition, 1995. 35: 431–435. 4. Kirsten, W.J., Automatic methods for the simultaneous determination of carbon, hydrogen, sulphur and sulphur alone in organic and inorganic materials. Analytical chemistry, 1979. 51: 1173–1179. 5. Mariotti, F., D. Tome, and P.P. Mirand, Converting nitrogen into protein—Beyond 6.25 and Jones’ factors. Critical Reviews in Food Science and Nutrition, 2008. 48: 177–184. 6. Isaksson, T. et al., Non-destructive determination of fat, moisture and protein in salmon fillets by use of near-infrared diff use spectroscopy. Journal of the Science of Food and Agriculture, 1995. 69: 95–100. 7. Uddin, M. et al., Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Food Chemistry, 2006. 96: 491–495. 8. Bock, J.E. and R.K. Connelly, Innovative uses of near-infrared spectroscopy in food processing. Journal of Food Science, 2008. 73: R91–R98. 9. Foegeding, E.A., T.C. Lanier, and H. Hultin, Characteristics of edible muscle tissue, in Food Chemistry, O.R. Fennema, Ed., Marcel Dekker: New York. 1996, pp. 879–942. 10. Haard, N.F., Control of chemical composition and food quality attributes of cultured fish. Food Research International, 1992. 25: 289–307. 11. Hultmann, L. and T. Rustad, Iced storage of Atlantic salmon (Salmo salar)—Effects on endogenous enzymes and their impact on muscle proteins and texture. Food Chemistry, 2004. 87: 31–41. 12. Anderson, M.L. and E.M. Ravesi, Relation between protein extractability and free fatty acid production in cod muscle aged in ice. Journal of Fisheries Research Board Of Canada, 1968. 25: 2025–2069. 13. Licciardello, J.J. et al., Time–temperature tolerance and physical-chemical quality tests for frozen Red Hake. Journal of Food Quality, 1982. 5: 215–234.
Peptides and Proteins ◾ 19 14. Martinez-Alvarez, O. and M.C. Gomez-Guillen, Effect of brine salting at different pHs on the functional properties of cod muscle proteins after subsequent dry salting. Food Chemistry, 2006. 94: 123–129. 15. Stefansson, G. and H.O. Hultin, On the solubility of cod muscle proteins in water. Journal of Agricultural and Food Chemistry, 1994. 42: 2656–2664. 16. Kelleher, S.D. and H.O. Hultin, Lithium chloride as a preferred extractant of fish muscle proteins. Journal of Food Science, 1991. 56: 315–317. 17. Eckhoff, K.M. et al., Collagen content in farmed Atlantic salmon (Salmo salar L.) and subsequent changes in solubility during storage on ice. Food Chemistry, 1998. 62: 197–200. 18. Sato, K. et al., Isolation of types I and V collagen from carp muscle. Comparative Biochemistry & Physiology, 1988. 90B: 155–158. 19. Yada, R.Y. et al., Analysis: Quantitation and physical characterization, in Food Proteins: Properties and Characterization, S. Nakai and H.W. Modler, Eds., VCH: New York, 1996. 20. Lowry, O.H. et al., Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 1951. 193: 265–275. 21. Peterson, G.L., Review of the Folin Phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Analytical Biochemitry, 1979. 100: 201–220. 22. Bradford, M.M., A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976. 72: 248–254. 23. Leach, A.A., Notes on a modification of the Neuman & Logan method for the determination of the hydroxyproline. Biochemistry Journal, 1960. 74: 70–71. 24. Almås, K.A., Muskelcellehylsteret hos torsk: Ultrastruktur og biokjemi, in Dep. Technicla Biochemistry. Norges Tekniske høgskole: Trondheim. 1981, p. 175. 25. Laemmli, U.K., Cleavage and structural proteins during assembly of the head of bacteriophage T4. Nature, 1970. 227: 680–685. 26. Adler-Nissen, J., Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry, 1979. 27(6): 1256–1262. 27. Taylor, W.H., Formol titration: An evaluation of its various modifications. Analyst, 1957. 82: 488–498. 28. Rohm, H. et al., Comparison of ethanol and trichloracetic acid fractionation for measurement of proteolysis in Emmental cheese. International Dairy Journal, 1996. 6: 1069–1077. 29. Bauchart, C. et al., Peptides in rainbow trout (Oncorhynchus mykiss) muscle subjected to ice storage and cooking. Food Chemistry, 2007. 100: 1566–1572. 30. Choe, E. and D.B. Min, Mechanisms and factors for edible oil oxidation. Comprehensive Reviews in Food Science and Food Safety, 2006. 5: 169–186. 31. Davies, M.J., The oxidative environment and protein damage. Biochimica Biophysica Acta, 2005. 1703: 93–109. 32. Baron, C.P. and H.J. Andersen, Myoglobin-induced lipid oxidation. A review. Journal of Agricultural and Food Chemistry, 2002. 50: 3887–3897. 33. Baron, C.P. et al., Protein and lipid oxidation during frozen storage of rainbow trout (Oncorhynchus mykiss). Journal of Agricultural and Food Chemistry, 2007. 55: 8118–8125. 34. Ellman, G.L., Tissue sulfhydryl groups. Archives in Biochemistry & Biophysics, 1959. 82: 70–78. 35. Sompongse, W., Y. Itoh, and A. Obtake, Effect of Cryoprotectants and a reducing reagent on the stability of actomyosin during ice storage. Fisheries Science, 1996. 62: 73–79. 36. Kinsella, J.E., Functional properties of food proteins: A review. CRC Critical Reviews Food Science & Nutrition, 1976, 7: 219–280. 37. Damodaran, S., Food Proteins: An Overview, in Food Proteins and their applications, S. Damodaran and A. Paraf, Eds., Marcel Dekker, Inc.: New York. 1997, pp. 1–24. 38. Hall, G.M., Ed., Methods for Testing Protein Functionality. Blackie Academic and Professional: London, U.K., 1996, p. 265.
Chapter 3
Proteomics Hólmfríður Sveinsdóttir, Ágústa Guðmundsdóttir, and Oddur Vilhelmsson Contents 3.1 Introduction ..................................................................................................................... 22 3.2 Proteome Analysis by 2DE ............................................................................................... 22 3.2.1 Sample Matrix Considerations .............................................................................. 22 3.2.1.1 Whole Larval Proteomes......................................................................... 22 3.2.1.2 Muscle Proteomes ................................................................................... 24 3.2.1.3 The Degradome ...................................................................................... 24 3.2.2 Basic 2DE Methods Overview ...............................................................................25 3.2.2.1 Sample Extraction and Cleanup ..............................................................25 3.2.2.2 First-Dimension Electrophoresis ..............................................................25 3.2.2.3 Equilibration .......................................................................................... 27 3.2.2.4 Second-Dimension Electrophoresis ......................................................... 27 3.2.2.5 Staining .................................................................................................. 28 3.2.2.6 Analysis .................................................................................................. 28 3.2.3 Protein Identification by Peptide Mass Fingerprinting .......................................... 28 3.3 Applications of 2DE in Seafood Analysis ..........................................................................31 3.3.1 Development .........................................................................................................31 3.3.2 Quality Involution ................................................................................................ 32 3.3.2.1 Protein Autolysis and Oxidation during Storage and Processing ............. 32 3.3.2.2 Aquaculture and Antemortem Effects on Quality and Processability ........33 3.3.3 Species Authentication .......................................................................................... 34 3.3.4 Allergen Identification .......................................................................................... 34 Acknowledgments ......................................................................................................................35 References ..................................................................................................................................35 21
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Handbook of Seafood and Seafood Products Analysis
3.1 Introduction As with all living matter, foodstuffs are in large part made up of proteins. This is especially true of fish and meat, where the bulk of the food matrix is constructed from proteins. Furthermore, the construction of the food matrix, both on the cellular and tissue-wide levels, is regulated and brought about by proteins. It stands to reason, then, that proteome analysis, also known as proteomics, is a tool that can be of great value to the food scientist, giving valuable insight into the composition of the raw materials, quality involution within the product before, during, and after processing or storage, the interactions of proteins with one another or with other food components, or with the human immune system after consumption. Proteomics, succinctly defined as “the study of the entire proteome or a subset thereof”1 is currently a highly active field possessing a wide spectrum of analytical methods that continue to be developed at a brisk pace. While high-throughput, gel-free methods, for example, based on liquid chromatography tandem mass spectrometry (LC–MS/MS),2 surface-enhanced laser desorption/ionization3 or protein arrays,4 hold great promise and are deserving of discussion in their own right, the “classic” process of two-dimensional (2D) gel polyacrylamide electrophoresis (2DE) followed by protein identification via peptide mass fingerprinting of trypsin digests (Figure 3.1) remains the workhorse of most proteomics work, largely because of its high resolution, simplicity, and mass accuracy. This chapter will therefore focus on 2DE.
3.2 Proteome Analysis by 2DE 2DE, the cornerstone of most proteomics research, is the simultaneous separation of hundreds, or even thousands, of proteins on a 2D polyacrylamide slab gel. The method most commonly used was originally developed by Patrick O’Farrell and is described in his seminal and thorough 1975 paper5 and briefly outlined, along with some of the main improvements that have developed since, in the following sections.
3.2.1
Sample Matrix Considerations
Unlike the genome, the proteome varies from tissue to tissue, as well as with time and in response to environmental stimuli. Selection of tissues for protein extraction is therefore an important issue that needs to be considered before a seafood proteomic study is embarked upon. Like other vertebrates, fish possess a number of tissues amenable to 2DE-based proteome analysis. Studies on whole larvae,6–8 liver,9–11 heart,12 kidney,12,13 skeletal muscle,14–18 gill,12 brain,12,19 intestine,12 and rectal gland12 have been reported. In the following sections, we present some issues and challenges related to sample matrices of particular interest to the seafood scientist.
3.2.1.1 Whole Larval Proteomes The production of good quality larvae is still a challenge in marine fish hatcheries. Several environmental factors can interfere with the protein expression of larvae leading to poor larval quality like malformations, growth depression, and low survival rate. Proteome analysis allows us to examine the effects of environmental factors on larval global protein expression,
Proteomics ◾
23
2D PAGE
Trypsin digestion
MS fingerprinting
MS/MS sequencing
Figure 3.1 An overview over the “classic approach” in proteomics. First, a protein extract (crude or fractionated) from the tissue of choice is subjected to 2D PAGE. Once a protein of interest has been identified, it is excised from the gel, subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry, yielding a peptide mass fingerprint. In many cases this is sufficient for identification purposes, but if needed, peptides can be dissociated into smaller fragment and small partial sequences obtained by MS/MS. See text for further details.
posttranslational modifications and redistribution of specific proteins within cells,20 all important information for controlling factors influencing the aptitude to continue a normal development until adult stages. Only a few proteome analysis studies on fish larvae have been published.6,7,21,22 Three of these publications have focused on the whole larval proteomes in Atlantic cod (Gadus morhua)6,22 and zebrafish (Danio rerio).7 These studies provided protocols for the production of high-resolution 2D gels. Peptide mass mapping using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed only on the cod larval proteins, allowing identification of ca. 85% of the of the selected protein spots.6,22 The advantage of working with whole larvae versus distinct tissues is the ease of keeping the sample handling to a minimum in order to avoid loss or modification of the proteins. Nevertheless, there are several drawbacks when working with the whole larval proteome, like the overwhelming presence of muscle and skin proteins. These proteins may mask subtle changes in proteins expressed in other tissues or systems, such as the gastrointestinal tract or the central nervous system. The axial musculature is the largest tissue in larval fishes as it constitutes approximately 40% of their body mass.23 This is reflected in our studies on whole cod larval proteome, where the majority of the highly abundant proteins were identified as muscle proteins.6,22 Also, cytoskeletal
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Handbook of Seafood and Seafood Products Analysis
proteins were prominent among the identified proteins. Removal of those proteins may increase detection of other proteins present at low concentrations. However, it may also result in a loss of other proteins, preventing identification of holistic alterations in the analyzed proteomes. Various strategies have been presented for the removal of highly abundant proteins24 or enrichment of lowabundant proteins.25,26
3.2.1.2 Muscle Proteomes In most seafood products, fish skeletal muscle is the main component. The fish muscle proteome is therefore likely to be of comparatively high interest to the seafood scientist. Structural proteins, such as actin and tubulin, are particularly abundant in the skeletal muscle proteome. An unfractionated 2DE map of the muscle proteome therefore tends to be dominated by comparatively few high-abundance protein spots, rendering analysis of low-abundance proteins difficult or impossible. Swamping of low-abundance spots by highly abundant ones may not be a problem for applications relating specifically to structural proteins, but for other applications low-abundance proteins, which include most regulatory proteins and many important metabolic enzymes, are of keen interest. No amplification method analogous to PCR exists for proteins, and simply increasing the amount of sample is usually not an option, as it will give rise to overloading artifacts in the gels.5 The remaining option, then, is fractionation of the protein sample in order to weed out the high-abundance proteins, allowing a larger sample of the remaining proteins to be analyzed. A myriad of methods suitable for subsequent 2DE exist for fractionating the proteome into defined subproteomes, such as those associated with individual organelles or cell compartments28 or by protein biochemical methods such as affi nity chromatography,29,30 preparative isoelectrofocusing31 or solubility in the presence of various detergents32 or chaotropes33 have been described. Fractionation methods for a variety of sample matrices have been reviewed recently.34–41
3.2.1.3 The Degradome The degradome may be a subproteome of particular interest to the food scientist, as many textural and other quality factors of muscle foods are related to proteolytic activity in the muscle tissue before, during and after processing. In addition to having a hand in controlling autolysis determinants, protein turnover is a major regulatory engine of cellular structure, function, and biochemistry. Cellular protein turnover involves at least two major systems: the lysosomal system and the ubiquitin–proteasome system.42,43 The 20S proteasome has been found to have a role in regulating the efficiency with which rainbow trout (Oncorhynchus mykiss) deposit protein.44 It seems likely that the manner, in which protein deposition is regulated, particularly in muscle tissue, has profound implications for quality and processability of the fish flesh. Protein turnover systems, such as the ubiquitin–proteasome or the lysosome systems, are suitable for rigorous investigation using proteomic methods. For example, lysosomes can be isolated and the lysosome subproteome queried to answer the question whether and to what extent lysosome composition varies among fish expected to yield flesh of different quality characteristics. Proteomic analysis on lysosomes has been successfully performed in mammalian (human) systems.45,46 An exploitable property of proteasome-mediated protein degradation is the phenomenon of polyubiquitination, whereby proteins are targeted for destruction by the proteasome by covalent
Proteomics ◾
25
binding to multiple copies of ubiquitin.43,47 By targeting these ubiquitin-labeled proteins, it is possible to observe the ubiquitin–proteasome “degradome,” i.e., which proteins are being degraded by the proteasome at a given time or under given conditions. Gygi and coworkers have developed methods to study the ubiquitin–proteasome degradome in the yeast Saccharomyces cerevisiae using multidimensional LC–MS/MS.2 Some proteolysis systems, such as that of the matrix metalloproteases, may be less directly amenable to proteomic study. Activity of matrix metalloproteases is regulated via a complex network of specific proteases.48–50 Monitoring of the expression levels of these regulatory enzymes, and how they vary with environmental or dietary variables, may be more conveniently carried out using transcriptomic methods.
3.2.2
Basic 2DE Methods Overview
O’Farrell’s original 2DE method first applies a process called isoelectric focusing (IEF), where an electric field is applied to a tube gel on which the protein sample and carrier ampholytes have been deposited. This separates the proteins according to their molecular charge. The tube gel is then transferred onto a polyacrylamide slab gel and the isoelectrically focused proteins are further separated according to their molecular mass by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), yielding a two-dimensional map (Figure 3.2) rather than the familiar banding pattern observed in one-dimensional (1D) SDS-PAGE. The map can be visualized and individual proteins quantified by radiolabeling or by using any of a host of protein dyes and stains, such as Coomassie blue, silver stains, or fluorescent dyes. Although a number of refinements have been made to 2DE since O’Farrell’s paper, most notably the introduction of immobilized pH gradients (IPGs) for IEF,51 the procedure remains essentially as outlined earlier. In the following sections, a general protocol is outlined briefly with some notes of special relevance to the seafood scientist. For more detailed, up-to-date protocols, the reader is referred to any of a number of excellent reviews and laboratory manuals.52–57
3.2.2.1
Sample Extraction and Cleanup
For most applications, sample treatment prior to electrophoresis should be minimal in order to minimize in-sample proteolysis and other sources of experimental artifacts. We have found direct extraction into the gel reswelling buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS [3-(3-chloramidopropyl)dimethylamino-1-propanesulfonate], 0.3% (w/v) DTT [dithiothreitol], 0.5% Pharmalyte ampholytes for the appropriate pH range) supplemented with a protease inhibitor cocktail to give good results for proteome extraction from whole Atlantic cod larvae6,22 and Arctic charr (Salvelinus alpinus) liver.58 Thorough homogenization is essential to ensure complete and reproducible extraction of the proteome. Cleanup of samples using commercial 2D sample cleanup kits may be beneficial for some sample types.
3.2.2.2 First-Dimension Electrophoresis The extracted proteins are first separated by IEF, which is most conveniently performed using commercial dry IPG gel strips. These strips consist of a dried IPG-containing polyacrylamide gel on a plastic backing. Ready-made IPG strips are currently available in a variety of linear and
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Handbook of Seafood and Seafood Products Analysis MW (kDa) 60
42
30
22
17
4
5
pl
6
7
Figure 3.2 A 2DE protein map of whole Atlantic cod (G. morhua) larval proteins with pI between 4 and 7 and molecular mass about 10–100 kDa. The proteins are separated according to their pI in the horizontal dimension and according to their mass in the vertical dimension. Isoelectrofocusing was by pH 4–7 IPG strip and the second dimension was in a 12% polyacrylamide slab gel.
sigmoidal pH ranges. This method is thus suitable for most 2DE applications and has all but completely replaced the older and less reproducible method of IEF by carrier ampholytes in tube gels. Broad-range linear strips (e.g., pH 3–10) are commonly used for whole-proteome analysis of tissue samples, but for many applications narrow-range and/or sigmoidal IPG strips may be more appropriate as these will give a better resolution of proteins in the fairly crowded pI 4–7 range. Narrow-range strips also allow for higher sample loads (since part of the sample will run off the gel) and thus may yield improved detection of low-abundance proteins. Before electrophoresis, the dried gel needs to be reswelled to its original volume. A recipe for a typical reswelling buffer is presented in Section 3.2.2.1. Reswelling is normally performed overnight at 4°C. Application of a low voltage current may speed up the reswelling process. Optimal conditions for reswelling are normally provided by the IPG strip manufacturer. If the protein sample is to be applied during the reswelling process, extraction directly into the reswelling buffer is recommended. IEF is normally performed for several hours at high voltage and low current. Typically, the starting voltage is about 150 V, which is then increased stepwise to about 3,500 V, usually totaling about 10,000–30,000 Vh, although this will depend on the IPG gradient and the length of the strip. The appropriate IEF protocol will depend not only on the sample and IPG strip, but also on
Proteomics ◾ 27
the equipment used. The manufacturer’s instructions should be followed. Görg et al.56 reviewed IEF for 2DE applications.
3.2.2.3 Equilibration Before the isoelectrofocused gel strip can be applied to the second-dimension slab gel, it needs to be equilibrated for 30–45 min in a buffer-containing SDS and a reducing agent such as DTT. During the equilibration step, the SDS–polypeptide complex that affords protein-size-based separation will form and the reducing agent will preserve the reduced state of the proteins. A tracking dye for the second electrophoresis step is also normally added at this point. A typical equilibrationbuffer recipe is as follows: 50 mM Tris–HCl at pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT, trace amount of bromophenol blue. A second equilibration step in the presence of 2.5% iodoacetamide and without DTT (otherwise identical buffer) may be required for some applications. This will alkylate thiol groups and prevent their reoxidation during electrophoresis, thus reducing vertical streaking.59
3.2.2.4 Second-Dimension Electrophoresis Once the gel strip has been equilibrated, it is applied to the top edge of an SDS-PAGE slab gel (Figure 3.3) and cemented in place using a molten agarose solution. Optimal pore size depends on the size of the target proteins, but for most applications gradient gels or gels of about 10% or 12% polyacrylamide are appropriate. Ready-made gels suitable for analytical 2DE are available commercially. While some reviewers recommend alternative buffer systems,60 the Laemmli method,61 using glycine as the trailing ion and the same buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at both electrodes, remains the most popular one. The gel is run at a constant current of 25 mA until the bromophenol blue dye front has reached the bottom of the gel.
Figure 3.3 Orientation and placement of an isoelectrofocused IPG strip onto the top of the second-dimension gel. Care must be taken that the (+) end of the strip is on the same side of all slab gels, that the gel side of the IPG strip faces the notched side of the glass plate, and that the strip is pressed gently onto the SDS gel, avoiding trapping air bubbles. This is best performed using a dentist’s tool or other appropriate implement, taking care to put the pressure on the IPG strip’s plastic backing rather than the gel itself.
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Handbook of Seafood and Seafood Products Analysis
3.2.2.5 Staining Visualization of proteins spots is commonly achieved through staining with colloidal Coomassie Blue G-250 due to its low cost and ease of use. A typical staining procedure includes fi xing the gel for several hours in 50% ethanol/2% ortho-phosphoric acid, followed by several 30 min washing steps in water, followed by incubation for 1 h in 17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, followed by staining for several days in 0.1% Coomassie Blue G-250/17% ammonium sulfate/34% methanol/2% ortho-phosphoric acid, and followed by destaining for several hours in water. There are, however, commercially available colloidal Coomassie staining kits that do not require fi xation or destaining. A great many alternative visualization methods are available, many of which are more sensitive than colloidal Coomassie and thus may be more suitable for applications where the visualization of low-abundance proteins is important. These include radiolabeling, such as with [35S] methionine, and staining with fluorescent dyes, such as the SYPRO or Cy series of dyes. Multiple staining with dyes fluorescing at different wavelengths offers the possibility of differential display allowing more than one proteome to be compared on the same gel, such as in difference gel electrophoresis (DIGE). Patton published a detailed review of visualization techniques for proteomics.62
3.2.2.6 Analysis Although commercial 2DE image analysis software, such as ImageMaster (Amersham), PDQuest (BioRad), or Progenesis (Nonlinear Dynamics), has improved by leaps and bounds in recent years, analysis of the 2DE gel image, including protein spot definition, matching, and individual protein quantification, remains the bottleneck of 2DE-based proteome analysis and still requires a substantial amount of subjective input by the investigator.63 In particular, spot matching between gels tends to be time-consuming and has proved difficult to automate.64 These difficulties arise from several sources of variation among individual gels, such as protein load variability due to varying IPG strip reswelling or protein transfer from strip to slab gel. Also, gene expression in several tissues varies considerably among the individuals of the same species, and therefore individual variation is a major concern and needs to be accounted for in any statistical treatment of the data. Pooling samples may also be an option and this depends on the type of experiment. These multiple sources of variation has led some investigators63–65 to cast doubt on the suitability of univariate tests, such as Student’s t-test, commonly used to assess the significance of observed protein expression differences. Multivariate analysis has been successfully used by several investigators in recent years.65–67
3.2.3
Protein Identification by Peptide Mass Fingerprinting
Identification of proteins on 2DE gels is most commonly achieved via mass spectrometry of trypsin digests. Briefly, the spot of interest is excised from the gel, digested with trypsin (or another suitable protease), and the resulting peptide mixture is analyzed by mass spectrometry. The most popular mass spectrometry method is MALDI-TOF mass spectrometry,68 where peptides are suspended in a matrix of small, organic, UV-absorbing molecules (such as 2,5-dihydroxybenzoic acid) followed by ionization by a laser at the excitation wavelength of the matrix molecules and acceleration of the ionized peptides in an electrostatic field into a flight tube where the time of flight of each peptide is measured and this gives its expected mass.
Peptides identified as those derived from Atlantic cod β-tubulin Trypsin autolysis peaks
0 741.0
1652.6
2108.4
2798.43
2506.36
2212.25
1974.00
1659.96 1697.90
1229.61 1272.70
1196.8
1822.98 1886.07
10
1131.56
20
856.50
30
1028.50
40
1575.35 1621.801616.86
50
1287.69
60
1061.54
Intensity
70
870.54
80
1960.06
1258.71
90
29
1159.63
1040.60
100
842.51
Proteomics ◾
2564.2
Mass (m/z)
Figure 3.4 A trypsin digest mass spectrometry fingerprint of an Atlantic cod larval protein spot, identified as b-2 tubulin. The open markers indicate mass peaks corresponding to trypsin self-digestion products and were, therefore, excluded from the analysis. The solid markers indicate the peaks that were found to correspond to expected b-2 tubulin peptides.
The resulting spectrum of peptide masses (Figure 3.4) is then used for protein identification by searching against expected peptide masses calculated from data in protein sequence databases, such as the National Centre for Biotechnology Information (NCBI) nonredundant protein sequences database, using the appropriate software. Several programs are available, many with a web-based open-access interface. The ExPASy Tools web site (http://www.expasy.org/tools) contains links to most of the available software for protein identification and several other tools. Attaining a high identification rate is problematic in fish and seafood proteomics due to the relative paucity of available protein sequence data for these animals. As can be seen in Table 3.1, this problem is surprisingly acute for species of commercial importance. To circumvent this problem, it is possible to take advantage of the available nucleotide sequences, which in many cases is more extensive than the protein sequences available, to obtain a tentative identity. How useful this method is will depend on the length and quality of the available nucleotide sequences. It is important to realize, however, that an identity obtained in this manner is less reliable than that obtained through protein sequences and should be regarded only as tentative in the absence of corroborating evidence (such as 2D immunoblots, correlated activity measurements, or transcript abundance). In their work on the rainbow trout liver proteome, Martin et al.10 and Vilhelmsson et al.9 were able to attain an identification rate of about 80% using a combination of search algorithms that included the open-access Mascot program69 and a licensed version of Protein Prospector MS-Fit70 by searching against both protein databases and a database containing all salmonid nucleotide sequences. In those cases where both the protein and nucleotide databases yielded results, 100% agreement was observed between the two methods.
30
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Handbook of Seafood and Seafood Products Analysis
Table 3.1 Families of Some Commercially Important Seafood Species and the Availability of Protein and Nucleotide Sequence Data as of March 27, 2008 Protein Sequences
Nucleotide Sequences
185,533
5,782,086
Anguilliformes (eels and morays)
1,680
2,208
Clupeiformes (herrings)
1,407
2,284
Cypriniformes (carps)
84,896
2,046,798
Siluriformes (catfishes)
3,845
81,845
10,063
999,489
3,122
130,353
268
287
26,424
170,381
2,442
45,626
237
138
2,344
16,210
3,592
768,007
Carcharhiniformes (ground sharks and dogfishes)
735
911
Lamniformes (mackrel sharks)
179
303
Rajiformes (skates and rays)
585
18,008
32,287
898,006
8,158
121,762
20,245
726,864
2,380
47,751
21,656
467,933
Caridea (shrimps, etc.)
2,585
3,871
Astacidea (lobsters and crayfishes)
1,237
32,138
Brachyura (short-tailed crabs)
2,203
36,557
Actinopterygii (Ray-Finned Fishes)
Salmoniformes (salmons and trout) Gadiformes (cod-likes, incl. cod, haddock, saithe, and pollock) Lophiiformes (anglerfishes, incl. monkfish) Perciformes (perch-likes, incl. sea bream, sea bass, mackrel, tuna, and wolffish) Pleuronectiformes (flatfishes, incl. halibut, turbot, sole, and plaice) Zeiformes (dories) Scorpaeniformes (scorpionfishes, incl. redfish and lumpfishes)
Chondrichthyes (Cartilagenous Fishes)
Mollusca (Mollusks) Bivalvia (mussels, scallops, etc.) Gastropoda (incl. whelks and abalone) Cephalopoda (squid and octopi)
Crustacea (Crustaceans)
Proteomics ◾ 31
A more direct, if rather more time-consuming, way of obtaining protein identities is by direct sequence comparison. Until recently, this was accomplished by N-terminal or internal (after proteolysis) sequencing by the Edman degradation of eluted or electroblotted protein spots.71,72 Today, the method of choice is tandem mass spectrometry (MS/MS). In the peptide mass fingerprinting discussed earlier, each peptide mass can potentially represent any of a large number of possible amino acid sequence combinations. The larger the mass (and longer the sequence), the higher the number of possible combinations. In MS/MS one or several peptides are separated from the mixture and dissociated into fragments that are then subjected to a second round of mass spectrometry, yielding a second layer of information. Correlating this spectrum with the candidate peptides identified in the first round narrows down the number of candidates. Furthermore, several short stretches of amino acid sequence will be obtained for each peptide, which, when combined with the peptide and fragment masses obtained, enhances the specificity of the method even further.73–75 Mass spectrometry methods in proteomics have been reviewed, for example, by Yates,76 Nyman,77 Damodaran et al.,78 Thiede et al.,79 Rappsilber et al.,80 Mo and Karger,81 Gygi and Aebersold,82 Lin et al.,83 and Delahunty and Yates.84
3.3 Applications of 2DE in Seafood Analysis The two-dimensional electrophoresis has been in use within food science for at least two decades. Early studies focused on relatively small, clearly defined subproteomes and included such applications as the characterization of bovine caseins,85 wheat flour baking quality factors,86 and soybean protein bodies.87 With the lower cost, improved reproducibility and resolving power of electrophoretic separation techniques, and vastly superior protein spot identification techniques, proteomic investigations on fish and seafood products, as well as in aquaculture, fish physiology, and development, have gained considerable momentum.88,89 A brief discussion of a few emerging areas within fish and seafood proteomics is given as follows.
3.3.1 Development Fishes go through different developmental stages (embryo, larva, and adult) during their life span that coincide with changes in the morphology, physiology, and behavior of the fish.23,90–92 The morphological and physiological changes that occur during these developmental stages are characterized by differential cellular and organelle functions.93 This is reflected in the variations of global protein expression and posttranslational modifications of the proteins that may cause alterations in protein function.94 Proteome analysis provides valuable information on the variations that occur within the proteome of organisms. These variations may, for example, reflect a response to biological perturbations or external stimuli9–11,95 resulting in different expression of proteins, posttranslational modifications, or redistribution of specific proteins within cells.20 To date few studies on fish development exist in which proteome analysis techniques have been applied. Recent studies on global protein expression during early developmental stages of zebrafish7 and Atlantic cod6 revealed that distinctive protein profiles characterize the developmental stages of these fishes even though abundant proteins are largely conserved during the experimental period. In both these studies, the identified proteins consisted mainly of proteins located in the cytosol, cytoskeleton, and nucleus. Proteome analyses in developing organisms have shown that many
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Handbook of Seafood and Seafood Products Analysis
of the identified proteins have multiple isoforms96 that reflect either different gene products97 or posttranslationally modified forms of these proteins.98 Different isoforms generated by posttranslational modifications are largely overlooked by studies based on RNA expression. This fact further indicates the importance of the proteome approach to understand cellular mechanisms that underlie fish development. Studies on various proteins have shown that during fish development sequential synthesis of different isoforms appear successively.8,21,94,99–107 In this context, developmental stage specific muscle protein isoforms have gained a special attention.8,21,99–107 The developmental changes in the composition of muscle protein isoforms have been tracked by proteome analysis in African catfish (Heterobranchus longifilis),102 common sole (Solea solea),21 and dorada (Brycon moorei).101 These studies demonstrated that the muscle shows the usual sequential synthesis of protein isoforms in the course of development. For example, in the common sole 2DE revealed two isoforms (larval and adult) of myosin light chain 2 and likewise in dorada larval and adult isoforms of troponin I were sequentially expressed during development. Proteomic techniques have thus been shown to be applicable for investigating cellular and molecular mechanisms involved in the morphological and physiological changes that occur during fish development. The major obstacle on the use of proteomics in embryonic fish has been the high proportion of yolk proteins. These interfere with any proteomic application that intends to target the cells of the embryo proper. In a recent study on the proteome of embryonic zebrafish, the embryos were deyolked to enrich the pool of embryonic proteins and to minimize ions and lipids found in the yolk prior to 2D gel analysis.7 Despite this undertaking, a large number of yolk proteins remained prominently present in the embryonic protein profiles. Link et al.27 published a method to efficiently remove the yolk from large batches of embryos without losing cellular proteins. The success in the removal of yolk proteins by Link et al.27 is probably due to dechorionation prior to the deyolking of the embryos. By dechorionation, the embryos fall out of their chorions facilitating the removal of the yolk.99,108
3.3.2 Quality Involution Degradation of proteins during chilled storage, and their oxidation during frozen storage, are among persistent quality problems in the seafood industry and have deleterious effects on fish flesh texture. Furthermore, several commercially important fish muscle processing techniques, such as curing, fermentation, and production of surimi and conserves occur under conditions conducive to endogenous proteolysis.109,110 Problems of this kind, where differences are expected to occur in the number, molecular mass, and pI of the protein present in a tissue, are well suited for investigation using 2DE-based proteomics. It is also worth noting that protein isoforms other than proteolytic ones, whether they be encoded in structural genes or brought about by posttranslational modification, usually have different molecular weight or pI and can, therefore, be distinguished on 2DE gels. Thus, specific isoforms of myofibrillar proteins, many of which are correlated with specific textural properties in seafood products, can be observed using 2DE or other proteomic methods.88,111
3.3.2.1 Protein Autolysis and Oxidation during Storage and Processing The specifics of fish muscle protein autolysis during storage and processing still remain in large part to be elucidated, although degradation of myofibrillar proteins by calpains and cathepsins112,113
Proteomics ◾ 33
and degradation of the extracellular matrix by the matrix metalloproteases and matrix serine proteases114,115 are thought to be among the main culprits. Whatever may be the mechanism, it is clear, that these quality changes are species dependent116,117 and, furthermore, appear to display seasonal variations.112,118 Several 2DE studies have been performed on postmortem changes in seafood flesh14–17, 67,111,117,119,120 and have demonstrated the importance and complexity of proteolysis and oxidative changes in seafood proteins during storage and processing. For example, Martinez et al.121 used a 2DE approach to demonstrate different protein composition of surimi made from prerigor versus postrigor cod and found that 2DE could distinguish between the two. Kjærsgård et al.17 used 2DE, 2D-immunoblots and LC–MS/MS to study changes in protein oxidation during frozen storage of rainbow trout. They found fish muscle proteins to be differentially carbonylated during frozen storage and were able to identify several carbonylated proteins using LC–MS/MS.
3.3.2.2
Aquaculture and Antemortem Effects on Quality and Processability
It is well known that an organism’s phenotype, including quality characteristics, is determined by environmental as well as genetic factors. Indeed, Huss noted in his review122 that product quality differences within the same fish species can depend on feeding and rearing conditions, differences that can affect postmortem biochemical processes in the product which, in turn, affect the involution of quality characteristics in the fish product. The practice of rearing fish in aquaculture, as opposed to wild fish catching, therefore raises the tantalizing prospect of managing quality characteristics of the fish flesh antemortem, where individual physiological characteristics, such as those governing gaping tendency, flesh softening during storage, etc., are optimized. To achieve that goal, the interplay between these physiological parameters and environmental and dietary variables needs to be understood in detail. With the ever increasing resolving power of molecular techniques, such as proteomics, this is fast becoming feasible. We are aware of two recent studies where Atlantic cod muscle proteomes have been compared between farmed and wild fish.15,123 Both studies indicated that several proteins are differentially expressed in farmed versus wild cod. Olsson et al.123 found these to comprise several members of the glycolytic and Krebs cycle pathways. In a recent study on the feasibility of substituting fish meal in rainbow trout diets with protein from plant sources, various quality characteristics of fillet and body were measured124,125 and the liver proteome was analyzed9,10,126 in fish fed with the experimental diets. The diet was found to have a marked effect on product texture, and the amount and composition of free amino acids in the fish flesh. Furthermore, the proteome analysis identified a number of metabolic pathways sensitive to plant protein substitution in rainbow trout feed, such as pathways involved in cellular protein degradation, fatty acid breakdown, and NADPH metabolism. In the context of this chapter, the effects on the proteasome are particularly noteworthy. The proteasome is a multisubunit enzyme complex that catalyzes proteolysis via the ATP-dependent ubiquitin–proteasome pathway which, in mammals, is thought to be responsible for a large fraction of cellular proteolysis.127,128 In rainbow trout, the ubiquitin–proteasome pathway has been shown to be downregulated in response to starvation129 and have a role in regulating protein deposition efficiency.44 The results led the authors to speculate that the difference in texture and postmortem amino acid-free pool development are affected by antemortem proteasome activity.1
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3.3.3
Handbook of Seafood and Seafood Products Analysis
Species Authentication
Processed fish products are increasingly common in the market and, as different fish species have different market values, this makes the issue of species authentication an area of increasing economic importance, as well as being relevant from a public health standpoint. While DNA-based species identification130–132 and isotope distribution techniques for determining geographical origin133 are powerful tools in this area and likely to remain the methods of choice in the near term, proteomics-based species identification methods are likely to develop rapidly and find commercial uses within this field, particularly for addressing questions on the health status of the fish in question, the presence of stress-factors or contamination levels at the place of breeding, and postmortem treatment.14 Unlike the genome, the proteome varies from tissue to tissue and with environmental conditions. Proteome analysis can therefore potentially yield more information than genomic methods, possibly indicating freshness and tissue information in addition to species. Martinez et al.134 recently reviewed proteomic and other methods for species authentication in foodstuffs. From early on, proteomic methods have been recognized as a potential way of fish species identification. During the 1960s, 1D electrophoretic techniques were developed to identify the raw flesh of various species,135–137 which was soon followed by methods to identify species in processed or cooked products.138,139 These early efforts were reviewed in 1980.140,141 More recently, 2DE-based methods have been developed to distinguish various closely related species, such as the gadoids or several flat fishes.18,142,143 Indeed, the proteomes of even closely related fish species are be easily distinguishable by eye from one another on 2D gels1 indicating that diagnostic protein spots may be used to distinguish closely related species. Piñeiro and coworkers have found that Cape hake (Merluccius capensis) and European hake (Merluccius merluccius) can be distinguished on 2D gels from other closely related species by the presence of a particular protein spot identified as corresponding to nucleoside diphosphate kinase.143 Lopez and coworkers, studying three species of European mussels: Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus, found that M. trossulus could be distinguished from the other two species on foot extract 2D gels by a difference in a tropomyosin spot. They found the difference to be due to a single T to D amino acid substitution.144 Martinez and Jakobsen Friis concluded that the identification of not only the species present, but also their relative ratios in mixtures of several fish species and muscle types14 would become viable once a suitable number of markers have been identified.
3.3.4 Allergen Identification Allergenic potential is food safety issue of particular concern to the seafood producer. Allergic reactions to seafood affect a significant part of the population. For example, about 0.5% of young adults are allergic to shrimp.145 Seafood allergies are caused by an immunoglobulin E-mediated response to particular proteins, including structural proteins such as tropomyosin.146 Proteome analysis can be a valuable tool for the identification and the characterization of allergens as exemplified by the study of Yu et al.147 at National Taiwan University. These authors, studying the cause of shrimp allergy in humans, performed a 2DE on crude protein extracts from the tiger prawn, Penaeus monodon, blotted the 2D gel onto a PVDF membrane, and probed the membranes with serum from confirmed shrimp allergic patients. The allergens were then identified by MALDITOF MS of tryptic digests. The allergen was identified as a protein with close similarity to arginine kinase. The identity was further corroborated by cloning and sequencing the relevant cDNA. A final proof was obtained by purifying the protein, demonstrating that it had arginine kinase
Proteomics ◾
35
activity and reacted to serum IgE from shrimp allergic patients and, furthermore, induced skin reactions in sensitized shrimp allergic patients.
Acknowledgments This work was supported by grants from the Icelandic Graduate Research Fund, the University of Iceland Research Fund, the University Research Fund of Eimskipafélag Íslands, and the AVS Research Fund.
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Proteomics ◾ 37 35. Cañas, B., Piñeiro, C., Calvo, E., López-Ferrer, D., and Gallardo, J. M., Trends in sample preparation for classical and second generation proteomics. Journal of Chromatography A 1153, 235–258, 2007. 36. Bodzon-Kulakowska, A., Bierczynska-Krysik, A., Dylag, T., Drabik, A., Suder, P., Noga, M., Jarzebinska, J., and Silberring, J., Methods for samples preparation in proteomic research. Journal of Chromatography B 849, 1–31, 2007. 37. Righetti, P. G., Castagna, A., Antonioli, P., and Boschetti, E., Prefractionation techniques in proteome analysis: The mining tools of the third millennium. Electrophoresis 26, 297–319, 2005. 38. Hollung, K., Veiseth, E., Jia, X., Færgestad, E. M., and Hildrum, K. I., Application of proteomics to understand the molecular mechanisms behind meat quality. Meat Science 77, 97–104, 2007. 39. Millea, K. M. and Krull, I. S., Subproteomics in analytical chemistry: Chromatographic fractionation techniques in the characterization of proteins and peptides. Journal of Liquid Chromatography and Related Technologies 26, 2195–2224, 2003. 40. Issaq, H. J., Conrads, T. P., Janini, G. M., and Veenstra, T. D., Methods for fractionation, separation and profiling of proteins and peptides. Electrophoresis 23, 3048–3061, 2002. 41. Dreger, M., Subcellular proteomics. Mass Spectrometry Reviews 22, 27–56, 2003. 42. Mortimore, G. E., Pösö, A. R., and Lardeaux, B. R., Mechanism and regulation of protein degradation in liver. Diabetes Metabolism Review 5, 49–70, 1989. 43. Hershko, A. and Ciechanover, A., The ubiquitin pathway for the degradation of intracellular proteins. Progress in Nucleic Acid Research and Molecular Biology 33, 19–56, 1986. 44. Dobly, A., Martin, S. A. M., Blaney, S., and Houlihan, D. F., Efficiency of conversion of ingested proteins into growth; protein degradation assessed by 20S proteasome activity in rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology A 137, 75–85, 2004. 45. Journet, A., Chapel, A., Kieffer, S., Louwagie, M., Luche, S., and Garin, J., Towards a human repertoire of monocytic lysosomal proteins. Electrophoresis 21, 3411–3419, 2000. 46. Journet, A., Chapel, A., Kieffer, S., Roux, F., and Garin, J., Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells. Proteomics 2, 1026–1040, 2002. 47. Ciechanover, A., The ubiquitin-proteasome proteolytic pathway. Cell 79, 13–21, 1994. 48. Okumura, Y., Sato, H., Seiki, M., and Kido, H., Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin. A possible cell surface activator. FEBS Letters 402, 181–184, 1997. 49. Brown, P. D., Kleiner, D. E., Unsworth, E. J., and Stetler-Stevenson, W. G., Cellular activation of the 72 kDa type IV procollagenase/TIMP-2 complex. Kidney International 43, 163–170, 1993. 50. Wang, M. and Lakatta, E. G., Altered regulation of matrix metalloproteinase-2 in aortic remodeling during aging. Hypertension 39, 865–873, 2002. 51. Görg, A., Postel, W., and Gunther, S., The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546, 1988. 52. Berkelman, T. and Stenstedt, T., 2-D Electrophoresis Using Immobilized pH Gradients. Principles and Methods, Amersham Biosciences, Uppsala, Sweden, 1998. 53. Link, A. J., 2-D proteome Analysis Protocols, Humana Press, Totowa, NJ, 1999, p. 601. 54. Walker, J. M., The Protein Protocols Handbook, 2nd edn., Humana Press, Totowa, NJ, 2002, p. 1176. 55. Westermeier, R. and Naven, T., Proteomics in Practice, Wiley-VCH, Weinheim, 2002, p. 318. 56. Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., and Weiss, W., The current state of two-dimensional elelctrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053, 2000. 57. Görg, A., Weiss, W., and Dunn, M. J., Current two-dimensional electrophoresis technology for proteomics. Proteomics 19, 3665–3685, 2004. 58. Coe, J. E. and Vilhelmsson, O., unpublished results. 59. Görg, A., Postel, W., Weser, J., Günther, S., Strahler, J. R., Hanash, S. M., and Somerlot, L., Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8, 122–124, 1987.
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60. Walsh, B. J. and Herbert, B. R., Casting and running vertical slab-gel electrophoresis for 2D-PAGE, in 2-D Proteome Analysis Protocols, Link, A. J., Ed., Humana Press, Totowa, NJ, 1999, pp. 245–253. 61. Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685, 1970. 62. Patton, W. E., Detection technologies in proteome analysis. Journal of Chromatography B 771, 3–31, 2002. 63. Barrett, J., Brophy, P. M., and Hamilton, J. V., Analysing proteomic data. International Journal for Parasitology 35, 543–553, 2005. 64. Wheelock, A. M. and Goto, S., Effects of post-electrophoretic analysis on variance in gel-based proteomics. Expert Review of Proteomics 3(1), 129–142, 2006. 65. Karp, N. A., Griffin, J. L., and Lilley, K. S., Application of partial least squared discriminant analysis to two-dimensional difference gel studies in expression proteomics, Proteomics 5, 81–90, 2005. 66. Gustafson, J. S., Ceasar, R., Glasbey, C. A., Blomberg, A., and Rudemo, M., Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data, Proteomics 4, 3791–3799, 2004. 67. Kjærsgård, I. V. H., Nørrelykke, M. R., and Jessen, F., Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis. Proteomics 6(5), 1606–1618, 2006. 68. Courchesne, P. L. and Patterson, S. D., Identification of proteins by matrix-assisted laser desorption/ ionization mass spectrometry using peptide and fragment ion masses, in 2-D Proteome Analysis Protocols, Link, A. J., Ed., Humana Press, Totowa, NJ, 1999, pp. 487–511. 69. Perkins, D. N., Pappin, D. J. C., Creasy, D. M., and Cottrell, J. S., Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567, 1999. 70. Clauser, K. R., Baker, P. R., and Burlingame, A. L., Role of accurate measurement (+/− 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Analytical Chemistry 71, 2871–2882, 1999. 71. Kamo, M. and Tsugita, A., N-terminal amino acid sequencing of 2-DE spots, in 2-D Proteome Analysis Protocols, Link, A. J., Ed., Humana Press, Totowa, NJ, 1999, pp. 461–466. 72. Erdjument-Bromage, H., Lui, M., Lacomis, L., and Tempst, P., Characterizing proteins from 2-DE gels by internal sequence analysis of peptide fragments, in 2-D Proteome Analysis Protocols, Link, A. J., Ed., Humana Press, Totowa, NJ, 1999, pp. 467–472. 73. Yu, Y. L., Huang, Z. Y., Yang, P. Y., Rui, Y. C., and Yang, P. Y., Proteomic studies of macrophagederived foam cell from human U937 cell line using two-dimensional gel electrophoresis and tandem mass spectrometry. Journal of Cardiovascular Pharmacology 42, 782–789, 2003. 74. Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M., Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379(6564), 466–469, 1996. 75. Chelius, D., Zhang, T., Wang, G. H., and Shen, R. F., Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry. Analytical Chemistry 75, 6658–6665, 2003. 76. Yates, J. R., Mass spectrometry and the age of the proteome. Journal of Mass Spectrometry 33, 1–19, 1998. 77. Nyman, T. A., The role of mass spectrometry in proteome studies. Biomolecular Engineering 18, 221–227, 2001. 78. Damodaran, S., Wood, T. D., Nagarajan, P., and Rabin, R. A., Evaluating peptide mass fingerprinting-based protein identification. Genomics, Proteomics and Bioinformatics 5, 152–157, 2007. 79. Thiede, B., Hohenwarter, W., Krah, A., Mattow, J., Schmid, M., Schmidt, F., and Jungblut, P. R., Peptide mass fingerprinting. Methods 35, 237–247, 2005. 80. Rappsilber, J., Moniatte, M., Nielsen, M. L., Podtelejnikov, A. V., and Mann, M., Experiences and perspectives of MALDI MS and MS/MS in proteomic research. International Journal of Mass Spectrometry 226, 223–237, 2003.
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81. Mo, W. and Karger, B. L., Analytical aspects of mass spectrometry and proteomics. Current Opinion in Chemical Biology 6, 666–675, 2002. 82. Gygi, S. P. and Aebersold, R., Mass spectrometry and proteomics. Current Opinion in Chemical Biology 4, 489–494, 2000. 83. Lin, D., Tabb, D. L., and Yates, J. R., Large-scale protein identification using mass spectrometry. Biochimica et Biophysica Acta 1646, 1–10, 2003. 84. Delahunty, C. and Yates, J. R., Protein identification using 2D-LC-MS/MS. Methods 35, 248–255, 2005. 85. Zeece, M. G., Holt, D. L., Wehling, R. L., Liewen, M. B., and Bush, L. R., High-resolution two-dimensional electrophoresis of bovine caseins. Journal of Agricultural and Food Chemistry 37, 378–383, 1989. 86. Dougherty, D. A., Wehling, R. L., Zeece, M. G., and Partridge, J. E., Evaluation of selected baking quality factors of hard red winter wheat flours by two-dimensional electrophoresis. Cereal Chemistry 67, 564–569, 1990. 87. Lei, M. G. and Reeck, G. R., Two dimensional electrophoretic analysis of isolated soybean protein bodies and of the glycosylation of soybean proteins. Journal of Agricultural and Food Chemistry 35, 296–300, 1987. 88. Piñeiro, C., Barros-Velázquez, J., Vázquez, J., Figueras, A., and Gallardo, J. M., Proteomics as a tool for the investigation of seafood and other marine products. Journal of Proteome Research 2, 127–135, 2003. 89. Parrington, J. and Coward, K., Use of emerging genomic and proteomic technologies in fish physiology. Aquatic Living Resources 15, 193–196, 2002. 90. Govoni, J. J., Boehlert, G. W., and Watanabe, Y., The physiology of digestion in fish larvae. Environmental Biology of Fishes 16, 59–77, 1986. 91. O’Connell, C. P., Development of organ systems in the northern anchovy Engraulis mordax and other teleosts. American Zoologist 21, 429–446, 1981. 92. Skiftesvik, A. B., Changes in behaviour at onset of exogenous feeding in marine fish larvae. Canadian Journal of Fisheries and Aquatic Sciences 49, 1570–1572, 1992. 93. Einarsdóttir, I. E., Silva, N., Power, D. M., Smáradóttir, H., and Björnsson, B. T., Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut. Anatomy and Embryology 211, 47–60, 2006. 94. Campinho, M. A., Sweeney, G. E., and Power, D. M., Regulation of troponin T expression during muscle development in sea bream Sparus auratus Linnaeus: The potential role of thyroid hormones. Journal of Experimental Biology 209, 4751–4767, 2006. 95. Anderson, N. L. and Anderson, N. G., Proteome and proteomics: New technologies, new concepts, and new words. Electrophoresis 19, 1853–1861, 1998. 96. Paz, M., Morin, M., and del Maso, J., Proteome profile changes during mouse testis development. Comparative Biochemistry and Physiology D 1, 404–415, 2006. 97. Guðmundsdóttir, Á., Guðmundsdóttir, E., Óskarsson, S., Bjarnason, J. B., Eakin, A. K., and Craik, C. S., Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen. European Journal of Biochemistry 217, 1091–1097, 1993. 98. Jensen, O., Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry. Current Opinion in Chemical Biology 8, 33–41, 2004. 99. Huriaux, F., Melot, F., Vandewalle, P., Collin, S., and Focant, B., Parvalbumin isotypes in white muscle from three teleost fish: Characterization and their expression during development. Comparative Biochemistry and Physiology B 113, 475–484, 1996. 100. Huriaux, F., Vandewalle, P., Baras, E., Legendre, M., and Focant, B., Myofibrillar proteins in white muscle of the developing catfish Heterobranchus longifilis (Siluriforms, Clariidae). Fish Physiology and Biochemistry 21, 287–301, 1999. 101. Huriaux, F., Baras, E., Vandewalle, P., and Focant, B., Expression of myofibrillar proteins and parvalbumin isoforms in white muscle of dorada during development. Journal of Fish Biology 62, 774–792, 2003.
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102. Focant, B., Melot, F., Collin, S., Chikou, A., Vandewalle, P., and Huriaux, F., Muscle parvalbumin isoforms of Clarias gariepinus, Heterobranchus longifilis and Chrysichthys auratus: Isolation, characterization, and expression during development. Journal of Fish Biology 54, 832–851, 1999. 103. Hall, T. H., Cole, N. J., and Johnston, I. A., Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic cod Gadus morhua L. Journal of Experimental Biology 206, 3187–3200, 2003. 104. Galloway, T. F., Kjørsvik, E., and Kryvi, H., Effect of temperature on viability and axial muscle development in embryos and yolk sac larvae of the Northeast Atlantic cod (Gadus morhua). Marine Biology 132, 547–557, 1998. 105. Galloway, T. F., Kjørsvik, E., and Kryvi, H., Muscle growth in yolk sac larvae of the Atlantic halibut as influenced by temperature in the egg and yolk sac stage. Journal of Fish Biology 55, 26–43, 1999. 106. Galloway, T. F., Bardal, T., Kvam, S. N., Dahle, S. W., Nesse, G., Randøl, M., Kjørsvik, E., and Anderson, Ø, Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: Transient asymmetric expression of MyoD. Journal of Experimental Biology 209, 2432–2441, 2006. 107. Campinho, M. A., Silva, N., Sweeney, G. E., and Power, D. M., Molecular, cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis. Cell and Tissue Research 327, 267–284, 2007. 108. Westerfield, M., The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th edn., University of Ohio Press, Eugene, 2000. 109. Pérez-Borla, O., Roura, S. I., Montecchia, C. L., Roldán, H., and Crupkin, M., Proteolytic activity of muscle in pre- and post-spawning hake (Merluccius hubbsi Marini) after frozen storage. Lebensmittelwissenschaft und -Technologie 35, 325–330, 2002. 110. Thorarinsdottir, K. A., Arason, S., Geirsdottir, M., Bogason, S. G., and Kristbergsson, K., Changes in myofibrillar proteins during processing of salted cod (Gadus morhua) as determined by electrophoresis and differential scanning calorimetry. Food Chemistry 77, 377–385, 2002. 111. Martinez, I., Ofstad, R., and Olsen, R. I., Electrophoretic study of myosin isoforms in white muscles of some teleost fishes. Comparative Biochemistry and Physiology B 96, 221–227, 1990. 112. Ladrat, C., Chaplet, M., Verrez-Bagnis, V., Noël, J., and Fleurence, J., Neutral calcium-activated proteases from European sea bass (Dicentrachus labrax L.) muscle: Polymorphism and biochemical studies. Comparative Biochemistry and Physiology B 125, 83–95, 2000. 113. Ogata, H., Aranishi, F., Hara, K., Osatomi, K., and Ishihara, T., Proteolytic degradation of myofibrillar components by carp cathepsin L. Journal of the Science of Food and Agriculture 76, 499–504, 1998. 114. Lødemel, J. B. and Olsen, R. L., Gelatinolytic activities in muscle of Atlantic cod (Gadus morhua), spotted wolffish (Anarhichas minor) and Atlantic salmon (Salmo salar). Journal of the Science of Food and Agriculture 83, 1031–1036, 2003. 115. Woessner, J. F., Matrix metalloproteases and their inhibitors in connective-tissue remodelling. FASEB Journal 5, 2145–2154, 1991. 116. Papa, I., Alvarez, C., Verrez-Bagnis, V., Fleurence, J., and Benyamin, Y., Post mortem release of fish white muscle a-actinin as a marker of disorganisation. Journal of the Science of Food and Agriculture 72, 63–70, 1996. 117. Verrez-Bagnis, V., Noël, J., Sautereau, C., and Fleurence, J., Desmin degradation in postmortem fi sh muscle. Journal of Food Science 64, 240–242, 1999. 118. Ingólfsdóttir, S., Stefánsson, G., and Kristbergsson, K., Seasonal variations in physicochemical and textural properties of North Atlantic cod (Gadus morhua) mince. Journal of Aquatic Food Product Technology 7, 39–61, 1998. 119. Morzel, M., Verrez-Bagnis, V., Arendt, E. K., and Fleurence, J., Use of two-dimensional electrophoresis to evaluate proteolysis in salmon (Salmo salar) muscle as affected by a lactic fermentation. Journal of Agricultural and Food Chemistry 48, 239–244, 2000.
Proteomics ◾ 41 120. Martinez, I., Jakobsen Friis, T., and Careche, M., Post mortem muscle protein degradation during ice-storage of Arctic (Pandalus borealis) and tropical (Penaeus japonicus and Penaeus monodon) shrimps: A comparative electrophoretic and immunological study. Journal of the Science of Food and Agriculture 81, 1199–1208, 2001. 121. Martinez, I., Solberg, C., Lauritzen, C., and Ofstad, R., Two-dimensional electrophoretic analyses of cod (Gadus morhua L.) whole muscle proteins, water soluble fraction and surimi. Effect of the addition of CaCl 2 and MgCl2 during the washing procedure. Applied and Theoretical Electrophoresis 2, 201–206, 1992. 122. Huss, H. H., Quality and Quality Changes in Fresh Fish, FAO, Rome, 1995. 123. Olsson, G. B., Friis, T. J., Jensen, E., and Cooper, M., Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua). Aquaculture Research 38, 1223–1227, 2007. 124. De Francesco, M., Parisi, G., Médale, F., Lupi, P., Kaushik, S. J., and Poli, B. M., Effect of long-term feeding with a plant protein mixture based diet on growth and body/fillet quality traits of large rainbow trout (Oncorhynchus mykiss). Aquaculture 236, 413–429, 2004. 125. Parisi, G., De Francesco, M., Médale, F., Scappini, F., Mecatti, M., Kaushik, S. J., and Poli, B. M., Effect of total replacement of dietary fish meal by plant protein sources on early post mortem changes in the biochemical and physical parameters of rainbow trout. Veterinary Research Communications 28, 237–240, 2004. 126. Martin, S. A. M., Vilhelmsson, O., and Houlihan, D. F., Rainbow trout liver proteome—Dietary manipulation and protein metabolism, in Progress in Research on Energy and Protein Metabolism, Souffrant, W. B. and Metges, C. C., Eds., Wageningen Academic Publishers, Wageningen, The Netherlands, 2003, pp. 57–60. 127. Craiu, A., Akopian, T., Goldberg, A., and Rock, K. L., Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proceedings of the National Academy of Science USA 94, 10850–10855, 1997. 128. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L., Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761–771, 1994. 129. Martin, S. A., Blaney, S., Bowman, A. S., and Houlihan, D. F., Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): Effect of food deprivation, Pflügers Archives European Journal of Physiology 445 (2), 257–66, 2002. 130. Sotelo, C. G., Piñeiro, C., Gallardo, J. M., and Pérez-Martín, R. I., Fish species identification in seafood products. Trends in Food Science and Technology 4, 395–401, 1993. 131. Mackie, I. M., Pryde, S. E., Gonzales-Sotelo, C., Medina, I., Pérez-Martín, R., Quinteiro, J., Rey-Mendez, M., and Rehbein, H., Challenges in the identification of species of canned fish. Trends in Food Science and Technology 10, 9–14, 1999. 132. Martinez, I., Jakobsen Friis, T., and Seppola, M., Requirements for the application of protein sodium dedecyl sulfate-polyacrylamide gel electrophoresis and randomly amplified polymorphic DNA analyses to product speciation. Electrophoresis 22, 1526–1533, 2001. 133. Campana, S. E. and Thorrold, S. R., Otoliths, increments, an elements: Keys to a comprehensive understanding of fish populations? Canadian Journal of Fisheries and Aquatic Sciences 58, 30–38, 2001. 134. Martinez, I., Aursand, M., Erikson, U., Singstad, T. E., Veliyulin, E., and van den Zwaag, C., Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs. Trends in Food Science and Technology 14, 489–498, 2003. 135. Cowie, W. P., Identification of fish species by thin slab polyacrylamide gel electrophoresis. Journal of the Science of Food and Agriculture 19, 226–229, 1968. 136. Mackie, I. M., Identification of fish species by a modified polyacrylamide disc electrophoresis technique. Journal of the Association of Public Analysts 5, 83–87, 1969. 137. Tsuyuki, H., Uthe, J. F., Roberts, E., and Clarke, L. W., Comparative electropherograms of Coregonis clupeoformis, Salvelinus namaycush, S. alpinus, S. malma and S. fontinalis from the family Salmonidae. Journal of the Fisheries Research Board of Canada 23, 1599–1606, 1966.
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138. Mackie, I. M., Some improvements in the polyacrylamide disc electrophoretic method of identifying species of cooked fish. Journal of the Association of Public Analysts 8, 18–20, 1972. 139. Mackie, I. M. and Taylor, T., Identification of species of heat-sterilized canned fish by polyacrylamide disc electrophoresis. Analyst 97, 609–611, 1972. 140. Mackie, I., A review of some recent applications of electrophoresis and isoelectric focusing in the identification of species of fish in fish and fish products, in Advances in Fish Science and Technology, Connell, J. J., Ed., Fishing News Books Ltd., Aberdeen, U.K., 1980. 141. Hume, A. and Mackie, I., The use of electrophoresis of the water-soluble muscle proteins in the quantitative analysis of the species components of a fish mince mixture, in Advances in Fish Science and Technology, Connell, J. J., Ed., Fishing News Books Ltd., Aberdeen, U.K., 1980. 142. Piñeiro, C., Barros-Velázquez, J., Sotelo, C. G., and Gallardo, J. M., The use of two-dimensional electrophoresis for the identification of commercial flat fish species. Zeitschrift für Lebensmitteluntersuchung und -forschung 208, 342–348, 1999. 143. Piñeiro, C., Vázquez, J., Marina, A. I., Barros-Velázquez, J., and Gallardo, J. M., Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis. Electrophoresis 22, 1545–1552, 2001. 144. Lopez, J. L., Marina, A., Alvarez, G., and Vazquez, J., Application of proteomics for fast identification of species-specific peptides from marine species. Proteomics 2, 1658–1665, 2002. 145. Woods, R. K., Thien, F., Raven, J., Walters, E. H., and Abramson, M., Prevalence of food allergies in young adults and their relationship to asthma, nasal allergies, and eczema. Annals of Allergy Asthma and Immunology 88, 183–189, 2002. 146. Lehrer, S. B., Ayuso, R., and Reese, G., Seafood allergy and allergens: A review. Marine Biotechnology 5, 339–348, 2003. 147. Yu, C.-J., Lin, Y.-F., Chiang, B.-L., and Chow, L.-P., Proteomics and immunological analysis of a novel shrimp allergen, pen m 2, The Journal of Immunology 170, 445–453, 2003.
Chapter 4
Seafood Genomics Astrid Böhne,* Delphine Galiana-Arnoux,* Christina Schultheis,* Frédéric Brunet, and Jean-Nicolas Volff Contents 4.1 Introduction ..................................................................................................................... 43 4.2 Genetics and Genomics.................................................................................................... 44 4.3 Genomic Resources and Genome Projects for Aquatic Species ..........................................45 4.4 Genomics, Fisheries, and the Management of Biodiversity ................................................47 4.5 Genomics and Aquaculture .............................................................................................. 49 4.6 Concluding Remarks ........................................................................................................51 Acknowledgments ......................................................................................................................52 References ..................................................................................................................................52 There the nets brought up beautiful specimens of fish: Some with azure fins and tails like gold, the flesh of which is unrivalled; some nearly destitute of scales, but of exquisite flavour; others, with bony jaws, and yellow-tinged gills, as good as bonitos; all fish that would be of use to us. Jules Verne, Twenty Thousand Leagues under the Sea
4.1 Introduction The development of high-throughput DNA sequencing methods has opened the era of genomics, which has revolutionized biology, medicine, and biotechnology over the last decade. The rise of * Equal contributors.
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genomics has generated an impressive wave of novel information concerning genome structure, function, and evolution. Massive analysis of functional gene variability in many organisms has allowed to better understand the molecular basis of biodiversity and disease. In the field of biotechnology, genomics is principally used to identify molecular markers, genes and alleles of zootechnical interest for the genetic improvement of economically important species, and to contribute to the management of biodiversity. Genomics has important applications for fisheries and aquaculture [1], which are reviewed in this chapter.
4.2 Genetics and Genomics Genetics can be defined as the science of heredity and variation in organisms. Heredity is based on genes, which are carried by chromosomes, which themselves constitute the genome. Most genes are located in the nucleus, but organelles (mitochondria and chloroplasts) have their own genome too. The science dealing with the analysis of genomes as a whole is called genomics. There are different but complementary ways to analyze genomes. One of them, called genetic mapping, consists in delineating intervals on the genome with genetic markers. This generates a genetic linkage map, with the distance between markers being directly proportional to the frequency of recombination between them. Genetic loci and genes of interest can then be mapped relative to these markers, providing an estimation of their localization in the genome. In addition, comparative mapping provides important information on the structure and evolution of genomes in different species. Genetic markers must be polymorphic to allow the analysis of their segregation, and therefore of their linkage. Different types of DNA markers are used for mapping, such as restriction fragment length polymorphisms (RFLPs, caused by sequence polymorphisms at restriction sites) [2]. DNA markers with a polymorphic number of tandem repeats are called minisatellites (repeat units up to 25 bp in length) and microsatellites (shorter repeat units, usually dinucleotides or tetranucleotides), the latter being of wide use in genotyping and mapping experiments. Other important markers are single nucleotide polymorphisms (SNPs), that is, one nucleotide differences within otherwise identical, generally orthologous sequences [3]. Since SNPs can occur not only in noncoding but also in coding sequences, they are likely to be less neutral than other markers from the functional point of view. SNP analysis can therefore uncover genes and residues that are targeted by evolution and lead to the identification of disease-associated genes. Random amplified polymorphic DNA (RAPD) markers are amplified enzymatically by polymerase chain reaction (PCR) using short arbitrary oligonucleotide primers. Amplified fragment length polymorphism (AFLP) markers combine the principle of RFLP with PCR: fragments cut with restriction enzymes are ligated with adaptors; DNA fragments are amplified enzymatically using primers matching both adaptor and restriction site. Finally, polymorphic insertions of retrotransposable elements, increasingly used for phylogenetic reconstructions [4], can also be used for mapping purposes. Such markers might be further developed in fish, which have genomes with very diverse transposable elements [5]. Molecular markers are not only useful for genome mapping but also represent important tools in other domains, for example, in population genetics. In order to investigate gene content, arrangement, and structure, nuclear and organelle genomes can be sequenced to (almost) completion, as done for the human genome [6,7]. Traditionally, genomes are sequenced using the “shotgun” strategy, with randomly sheared pieces of DNA massively cloned, sequenced, and subsequently assembled in “contigs” in silico. Gene regulatory and coding sequences are then predicted through bioinformatic analysis involving sequence prediction and database comparisons. The development of efficient methods in bioinformatics is a condition sine qua non for progresses in the field of genomics.
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A clone-by-clone approach can be used as an alternative to, or even better in combination with, shotgun sequencing. Parts of the genome, cloned in a bacterial vector and constituting a so-called genomic library, can be sequenced either to completion or from their ends. Such an approach is, for example, useful in the case of regions rich in repetitive sequences posing problems to assembly after whole genome shotgun sequencing. Bacterial artificial chromosomes (BACs) accepting inserts from several hundreds of kilobases are frequently used as vectors. The overlapping between these clones and their relative arrangement in the genome can be determined through fingerprint analysis (e.g., through the identification of common restriction fragments). This provides a physical map respecting the “real” base pair distance between genes and markers, which can be very useful to precisely determine the relative position of sequence contigs assembled “in silico” from whole genome shotgun sequencing data. The relative position of two contigs can also be estimated cytogenetically using double fluorescent in situ hybridization [8]. Probes specific to each contig marked with different fluorochromes are cohybridized on chromosome preparations to test if they are located on the same or on different chromosomes. Physical maps can also be constructed by analyzing the segregation of genomics markers (also called STSs for sequence-tagged sites) in randomly fragmented parts of the genome. These fragments are either integrated in the genome of a host cell line from a different organism in radiation hybrid (RH) mapping [9] or diluted to give aliquots containing approximately one haploid genome equivalent (HAPPY mapping [10]). Importantly, a new revolution of large-scale sequencing is ushering in a second era of genomics, with novel methods allowing very rapid and much cheaper sequencing of large amounts of DNA [11–13]. Sequence data can be used among others to identify similarities and differences between species and study genome evolution (comparative genomics [14]) or to infer reliable phylogenetic relationships between organisms (molecular phylogenetics and phylogenomics [15]). A method called “DNA barcoding” should help to identify species and phylogenetic units, hereby contributing to species conservation and management of global fish biodiversity (http://www.fishbol.org/). Barcoding is based on a sequence of short standard parts of the genome. Generally, a 650 bp fragment of the 5′ end of the mitochondrial gene cytochrome c oxidase I is used as a global standard in fish and other animals (for review, see Ref. [16]). Additional approaches are required to study gene expression (transcriptomics, proteomics) and function (functional genomics) as well as interactions with the environment (environmental genomics). Large-scale expression studies at the transcriptional level are generally performed using microarrays or other methods of high-throughput expression profiling. Of particular interest are expressed sequence tags (ESTs), obtained through sequencing of complementary DNA (cDNA) libraries. EST analysis not only provides important data on genes expressed in particular tissues/ organs or at specific stages of development but also allows the characterization of gene structure through comparison with genomic sequences. ESTs can also be used, for instance, for SNP detection and phylogenetic reconstructions.
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Genomic Resources and Genome Projects for Aquatic Species
Genetic and genomic resources have been generated for many aquatic species of economical interest. In addition, aquatic model organisms of insignificant importance such as seafood have been developed for other scientific purposes and have been targeted for whole genome-sequencing projects [17]. For example, zebrafish and medaka are two complementary fish models to study
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vertebrate development [18]. These models are nevertheless useful to decipher gene content in species targeted by fisheries and aquaculture through comparative genomics [19,20]. Compared with agricultural plants and terrestrial livestock, genomic studies on aquatic species are relatively recent. SNPs and other polymorphic markers as well as linkage maps have now been generated for many aquaculture species, including fish (sea bream, sea bass, Atlantic salmon, rainbow trout, and other salmonids, tilapia, carp, catfish, Japanese flounder, and others) and invertebrates (oyster, abalone, mussel, scallop, sea urchin, shrimp, and others) (for review, see Refs. [1,21]). Expressed sequence tags are also available for many fish species, providing useful information on gene sequence and expression in different tissues and organs or at different stages of development (http://www.ncbi.nlm.nih.gov/dbEST/). Aquatic invertebrate species with well-developed EST resources include scallop and oyster (mollusks) as well as blue/green crabs, shrimp, and lobster (crustaceans). A variety of genomic libraries, particularly BAC libraries, as well as RH panels and cDNA microarrays have been constructed for aquatic organisms; physical maps are available for species such as Nile tilapia, Atlantic salmon, and channel catfish [22–28]. For some species like the rainbow trout, assignment of linkage groups to specific chromosomes has been performed through fluorescent in situ hybridization [29]. Most genome drafts available so far are for aquatic model species without any real economic importance (for review, see Ref. [17]). However, these sequencing projects have provided valuable general information on the structure, evolution, and gene content of fish genomes. Particularly, they have revealed some evolutionary peculiarities possibly linked to biodiversity, such as the high diversity of transposable elements and presence of numerous duplicated genes that are remnants of an ancestral whole genome duplication [30–33]. Fishes with sequenced genomes include the pufferfish species Takifugu rubripes ([34]; http:// www.fugu-sg.org/) and Tetraodon nigroviridis [35]. Both species have an extremely compact genome with low repeat content and short intronic and intergenic sequences and have been useful to identify conserved genes and noncoding sequences in the human genome [36]. Other species with advanced or completed genome projects include the medaka Oryzias latipes [37,38], the three-spined stickleback Gasterosteus aculeatus (http://www.ensembl.org/Gasterosteus_ aculeatus/), and the zebrafish Danio rerio (http://www.ensembl.org/Danio_rerio/). A genomesequencing project is underway for the tilapia Oreochromis niloticus, an aquaculture species of high economical value, in association with low-coverage sequencing projects for three additional cichlids (http://www.genome.gov/10002154). For Atlantic salmon and other salmonids, no draft genome is available now, but many other genomic resources have been developed, particularly by the Genomics Research on All Salmon Project consortium (cGRASP) (http://web.uvic.ca/grasp/). Atlantic salmon genome should be sequenced soon, possibly followed by the genome of the rainbow trout. Other projects aim to enhance genomic resources for economically important species, for example, for the Atlantic cod (Cod Genomics and Broodstock Development Project, http:// codgene.ca/index.php). For cartilaginous fish, the genome of the elephant shark Callorhinchus milii, which is relatively compact, has been sequenced at low coverage [39,40]; http://esharkgenome.imcb.a-star.edu.sg/). A genome project is in the pipeline for another cartilaginous fish, the little skate Leucoraja erinacea. (http://www.mdibl.org/research/skategenome.shtml). Further projects aim to sequence the genome of coelacanth, gar, skate, lamprey, and hagfish, which occupy strategic taxonomic positions within and relative to vertebrates (http://www.genome.gov/10002154). The genome of an echinoderm, the purple sea urchin Strongylocentrotus purpuratus, has been sequenced [41]. Beside the genome of the zooplankton Daphnia pulex (water flea; http://wfleabase. org/), the sequencing of the genome of other crustaceans is planned, including the amphipod
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crustacean Jassa slatteryi (http://www.genome.gov/10002154) as well as the genome of the Atlantic horseshoe crab (chelicerate) (http://www.jgi.doe.gov/sequencing). Genome projects are performed for the cnidarian species Hydra magnipapillata (green hydra) and Nematostella vectensis (sea anemone) (http://hydrazome.metazome.net/; http://genome.jgi-psf.org/Nemve1/Nemve1.home.html). Genome sequencing should follow for many other aquatic animal species of economical interest, for example, the Pacific oyster [42]. Seaweed, constituted by several groups of multicellular algae (red algae, green algae, and brown algae), is used as food by coastal populations, particularly in East Asia. Organelle genome sequences and EST resources are available for many algal species, for example, for the red alga Porphyra yezoensis (http://est.kazusa.or.jp/en/plant/porphyra/EST/). Genome drafts have been generated for the red alga Cyanidioschyzon merolae, the green algae or chlorophytes Chlamydomonas reinhardtii and Volvox carteri, the marine picoeukaryote Ostreococcus tauri, the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, and the haptophyte Emiliania huxleyi (for review, see Ref. [43]).
4.4 Genomics, Fisheries, and the Management of Biodiversity Many aquatic populations have been overexploited through overfishing or collapsed and even become extinct through other factors such as pollution, habitat degradation and loss, introduction of exogenous species, climate change, and perturbations of ocean biogeochemistry [44–47]. About 30% of seafood stocks available in 1950 have already collapsed; it has been predicted that all commercial fish and seafood species will have done so by 2048 [48]. Harvesting and other forms of stress can cause strong alterations in population structure as well as a reduction in biodiversity. In addition, exploitation can act as a selective pressure and induce phenotypical shifts as evolutionary responses. For example, fisheries targeting large individuals will select for early maturation at smaller sizes, leading to a reduction of fisheries’ yield [49,50]. Biodiversity decline is associated with a collapse of seafood resource and a reduction in species stability and recovery potential, as well as with a decrease in water quality. Restoration of biodiversity increases fisheries productivity. Hence, the loss of marine biodiversity impairs the ability of ocean to provide food, to maintain water quality, and to recover from perturbations [48]. Consequently, description, monitoring, and conservation of biodiversity of aquatic organisms are now high priorities, with a major role for genomics, particularly in the assessment and follow-up of biodiversity in wild stocks, the estimation of fisheries-induced evolution, and the definition of conservation units and priorities for sustainable fishery management. Genetic monitoring, that is, the quantification of temporal changes in populations using molecular markers, provides information relevant to both the ecological and evolutionary time frame [51]. Important demographic and evolutionary parameters to be considered include organism abundance and vital rates, population structure and interactions, site occupancy, reproductive structure and behavior, pedigrees and social structure, gene flow, and hybridization, and invasion of disease and invasive species [51,52]. Nuclear and mitochondrial molecular markers can be used to identify units of management for fisheries and priorities for the conservation of biodiversity. Populations and ecosystems, with their particular adaptations and contributions to biodiversity, can be considered as conservation units [52]. Characterization of minimum viable population size is required to assess if they are facing a risk of extinction [45]. Population genetics is determined using various polymorphic genetic markers, including mitochondrial DNA polymorphisms, micro/minisatellites, AFLP and
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RAPD markers, SNPs, and others (for review, see Ref. [1]). For example, multiple SNPs have been generated for Atlantic cod. Populations of North-East Arctic cod and Norwegian coastal cod have been analyzed, thereby identifying loci potentially influenced by natural selection [53]. Different types of markers have been used for the estimation of natural population and the determination of conservation genetic parameters in salmonids [54] and to estimate quantitative genetic parameters under wild conditions [55]. For several species, including Atlantic herring, Atlantic salmon, brown trout, European eel, turbot, and pike, sufficient genetic data might be available to provide at least basic information on genetic structure and genetic units for biologically sustainable use [56]. In contrast, the available population genetic information is insufficient for most other species. Genetic monitoring of diversity using polymorphic markers allows monitoring population size and diversity over time. For example, microsatellite data indicated marked genetic changes in declining North Sea cod [57]. Population genomics is a form of population genetics extending the analysis of genetic variation in natural populations to the scale of the genome itself, heralding a new era in the analysis of adaptive evolution and functional variation [58,59]. With the development of much faster and cheaper high-throughput sequencing methods, this field will certainly be of major importance in the future of fisheries management and biodiversity conservation. Genome-wide gene expression profiling can also be used to detect variations in gene expression within and among natural populations [60]. This approach has already been used to identify adaptive differences between natural populations in several species, including the European flounder and the brown trout [61,62]. Beside populations, taxa can also be considered as conservation units, with poorly represented phylogenetic groups receiving high conservation priorities [52]. Accordingly, phylogenetics and phylogenomics are of major importance for the recognition of endangered taxa from the systematic point of view, with the discovery of new groupings and the determination of divergence times and molecular clocks [63]. Finally, conservation efforts could focus on the preservation of genetic diversity allowing biota to adapt to new conditions. For example, species-rich groups such as the East African cichlids [64] might be preserved with priority since their evolution potential might predispose them to serve as progenitors of future biodiversity [52]. Quantitative genetics as well as evolutionary genetics and genomics can help to identify such groups of high evolvability and to study the mechanisms driving their adaptability and speciation, with possible detection of DNA sequences promoting evolution in their genomes [17]. Evolutionary genetics and genomics might also help to understand the interplay between fishing and natural selection on population and species targeted by fisheries [65]. DNA barcoding and other methods have applications not only for species identification and molecular phylogenies but also in the field of population genetics to describe genetic diversity within species [16]. Molecular markers can be used to monitor the efficiency of programs aiming to supplement declining wild populations through individuals reared in captivity. Through pedigree reconstruction with microsatellite markers, it has been, for example, observed that reintroduced steelhead trout presented reduced reproductive capabilities caused by genetic effects of domestication [66]. Genomics and transcriptomics can allow assessing the genetic and functional consequences of interbreeding between farmed and wild fish. This type of study has been performed on Atlantic salmon, for which large annual escapees of farmed Atlantic salmon enhance the risk of extinction of wild populations. Gene transcription profiling suggested that interbreeding of fugitive farmed salmon and wild individuals can substantially modify gene transcription in natural populations exposed to high migration from fish farms, resulting in potentially detrimental effects on survival of these populations [67]. The effects of stress factors contributing to species collapse and
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extinction, for example, pollution (ecotoxicogenomics), as well as the development of resistance mechanisms by the targeted species can be studied using transcriptomics [25,68].
4.5 Genomics and Aquaculture Fish consumption has doubled over the past 50 years and would need to double again over the next 25 years ([69] and references therein). In order to reduce the ecological disaster of overfishing and contribute to solve the problem of global feeding, especially in developing countries, aquaculture including marine aquaculture (mariculture) has increased its production by a 20-fold factor over the last 30 years. Aquaculture needs to be further developed in the future; diversification and genetic improvement of cultivated species should lead to both a reduction in production costs and an increase in fish production. The genetic basis of important zootechnical traits, such as resistance to viral and bacterial diseases, fillet quality (color, texture, and fat deposition), growth and feed efficiency, sexual development, and others must be analyzed to allow efficient breeding and management programs. Significant improvements have been obtained through efficient breeding programs for several species such as farmed salmon and trout. Molecular methods have contributed to the significant increase in aquaculture production worldwide, but genetics and genomics remain poorly developed for aquaculture species compared with crops and livestocks [70]. Genomic sequences, particularly polymorphic DNA markers such as microsatellites, can be used for parental assignment and construction of DNA pedigrees to analyze the heritability of zootechnical parameters and reproductive success or to avoid inbreeding and estimate genetic diversity [71]. These methods are particularly useful when classical individual tagging is difficult or when individual tanks are not available to separate families. Linkage maps are used to map onto genomes genetic loci such as quantitative trait loci (QTLs) influencing traits of economical interest in aquaculture fish species. Linkage analysis allows determining the segregation of a trait of interest relative to polymorphic molecular markers. Examples include the mapping of QTLs involved in development rate, body weight and size, disease resistance and thermal tolerance in salmonids [72–78], cold tolerance, innate immunity, response to stress, biochemical parameters of blood and fish size in tilapia [79–81] and growth-related traits in sea bass [82], as well as in growth-related traits in the Pacific abalone [83], disease resistance in oyster [84], body weight and length in the Kuruma prawn [85], and virus resistance in shrimp [86]. DNA markers linked to a locus of zootechnical interest can subsequently be used to perform marker-assisted selection (MAS). Marker-assisted selection is an indirect process based on the selection of a DNA marker linked to a trait of interest to choose animals for selective breeding programs instead of selecting on the trait itself. Selection against an allele, conferring for example a disease, is also feasible with this method (for review, see Ref. [2]). MAS can be performed at early stages of development and is particularly appropriate for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development. This method also allows monitoring the transfer of genes that control desired phenotypes between breeds, for example, a gene conferring disease resistance into a strain selected for production. In this case, individuals backcrossed with the “production” parent will be selected for the presence of a molecular marker linked to the resistance locus. The efficiency of the method depends on the predictability provided by the marker, that is, on its linkage with the locus of interest. Accordingly, the most effective markers to perform this method of selection are the functional mutations within the trait genes (“direct” markers). A variation of MAS using markers covering the whole genome to assess the status of multiple QTLs is called genomic selection
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[87]. For the great majority of aquaculture species, MAS has not been used so far, particularly due to the lack of high-resolution genetic maps [1]. A trait of particular interest for aquaculture is sex determination. In order to avoid overcrowding and stress induced by sexual maturation and exploit advantageous sex-linked traits (growth rate, flesh quality, behavior, etc.), monosex cultures (either all-male or all-female populations, depending on the species) are frequently used in fish farming. Such monosex populations can be obtained with parents sex-reversed through hormone treatment or produced by androgenesis or gynogenesis. Molecular sexing of individuals at early stages of their development using sex-specific markers would allow the early selection of breeders of a chosen genotype for the production of monosex populations and the rapid analysis of breeding, androgenesis, and gynogenesis products. A better knowledge of sex determination is also required for environment-friendly manipulation of phenotypic sex, for example through temperature, as an alternative to exogenous hormone treatment. Interestingly, in contrast to the situation observed for example in birds and mammals, sex determination is hypervariable in fish [88]. Several hundreds of fish species are sequential hermaphrodites and develop either first as a male and subsequently as a female (protandrous) or vice versa (protogynous). Synchronous hermaphrodites also exist in fish. In gonochoristic (with distinct sexes) species, all possible forms of genetic sex determination have been observed, from male and female heterogamety with or without influence of autosomal loci to more complicated systems involving several loci but without sex chromosomes (polyfactorial sex determination) or more than two sex chromosomes and even several pairs of sex chromosomes. In numerous species, sex determination can be influenced by temperature and other environmental factors such as the pH of water and even social parameters [89]. Phenotypic sex can frequently be fully reversed by hormone treatment, a method largely used in aquaculture to control fish reproduction. Interestingly, even closely related fish species can have very different mechanisms of sex determination, thus reflecting a frequent switching between sex determination systems during evolution. Due to this variability, sex-linked markers for molecular sexing at early stages of development are generally restricted to a single species or are even population-specific within a same species. Sex-specific molecular markers linked to the master sex-determining gene on the sex chromosomes have been identified in many aquaculture fish species, including salmonids, tilapia, and African catfish [90–97]. The only master sex-determining gene identified so far in fish, dmrt1bY from the medaka fish Oryzias latipes [98,99], is not present in any fish species of economical interest. Once DNA markers linked to a locus controlling a trait of economical interest have been identified, the gene itself and the sequence polymorphism involved in phenotypic variation can be identified through positional cloning. When a genomic library is available, for example, a BAC library, genomic clones containing markers linked to the locus can be isolated from the library and sequenced to determine their gene content. When a physical map is available, sequencing can be performed on the tilling path, the minimal set of overlapping clones covering the region of interest. Genes identified through sequencing can be chosen for further analysis according to their described function or their pattern of expression. Sequencing of genomic clones covering a region of interest can also provide new DNA markers that can be used to refine the mapping of the locus, thereby reducing the number of genes to be tested. Alternatively, gene candidates with described functions related to the trait of interest can be directly mapped on the linkage map, with the hope of revealing a colocalization with the locus itself. Sequencing and sequence comparison of the different versions of the gene in individuals polymorphic for the phenotypes studied can allow the identification of the sequence variation at the origin of phenotype differences. Further characterization can be performed at the functional level in vitro or in vivo. Gene candidates with potentially interesting functions can be also directly sequenced in different families without
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mapping in order to test for associations between sequence and phenotype variation. One example is the identification of associations between SNPs in candidate genes and the growth rate in Arctic charr [100]. The detection of genes of zootechnical interest can also be performed through large-scale transcriptional analysis (transcriptomics). EST- and microarray-based transcription profiling for specific tissues, organs and stages of development has been performed in a variety of aquaculture species (for review, see Wenne et al. [1]). Transcriptomics is useful to detect genes differentially expressed in different genetic backgrounds or conditions. For example, genes differentially expressed in progenies exhibiting opposed susceptibility to summer mortality have been identified by suppression subtractive hybridization in oyster [101]. The effect of artificial selection on gene expression has been monitored through transcriptome analysis in Atlantic salmon [102]. The effect of dietary fish oil and fishmeal replacement by vegetable oils and plant proteins on farmed fish metabolism has been investigated in juvenile rainbow trout through hepatic gene expression profiling (nutrigenomics [103]). Phosphorus-responsive genes have been identified through transcriptomics in rainbow trout [104]. The effects of hormone treatments can be also monitored using microarrays [105–107]. Transcriptomics is frequently used to analyze disease and other stress response gene expression and identify resistance gene candidates. Immune response genes downregulated in the gills of amoebic gill disease-affected Atlantic salmons have been found through transcriptome analysis [108], and stress response genes have been investigated in the gilthead sea bream [109]. Microarray analysis of gene expression changes in catfish liver after infection with the gram-negative bacterium Edwardsiella ictaluri indicated a strong upregulation of several pathways involved in the inflammatory immune response and potentially in innate disease resistance [110]. Genes expressed in response to infection with white spot syndrome virus have been identified in shrimp [111].
4.6 Concluding Remarks In the future, seafood genetics and genomics might revolutionize fisheries management and aquaculture development. From systematic, ecological, and evolutionary perspectives, genomics has important applications in biodiversity analysis, exploitation, and conservation, with strong consequences on fisheries productivity. In this domain, much work is still to be done, since information on resource status and extinction risk is available for only a minority of marine fish species [45]. In aquaculture, a better knowledge of genes involved in the control of economically important traits will contribute to improve the production and reduce the costs for current aquaculture species and to identify and develop new potential target species for aquaculture. Such new species might include halibut, cod, wolf fish, flounder, bream, jack, dolphin fish, cobia, and grouper for marine species, and Arctic char, hybrid striped bass, and Australian Murray cod for fresh water species [69]. Genomics will also help to improve and control transgenesis and other methods of modification of gene expression, with the potential of increasing growth, environmental tolerance, and disease resistance ([69]; but see Ref. [112]). Comparative genomics will need to be further developed to increase the transfer of knowledge from models to aquaculture. Importantly, selection methods based on molecular makers remain extremely underdeveloped for aquatic species and will require further exploration based on denser genetic maps. Finally, genomics will boost the discovery of new bioactive molecules in aquatic organisms [113,114] and will be further developed for the identification/authentication of the composition of sea food products put on the market [115].
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Accordingly, many collaborative projects dealing with marine and aquaculture genomics have been or are currently funded by various agencies. For example, the European Union supports different projects. “Aquafirst” aims to combine genetic and functional genomic approaches for stress and disease resistance MAS in fish and shellfish (http://aquafirst.vitamib.com/). “Marine Genomics” is a network of excellence devoted to the development, utilization, and spreading of high-throughput approaches for the investigation of the biology of marine organisms (http:// www.marine-genomics-europe.org/). “Bridgemap” (http://www.bridgemap.tuc.gr/) develops an integrated genomic approach toward the improvement of aquacultured fish species. “AquaFunc” wants to generate an integrated knowledge on functional genomics in sustainable aquaculture (http://genomics.aquaculture-europe.org/index.php?id = 3). Finally, “AquaGenome” aims to coordinate the ongoing and future national and international research projects in the field of genomics in fish and shellfish European aquaculture and support diff usion of genomic approaches within research laboratories. Importantly, recent impressive progresses in large-scale DNA sequencing technology are currently re-revolutionizing the field of genomics (next generation rapid sequencing technology; for review, see Refs. [11–13]). New sequencing platforms allow rapid and much cheaper sequencing of large amounts of DNA, with major applications in genome sequencing, SNP analysis, and most other aspects of genomics. The first full human genome to be sequenced using next generation rapid-sequencing technology has been already published [116]. Genomics is a fast evolving discipline, with a strong potential impact of such new technologies on seafood production for the future.
Acknowledgments Our work is supported by grants from the Association pour la Recherche contre le Cancer (ARC), the Fondation de la Recherche Médicale (FRM), the Centre National de la Recherche Scientifique (CNRS), and the Institut National de la Recherche Agronomique (INRA).
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68. Fisher M.A. and Oleksiak, M.F., Convergence and divergence in gene expression among natural populations exposed to pollution, BMC Genomics, 8, 108, 2007. 69. Muir, J., Managing to harvest? Perspectives on the potential of aquaculture, Philos. Trans. R. Soc. Lond. B Biol. Sci., 360, 191, 2005. 70. Melamed, P., The potential impact of modern biotechnology on fish aquaculture, Aquaculture, 204, 255, 2002. 71. Jones, A.G. and Ardren, W.R., Methods of parentage analysis in natural populations, Mol. Ecol., 12, 2511, 2003. 72. Reid, D.P. et al., QTL for body weight and condition factor in Atlantic salmon (Salmo salar): Comparative analysis with rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus), Heredity, 94, 166, 2005. 73. Robison, B.D. et al., Composite interval mapping reveals a major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss), J. Hered., 92, 16, 2001. 74. Moen, T. et al., Mapping of a quantitative trait locus for resistance against infectious salmon anemia in Atlantic salmon (Salmo salar): Comparing survival analysis with analysis on affected/resistant data, BMC Genet., 8, 53, 2007. 75. Perry, G.M. et al., Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss), Heredity, 86, 333, 2001. 76. Perry, G.M. et al., Sex-linked quantitative trait loci for thermotolerance and length in the rainbow trout, J. Hered., 96, 97, 2005. 77. Nichols, K.M. et al., Quantitative trait loci x maternal cytoplasmic environment interaction for development rate in Oncorhynchus mykiss, Genetics, 175, 335, 2007. 78. Houston, R.D. et al., Major quantitative trait loci affect resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar), Genetics, 178, 1109, 2008. 79. Agresti, J.J. et al., Breeding new strains of tilapia: Development of an artificial center of origin and linkage map based on AFLP and microsatellite loci, Aquaculture, 185, 43, 2000. 80. Cnaani, A. et al., Genome-scan analysis for quantitative trait loci in an F2 tilapia hybrid, Mol. Genet. Genomics, 272, 162, 2004. 81. Charo-Karisaa, H. et al., Heritability of cold tolerance in Nile tilapia, Oreochromis niloticus, juveniles, Aquaculture, 249, 115, 2005. 82. Wang, C.M. et al., A genome scan for quantitative trait loci affecting growth-related traits in an F1 family of Asian seabass (Lates calcarifer), BMC Genomics, 7, 274, 2006. 83. Liu, X., Liu, X. and Zhang, G., Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino, Aquac. Res., 38, 789, 2007. 84. Yu, Z. and Guo, X., Genetic linkage map of the Eastern oyster Crassostrea virginica Gmelin, Biol. Bull., 204, 327, 2006. 85. Li, Y. et al., QTL detection of production traits for the Kuruma prawn Penaeus japonicus (Bate) using AFLP makers, Aquaculture, 258, 198, 2006. 86. Hizer, S.E. et al., RAPD markers as predictors of infectious hypodermal and hematopoietic necrosis virus (IHHNV) resistance in shrimp (Litopenaeus stylirostris), Genome, 45, 1, 2002. 87. Goddard, M.E. and Hayes, B.J., Genomic selection, J. Anim. Breed. Genet., 124, 323, 2007. 88. Volff, J.-N. et al., Governing sex determination in fish: Regulatory putsches and ephemeral dictators, Sex. Dev., 1, 85, 2007. 89. Baroiller, J.F. and D’Cotta, H., Environment and sex determination in farmed fish, Comp. Biochem. Physiol. C Toxicol. Pharmacol., 130, 399, 2001. 90. Devlin, R.H., Biagi, C.A., and Smailus, D.E., Genetic mapping of Y-chromosomal DNA markers in Pacific salmon, Genetica, 111, 43, 2001. 91. Du, S.J., Devlin, R.H., and Hew, C.L., Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): Presence of two functional genes, GH-I and GH-II, and a malespecific pseudogene, GH-psi, DNA Cell Biol., 12, 739, 1993.
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92. Kovács, B. et al., Male-specific DNA markers from African catfish (Clarias gariepinus), Genetica, 110, 267, 2000. 93. Woram, R.A. et al., Comparative genome analysis of the primary sex-determining locus in salmonid fishes, Genome Res., 13, 272, 2003. 94. Lee, B.Y., Hulata, G., and Kocher, T.D, Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus), Heredity, 92, 543, 2004. 95. Ezaz, M.T. et al., Isolation and physical mapping of sex-linked AFLP markers in nile tilapia (Oreochromis niloticus L.), Mar. Biotechnol., 6, 435, 2004. 96. Cnaani, A. et al., Genetics of sex determination in tilapiine species, Sex. Dev., 2, 43, 2008. 97. Devlin, R.H., Nagahama, Y., Sex determination and sex differentiation in fish: An overview of genetic, physiological, and environmental influences, Aquaculture, 208, 191, 2002. 98. Matsuda, M. et al., DMY is a Y-specific DM-domain gene required for male development in the medaka fish, Nature, 417, 559, 2002. 99. Nanda, I. et al., A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes, Proc. Natl. Acad. Sci. U.S.A., 99, 11778, 2002. 100. Tao, W.J. and Boulding, E.G., Associations between single nucleotide polymorphisms in candidate genes and growth rate in Arctic charr (Salvelinus alpinus L.), Heredity, 91, 60, 2003. 101. Huvet, A. et al., The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality, Gene, 343, 211, 2004. 102. Roberge, C. et al., Rapid parallel evolutionary changes of gene transcription profiles in farmed Atlantic salmon, Mol. Ecol., 15, 9, 2006. 103. Panserat, S. et al., Hepatic gene expression profiles in juvenile rainbow trout (Oncorhynchus mykiss) fed fishmeal or fish oil-free diets, Br. J. Nutr., 2008. [Epub ahead of print.] 104. Kirchner, S. et al., Salmonid microarrays identify intestinal genes that reliably monitor P deficiency in rainbow trout aquaculture, Anim. Genet., 319, 2007. 105. Baron, D. et al., Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles, BMC Genomics, 8, 357, 2007. 106. Baron, D. et al., Expression profiling of candidate genes during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens, Gen. Comp. Endocrinol., 156, 369, 2008. 107. Gahr, S.A. et al., Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss), Physiol. Genomics, 32, 380, 2008. 108. Young, N.D. et al., Coordinated down-regulation of the antigen processing machinery in the gills of amoebic gill disease-affected Atlantic salmon (Salmo salar L.), Mol. Immunol., 45, 2581, 2008. 109. Sarropoulou, E. et al., Gene expression profi ling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology, Physiol. Genomics, 23, 182, 2005. 110. Peatman, E. et al., Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri, Mol. Immunol., 45, 553, 2008. 111. Robalino, J. et al., Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: Tissue-specific expression profiles and transcriptomic responses to immune challenge, Physiol. Genomics, 29, 44, 2007. 112. Devlin, R.H. et al., Growth of domesticated transgenic fish, Nature, 409, 781, 2001. 113. Rasmussen, R.S. and Morrissey, M.T., Marine biotechnology for production of food ingredients, Adv. Food Nutr. Res., 52, 237, 2007. 114. Blunt, J.W. et al., Marine natural products. Nat. Prod. Rep., 25, 35, 2008. 115. Teletchea, F., Maudet, C., and Hänni, C., Food and forensic molecular identification: Update and challenges, Trends Biotechnol., 23, 359, 2005. 116. Wheeler, D.A. et al., The complete genome of an individual by massively parallel DNA sequencing, Nature, 452, 872, 2008.
Chapter 5
Nucleotides and Nucleosides M. Concepción Aristoy, Leticia Mora, Aleida S. Hernández-Cázares, and Fidel Toldrá Contents 5.1 Introduction ......................................................................................................................57 5.2 Chemical Structure of Main Seafood Nucleosides and Nucleotides ..................................59 5.3 Analysis of ATP-Related Compounds ...............................................................................59 5.3.1 Extraction of Nucleotides and Nucleosides ........................................................... 60 5.3.2 Nucleotides and Nucleosides Determination .........................................................61 5.3.2.1 31Phosphorous-Nuclear Magnetic Resonance Spectroscopy .....................61 5.3.2.2 Capillary Electrophoresis .........................................................................61 5.3.2.3 Chromatography......................................................................................61 5.3.2.4 Enzymatic Analysis................................................................................. 64 References ..................................................................................................................................65
5.1
Introduction
Bacterial growth is the main factor limiting fish commercial life by producing its alteration and unpleasant flavor. Nevertheless, the autolytic process derived from tissue enzymatic activity and lipid oxidations also contributes to fish maturation and subsequent spoilage. Sensory methods to evaluate fish quality are subjective and difficult to use in the evaluation of processed (fillets, beheaded, or eviscerated fish) or canned fish. Thus, objective methods for freshness determination are required and the determination of the biochemical changes occurring in early postmortem in fish constitute a helpful tool. The first autolytic process taking place in fish affects carbohydrates and nucleotides. After death, the adenosine triphosphate (ATP) regeneration that occurs in vivo stops and ATP is degraded until 57
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rigor mortis is reached. This process involves a series of reactions commonly represented according to the sequence shown in Figure 5.1. As a result of endogenous enzymes action, ATP molecule is rapidly degraded to adenosine monophosphate (AMP) and afterward to inosine monophosphate (IMP), which is accumulated in postharvest fish.1 The following IMP dephosphorylation to obtain inosine is mainly autolytic and occurs at a slower rate during the first stage of cold storage, although it might be accelerated by the action of different bacteria. IMP degradation to inosine (Ino) and its disappearance have been correlated with lack of freshness in some fish species. Inosine is transformed to hypoxanthine (Hx) by the action of the enzyme nucleoside phosphorylase (NP), which is oxidized to xanthine (Xa) and uric acid in the presence of xanthine oxidase (XO) enzyme. This enzyme is mainly generated in muscle from biochemical processes of microorganisms. The speed of each step in this reaction chain and especially in the Ino to Hx and Hx to Xa conversion depends on the fish species. IMP is the main nucleotide present in fish species, whereas AMP remains major in crustaceans.2 Howgate et al. (2006) published a review of the concentration of IMP, Ino, and Hx in the flesh of some species of fish during chilled storage.3 In all cases, Ino and Hx concentrations increased during storage, and either of the two may be used as freshness indicators.4 However, the use of a single compound as freshness indicator is not always advisable, because many factors can affect
O HO
N H 2N
N N
P O
N
OH O P
O
O
N
O
P
N
N
O P
O O
Pi
OH HO
HO
OH ADP Myokinase
Pi
N
N Pi
N N
N OH
HO
N N
HO
O
N
OH O
OH
OH ATP
HO
O P
ATP ase
OH O
HO
N
H2N
NH3
OH O P OH AMP deaminase
Nucleotidase HO
OH Ino
N
O
O
N
H2N N
O
O
N
O
OH
HO
IMP
OH AMP
Pi Nucleosidase phosphorilase Ribose 1-phosphate O N
HN
N H
N Hx
Figure 5.1
O
O2
O H2O2
N
HN
Xanthine oxidase
O
O2
N H
N H Xa
Degradation of ATP in postmortem fish muscle.
O H2O2
H N
HN
Xanthine oxidase
O N H UA
N H
P
OH
OH
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nucleotide degradation such as the type of spoilage bacteria and mechanical handling of fish,5 and, also, the disappearance of the degradation products differs from one species to another3 as mentioned here. This is the main reason for the use of indexes with more than one compound from the ATP-degradation chain. Some of them are briefly described here. K value is defined as the ratio of Ino and Hx to the sum of ATP and related compounds expressed as a percentage.6 This value has been used as one of the freshness indexes to evaluate the quality change of postharvest fish.2,7–9 Nevertheless, ATP, adenosine diphosphate (ADP), and AMP disappear early postmortem, generally within 1 day of storage in ice after death in all fish species,10,11 and, consequently, a revised K value, often designed K ′ value or Ki index, is more often considered as monitoring the loss of IMP and is defined as the ratio of Ino and Hx to the sum of IMP, Ino, and Hx expressed as percentage.12 However, for several species, a high accumulation of Ino occurs during ATP degradation, making K value inadequate as a freshness indicator. For this reason, a hypoxanthine ratio or H value (Hx/(IMP + Ino + Hx) × 100) was considered as a better indicator of fish freshness in this type of species. The ratio Hx/AMP was considered an adequate alternative to characterize fish freshness due to its constant increment with time.13,14 Measurement of ATP-related compounds is also useful for the quality control of retorted fishes, as shown when comparing high-temperature short-time process at 125°C for 9 min with a common retort process at 115°C for 90 min.15,16 Another suggestion to use nucleotide compounds as a measurement of seafood quality is their relation with sensory attributes. In this way, a high content of Hx is related with the bitter off taste of spoiled fish, whereas IMP evokes a fresh meaty taste sensation.16,17
5.2
Chemical Structure of Main Seafood Nucleosides and Nucleotides
To a better understanding of the methods of analysis of these compounds, the knowledge of their molecular structure is important. Nucleosides are glycosylamines that are formed when a nucleobase (purine or pyrimidine base) attaches to a ribose or deoxyribose ring. Nucleosides currently analyzed in seafoods are those in which a purine ring, adenine, or hypoxanthine is attached to a ribose, forming the adenosine or inosine, respectively. Nucleotides are o-phosphoric acid esters of the nucleosides, and thus, AMP or adenylic acid is derived from the adenosine in which a phosphate group is attached at the 5-ribose carbon. ADP and ATP are derived from the AMP, to which one or two additional phosphate groups are attached through pyrophosphate bonds (∼P) (Figure 5.2). On the other hand, IMP is derived from the inosine in which a phosphate group is attached to the 5-ribose carbon.
5.3 Analysis of ATP-Related Compounds The correct analysis of ATP-related compounds must take into account that early postmortem fish muscle is very sensitive to temperature. ATP-chain degradation occurs very fast, even at refrigeration temperatures, and it is important to stop this reaction drastically at the sampling time. This is achieved by immediately freezing the excised muscle under liquid nitrogen to stop all enzymatic reactions. In order to achieve this rapid freezing, it is advisable to collect small tissue samples and immerse them into liquid nitrogen. These cold conditions must be held along the sample preparation.18 After this, nucleotides and nucleosides should be extracted and analyzed.
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N NH2
N
OH
OH HO
P O
O
P O
O
O O
P O
N
N
OH OH
HO
Ribose
Adenine purine base
AMP ADP ATP Adenosine nucleotide
Figure 5.2
5.3.1
Structure of adenosine-derived nucleotides.
Extraction of Nucleotides and Nucleosides
A typical extraction procedure for the analysis of fish samples by reversed-phase chromatography, with or without employing an ion-pairing agent, is the following: 5 g or less of muscle tissue are excised and quickly frozen with liquid nitrogen. The frozen tissue is minced, avoiding any thawing, 3–5 vol. cold 0.6 M perchloric acid is added, and the tissue is homogenized with a stomacher-type homogenizer for a few minutes under cold conditions. Once the extract is centrifuged (15,000 g for 20 min), the supernatant is filtered through glass wool and neutralized to pH 6.5–6.8 by adding solid potassium carbonate or 1 M potassium hydroxide. This neutralized extract is kept in an ice bath for 15 min and centrifuged again (15,000 g for 10 min). The supernatant is filtered through a 0.2 μm membrane filter and stored under frozen storage at temperatures below −20°C until analysis, although storage at −18°C has been demonstrated to be enough to preserve fish samples and fish extracts for the analysis of IMP, Ino, and Hx.17 Other extraction methods consist in the homogenization of 2.5 g of fish sample with 10% trichloroacetic acid and, after centrifugation (27,000 g for 15 min), they are neutralized with 2 M sodium hydroxide. The neutralized extract must be made up to 5 mL with 20 mM phosphate buffer pH 7.8 and then filtered with a 0.45 mm membrane. These fish extracts are used in enzymatic assays with biosensors19,20 and/or spectrophotometers as well as in capillary electrophoresis (CE)21 or ion chromatography (IC).22
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In the development of biosensor analysis, some authors have described extraction methods that consisted of heated fish sample. In this way, both a microwave oven at 500 V for 5 s and heating at 100°C for 60 min have been used.23,24
5.3.2
Nucleotides and Nucleosides Determination
Several methods have been used to measure nucleotides and nucleosides, including nuclear magnetic resonance spectroscopy (NMR), high-performance capillary electrophoresis (HPCE), radioimmunoassay,25 thin-layer chromatography (TLC),26 reversed-phase high-performance liquid chromatography (RP-HPLC) with and without ion pair, ion-exchange HPLC,27 IC,22 and enzymatic assays.
5.3.2.1
31
Phosphorous-Nuclear Magnetic Resonance Spectroscopy
31
The phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) technique makes it possible to perform multiple determinations of high-energy phosphates in vivo in the same muscle sample. Thus, in vivo 31P-NMR spectroscopy has been used as a powerful technique to characterize the biochemical changes that occur in live, intact fishes after being submitted to physical and chemical stressors such as hypoxia.28 Also in vitro 31P-NMR spectroscopy has been applied to both excised tissue and perchloric acid extracts of fish muscle.
5.3.2.2 Capillary Electrophoresis CE is a powerful separation technique that can provide high separation efficiency and high sample throughput with minimal sample volume and buffer consumption. Capillary electrophoresis has been used in many nucleotide analysis applications as in the study of nucleotide degradation in fish tissues. In the analysis of complex biological samples, including fish extract, this technique can present problems in reproducibility, because these samples usually contain significant amounts of ions, which may be adsorbed on capillary walls. However, the reconditioning of the capillary surface is ensured by washing 1 min with 1M NaOH, followed by 2 min of the running buffer used.21 Typical conditions to get a good separation of IMP, inosine, and hypoxanthine would be a potential of 416 V/cm of capillary using 100 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer, pH 11.19
5.3.2.3
Chromatography
At present, among other chromatographic techniques, HPLC has been shown to be the most widely used technique to analyze nucleotides and nucleosides. In particular, RP-HPLC and ion-paired reverse-phase are the methods of choice for this analysis. The mode of separation will depend on the analyte of interest. Thus, to analyze nucleotides, the addition of an ion-pair to the mobile phase greatly improves the separation by increasing the retention time of charged molecules (ATP, ADP, AMP). Nevertheless, nucleotides will disappear at the rigor mortis state (normally 1 day after catch), and the K′ or K i index will be usually enough to characterize fish
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freshness or quality. Then, a simple RP-HPLC with a phosphate buffer as mobile phase will be adequate.
5.3.2.3.1 Reversed-Phase HPLC The chromatographic analysis should be performed in a liquid chromatograph equipped with an UV detector (254 nm). The column used is an analytical reversed-phase RP-18 column. There are many approaches to analyze nucleotides and nucleosides by this technique, which differ mainly in the pH of the mobile phase. All of them use a phosphate buffer as the mobile phase and a gradient with methanol or acetonitrile should be accomplished to improve the Ino resolution and reduce the chromatogram time.17,29 With buffer pH 7,29 phosphorylated metabolites are also well separated in the chromatogram.15 The identification of the chromatographic peaks can be performed by comparing the peak retention times and spectral characteristics (if a diode array detector is available) with those of standards. Quantitative analysis can be performed by external or internal standard method. In Figure 5.3 both chromatograms of standards and hake nucleosides and nucleotides are shown. The separation was achieved with an RP C-18 column at 35°C and a gradient between phosphate buffer at pH 7 and acetonitrile. 1000
(a) 6
800
600
Absorbance at 254 nm (mAU)
400
2 1
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4 5
3
0 (b)
1200
1 1000 6
800 600 400
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2 3
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0 0
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Figure 5.3 RP-HPLC chromatograms of standards (a) and hake (b) ATP-derived compounds. (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) hypoxanthine, and (6) inosine.
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5.3.2.3.2 Ion-Pair RP-HPLC The most common technique used for the separation of nucleotides is ion-pair RP-HPLC, which is especially useful in separating mixtures of charged and uncharged molecules. The separation is achieved in a reversed-phase column, and the key is to add an ion pair (an ion of charge opposite to that of the analyte molecule). Due to the negative charge of the phosphorylated groups of nucleotides, the ion pair should be a positive ion with a hydrophobic rest to improve the affinity with the stationary phase. Thus, either tetrabutylammonium hydrogen sulfate or phosphate is the ion pair most used.17,30 This ion-paired technique is especially useful when di- and tri-nucleotides have to be analyzed, because the ion pair enhances the retention time and separation, making it less dependant on the type of column, as well as the resolution, due to the ionic nature of the phosphate esters that facilitates strong interactions with the ion-pair reagent at the appropriate pH. Nevertheless, this method is more expensive than the more simple technique previously described. Figure 5.4 shows an ion-paired chromatogram of a 48 h postmortem sardine extract.
1400
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Figure 5.4 Ion-paired HPLC chromatograms of salmon (a) and sardine (b). ATP-derived compounds. (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) hypoxanthine, and (6) inosine.
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5.3.2.4 Enzymatic Analysis The use of enzymatic methods to analyze nucleotides in seafood is widespread due to their high specificity. These assays may be carried out with the enzymes in solution31,32 or immobilized, constituting what is known as enzyme sensors o biosensors.20,33,34 A biosensor is a system composed of a biological recognition element and a biochemical or physical transducer in intimate contact or in close proximity with each other in order to relate the concentration of an analyte to a measurable signal.35 Some details about the use of different biomaterials in order to select the best recognition elements and the most adequate methods for the enzyme immobilization have been described.36–38 The most used biosensors for the nucleotide-related compound analysis are electrochemical sensors, in which an enzyme or a group of enzymes are immobilized in a membrane or other supports, which is further coupled to a chemical transducer. These enzymes act by oxidizing the substrates (analyte) while consuming oxygen or producing hydrogen peroxide or uric acid. The depletion of oxygen or the formation of hydrogen peroxide or uric acid may be detected amperometrically. This option offers some advantages in relation to the free enzyme, due to its specificity, simplicity, and rapid response.36 Nevertheless, although biosensors have shown its utility in some applications such as clinical, environmental, agricultural, and biotechnology, the application in the food industry is still restricted36 mainly due to critical stages such as enzyme immobilization or sample preparation for analysis. Indeed, all the approaches to date need the sample preparation described earlier. In addition, the use of commercial kits or disposals presents some problems, because the denaturalization of the enzymes with time, and thus test kits, enzyme-coated strips, electrodes, or sensors have a limited shelf life.
5.3.2.4.1 Enzymatic Methods with the Enzyme in Solution The concentration of Hx, Ino, and IMP may be determined spectrophotometrically by a sequential addition of XO, NP, and 5′-nucleotidase (NT) into a reaction phosphate buffer containing the fish extract sample at pH 7.6–7.8 and 30°C–37°C. In these conditions, IMP, inosine, and hypoxanthine will be oxidized to uric acid and H2O2, which will be further quantified by measuring the absorbance at 290 nm and by polarimetry, respectively.20,39 This procedure was also used to analyze ATP and related compounds in fish sauces with very good results, because no interference of salt in the medium was observed here as was in the case using the HPLC method.31 Another possibility consisted in monitoring the oxygen consumption after these enzymatic reactions with an amperometric-type sensor (oxymeter), although these applications used to be achieved with at least one of these enzymes immobilized as described earlier.
5.3.2.4.2 Enzymatic Methods with Immobilized Enzymes In this case, the analysis may be performed with one or more enzymes, but they remain immobilized in different supports. Prodomidis and Karayannis85 reported a review on enzyme-based amperometric sensors applied to food analysis in which the principles and materials commonly used for the construction of the electrodes are described.35 The most used is the biosensor based on the measure of hypoxanthine. This sensor has been developed mainly for assessing the freshness of fish meat40,41 or for the evaluation of chicken32 and beef meat33 aging. In this sensor, XO enzyme, which is immobilized in a membrane fi xed in the sensing area of the electrode, oxidizes the hypoxanthine to xanthine and uric acid, while the depletion of oxygen is measured by a Clark-type elec-
Nucleotides and Nucleosides ◾
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trode at a platinum cathode (−0.6 to −0.9 V) vs. an Ag/AgCl reference electrode. The consumed oxygen produces a current decrease that can be correlated to the concentration of Hx. Both Hx and X are substrates for the XO action and will be oxidized either simultaneously or sequentially. The proposed relation 1 Hx for ½ X for each oxygen molecule formed must be taken into account to quantify the Hx,33 although this relation should be confirmed in each particular system. In the measurement of hypoxanthine, different supports have been used for the immobilization of the XO enzyme. Most of these supports have been developed with the aim of eliminating interferences due to ascorbic acid, uric acid, or H2O2, which can be present in the sample or formed during the enzymatic reaction. In this way, cellulose triacetate,41 preactivated nylon,18 and a silk fibron membrane in combination with a cellulose acetate membrane42 or a nylon net43 have been used. Most recent approaches to determine Hx are based on the incorporation of the XO enzyme in a graphite/Teflon matrix,44 a polyaniline film by electropolimerization,45 a nafion-coated platinum disc electrode,46 or even in a carbon paste electrode modified with electrodeposited gold nanoparticles.47 On the other hand, specific biosensors to determine AMP,48 IMP,49 and Ino50 and a multienzymatic sensor to analyze simultaneously AMP, IMP, Ino, and Hx using a cellulose triacetate membrane have been described.51 The use of multienzymatic biosensors to measure fish freshness has been very helpful for the simultaneous determination of AMP, IMP, Ino, and Hx amounts, necessary to obtain K, K i, and H values. Thus, Luong and Male20 used a multienzymatic biosensor system to determine the H value as a fish freshness indicator. An immobilized NT was used for the previous conversion of IMP to Ino, and then after adding a soluble NP, Ino was converted to Hx. Formed Hx was measured with an amperometric sensor that detected uric acid + hydrogen peroxide in an additive matter, and, thus, 1 mol of Hx would be converted to 1 mol of uric acid and 2 mol of hydrogen peroxide. This method was patented by Luong, Male, and Nguyen52 and afterward it has been commercialized as a Freshness Meter KV-101 (Oriental Electric Co. Ltd., Japan). Comparable results to that of HPLC were reported. A similar application was proposed12,34 to obtain the Ki parameter as a freshness indicator. Flow injection analysis (FIA) has been widely used in the development of these multienzymatic biosensors constituting different types of reactors in which different enzyme combinations can be immobilized as well as introduced as soluble enzyme.53,54 In fact, some authors have described this type of biosensor coupled with an oxygen electrode.23,53,55,56
References 1. Massa, A.E.; Palacios, D.L.; Paredi, M.E. et al. Postmortem changes in quality indices of ice-stored flounder (Paralichthys patagonicus). J. Food Biochem. 29: 570–590, 2005. 2. Mendes, R.; Quinta, R.; Nunes, M.L. Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast. Eur. Food Res. Technol. 212: 141–146, 2001. 3. Howgate, P. A review of the kinetics of degradation of inosine monophosphate in some species of fish during chilled storage. Int. J. Food Sci. Technol. 41: 341–353, 2006. 4. Özogul, F.; Özogul, Y.; Kuley, E. Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C. J. Fish. Sci. 1: 13–19, 2007. 5. Surette, M.E.; Gill, T.A.; LeBlanc, P.J. Biochemical basis of postmortem nucleotide catabolism in cod (Gadus morhua) and its relationship to spoilage. J. Agric. Food Chem., 36: 19–22, 1988. 6. Saito, T., Arai, K., Matsuyoshi, M. A new method for estimating the freshness of fish. Bull. Jpn. Soc. Sci. Fish., 24: 749–750, 1959. 7. Pacheco-Aguilar, R.; Lugo-Sánchez, M.E.; Robles-Burgueno, M.R. Postmortem biochemical and functional characteristic of Monterey sardine muscle stored at 0°C. J. Food Sci. 65: 40–47, 2000.
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8. Aubourg, S.P.; Quitral, V.; Larrain, M.A. et al. Autolytic degradation and microbiological activity in farmed Coho salmon (Oncorhynchus kisutch) during chilled storage. Food Chem. 104: 369–375, 2007. 9. Castillo-Yanez, F.J.; Pacheco-Aguilar, R.; Marquez-Rios, E. et al. Freshness loss in sierra fish (Scomberomorus sierra) muscle stored in ice as affected by postcapture handling practices. J. Food Biochem. 31: 56–67, 2007. 10. Jones, N.R. Meat and fish flavors—Significance of ribomononucleotides and their metabolites. J. Agric. Food Chem. 17: 712, 1969. 11. Jones, N.R.; Murray, J. Degradation of adenine- and hypoxanthine nucleotide in the muscle of chillstored, trawled cod (Gadus callarius). J. Sci. Food Agric. 13: 475–480, 1962. 12. Karube, I.; Matsuoka, H.; Suzuki, S. et al. Determination of fish freshness with an enzyme sensor system. J. Agric. Food Chem. 32: 314–319, 1984. 13. Massa, A.E.; Paredi, M.E.; Crupkin, M. Nucleotide catabolism in cold stored adductor muscle of scallop (Zygochlamys patagonica). J. Food Biochem. 26: 295–305, 2002. 14. Márquez-Rios, E.; Moran-Palacio, E.F.; Lugo-Sanchez, M.E. et al. Postmortem biochemical behavior of giant squid (Dosidicus gigas) mantle muscle stored in ice and its relation with quality parameters. J. Food Sci. 72: C356–C362, 2007. 15. Kuda, T.; Fujita, M.; Goto, H. et al. Effects of freshness on ATP-related compounds in retorted chub mackerel Scomber japonicus. LWT-Food Sci. Technol. 40: 1186–1190, 2007. 16. Kuda, T.; Fujita, M.; Goto, H. et al. Effects of retort conditions on ATP-related compounds in pouched fish muscle. LWT-Food Sci. Technol. 41: 469–473, 2008. 17. Veciana-Nogués, M.T.; Izquierdo-Pulido, M.; Vidal-Carou, M.C. Determination of ATP related compounds in fresh and canned tuna fish by HPLC. Food Chem. 59: 467–472, 1997. 18. Aristoy, M.C.; Toldra´, F. Nucleotides and its derived compounds. In Nollet L.M.L.; Toldra´, F. (Eds.), Handbook of Muscle Foods Analysis, CRC Press, Boca Raton, FL, pp. 279–288, 2009. 19. Luong, J.H.T.; Male, K.B.; Masson, C. et al. Hypoxanthine ratio determination in fish extract using capillary electrophoresis and immobilized enzymes. J. Food Sci. 57–1: 77–81, 1992a. 20. Luong, J.H.T.; Male, K.B. Development of a new biosensor system for the determination of the hypoxanthine ratio, an indicator of fish freshness. Enzyme Microb. Technol. 14: 125–130, 1992b. 21. Nguyen, A.L.; Luong, J.H.T.; Masson, C. Determination of nucleosides in fish tissues using capillary electrophoresis. Anal. Chem. 62: 2490–2493, 1990a. 22. Gao, R.; Xue, C.; Yuan, L. et al. Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography. J. Chromatogr. A 1118: 278–280, 2006. 23. Okuma, H.; Watanabe, E. Flow system for freshness determination based on double multi-enzyme reactor electrodes. Biosens. Bioelectron. 17: 367–372, 2002. 24. Watanabe, E.; Tamada, Y.; Hamada-Sato, N. Development of quality evaluation sensor for fish freshness control based on K1 value. Biosens. Bioelectron. 21: 534–538, 2005. 25. Roberts, B.; Morris, B.A.; Clifford, M.N. Comparison of radioimmunoassay and spectrophotometric analysis for the quantitation of hypoxanthine in fish muscle. Food Chem. 42: 1–17, 1991. 26. Dingle, J.R.; Hines, J.A.; Fraser, D.I. Post-mortem degradation of adenine nucleotides in muscle of the lobster, Homarus americanus. J. Food Sci. 33: 100–103, 1968. 27. Borgese, T.A.; Nagel, R.L.; Roth, E. et al. Guanosine triphosphate (GTP): The major organic phosphate in the erythrocytes of the elasmobranch Mustelus canis (smooth dogfish). Comp. Biochem. Physiol. 60: 317–321, 1977. 28. Van der Thillart, G.; Van Waarde, A.; Muller, H.J. et al. Determination of high-energy phosphate compounds in fish muscle: 31P-NMR spectroscopy and enzymatic methods. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 95(4): 789–795, 1990. 29. Özogul, F.; Taylor, K.D.A.; Quantick, P.C. et al. A rapid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmosphere. Int. J. Food Sci. Technol. 35: 549–554, 2000.
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30. Meynial, I.; Paquet, V.; Combes, D. Simultaneous separation of nucleotides and nucleotide sugars using an ion-pair reversed-phase HPLC: Application for assaying glycosyltransferase activity. Anal. Chem. 67: 1627–1631, 1995. 31. Cho, Y.J.; Im, Y.S.; Kim, S.M. et al. Enzymatic method for measuring ATP related compounds in fish sauces. J. Korean Fish. Soc. 32: 385–390, 1999. 32. Fujita, T., Hori, Y., Otani, T. et al. Applicability of the K0 value as an index of freshness for porcine and chicken muscles. Agric. Biol. Chem. 52: 107–112, 1988. 33. Yano, Y.; Kataho, N.; Watanabe, M. et al. Evaluation of beef ageing by determination of hypoxanthine contents: Application of a xanthine sensor. Food Chem. 52: 439–445, 1995. 34. Volpe, G.; Mascini, M. Enzyme sensors for determination of fish freshness. Talanta 43: 283–289, 1996. 35. Prodomidis, M.I.; Karayannis, M.I. Enzyme based amperometric biosensors for food analysis. Electroanalysis 14: 214–261, 2002. 36. Mello, L.D.; Kubota, L.T. Review of the use of biosensors as analytical tools in the food and drink industries. Food Chem. 77: 237–256, 2002. 37. Venugopal, V. Biosensor in fish production and quality control. Biosens. Bioelectron. 17: 147–157, 2002. 38. Sharma, S.K.; Sehgal, N.; Kumar, A. Biomolecules for development of biosensors and their applications. Curr. Appl. Phys. 3: 307–316, 2003. 39. Luong, J.H.T.; Male, K.B.; Nguyen, A.L. Application of polarography for monitoring the fish post-mortem metabolite transformation. Enzyme Microb. Technol. 11: 277–282, 1989. 40. Nguyen, A.L.; Luong, H.T.; Yacynych, A.M. Retention of enzyme by electropolymerized film: A new approach in developing a hypoxanthine biosensors. Biotech. Bioeng. 37: 729–735, 1990b. 41. Watanabe, E.; Ando, K.; Karube, I. et al. Determination of hypoxanthine in fish meat with an enzyme sensor. J. Food Sci. 48: 496–500, 1983. 42. Qiong, Q.; Tuzhi, P.; Liju, Y. Silk fibroin/cellulose acetate membrane electrodes incorporating xanthine oxidase for the determination of fish freshness. Anal. Chim. Acta 369: 245–251, 1998. 43. Shen, L.; Yang, L.; Peng, T. Amperometric determination of fish freshness by hypoxanthine biosensor. J. Sci Food Agric. 70: 298–302, 1996. 44. Cayuela, G.; Peña, N.; Reviejo, A.J.; Pingaron, J.M. Development of a bioenzymatic graphite-Teflon composite electrode for the determination of hypoxanthine in fish. Analyst 123: 371–377, 1998. 45. Hu, S.; Xu, C.; Luo, J. et al. Biosensor for detection of hypoxanthine based on xanthine oxidase immobilized on chemically modified carbon paste electrode. Anal. Chim. Acta 412: 55–61, 2000. 46. Nakatani, H.S.; Viera dos Santos, L.; Pelegrine, C.P. et al. Biosensor based on xanthine oxidase for monitoring hypoxanthine in fish meat. Am. J. Biochem. Biotechnol. 1(2): 85–892, 2005. 47. Agüí, L.; Manso, J.; Yañez-Sedeño, P. et al. Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on nanocrystal gold-carbon paste electrodes. Sens. Actuators B 113: 272–280, 2006. 48. Watanabe, E.; Ogura, T.; Toyama, K. Determination of adenosine 5′-monophosphate in fish and shellfish using an enzyme sensor. Enzyme Microb. Technol. 6: 207–211, 1984a. 49. Watanabe, E.; Toyama, T.; Karube, I. et al. Determination of inosine-5-monophosphate in fish tissue with an enzyme sensor. J. Food Sci. 48: 114–116, 1984b. 50. Watanabe, E.; Toyama, T.; Karube, I. et al. Enzyme sensor for hypoxanthine and inosine determination in edible fish. Appl. Microbiol. Biotechnol. 19(1): 18, 1984c. 51. Watanabe, E.; Tokimatsu, S.; Toyama, K. Simultaneous determination of hypoxanthine, inosine-5′ phosphate and adenosine 5′-phosphate with a multielectrode enzyme sensor. Anal. Chim. Acta 164: 139–146, 1984d. 52. Luong, H.T.; Male, K.B.; Nguyen, A.L. 1994. http://www.freepatentsonline.com/5288613.html. 53. Okuma, H.; Takahashi, H.; Yazawa, S. et al. Determination of a system with double enzyme reactors for the determination of fish freshness. Anal. Chim. Acta 260: 93–98, 1992.
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54. Carsol, M.-A.; Volpe, G.; Mascini, M. Amperometric detection of uric acid and hypoxanthine with xanthine oxidase immobilized and carbon based screen-printed electrode. Application for fish freshness determination. Talanta 44: 2151–2159, 1997. 55. Park, I.-S.; Kim, N. Simultaneous determination of hypoxanthine, inosine and inosine 5′-monophosphate with serially connected three enzyme reactors. Anal. Chim. Acta 394: 201–221, 1999. 56. Park, I.-S.; Cho, Y.-J.; Kim, N. Characterization of meat freshness application of a serial three-enzyme reactor system measuring ATP-degradative compounds. Anal. Chim. Acta 404: 75–81, 2000.
Chapter 6
Lipid Compounds Santiago P. Aubourg Contents 6.1 Introduction ..................................................................................................................... 70 6.1.1 General Aspects of Lipid Compounds .................................................................. 70 6.1.2 Marine Lipid Characteristics ................................................................................ 70 6.1.3 Lipid Analysis in Marine Products ........................................................................ 71 6.2 First Steps in Marine Lipid Analysis ................................................................................. 72 6.2.1 Isolation of Lipids from Tissues ............................................................................ 72 6.2.2 Removal of Nonlipid Contaminants ..................................................................... 72 6.2.3 Lipid Manipulation and Storage ........................................................................... 73 6.2.4 Lipid Quantification ............................................................................................. 73 6.3 Marine Fatty Acid Analysis .............................................................................................. 73 6.3.1 Fatty Acid Methyl Esters Preparation ................................................................... 73 6.3.1.1 Acid-Catalyzed Esterification and Transesterification ............................. 73 6.3.1.2 Base-Catalyzed Transesterification ......................................................... 75 6.3.2 GLC Analysis of FAME........................................................................................ 75 6.3.2.1 Qualitative Analysis of Fatty Acid Composition ..................................... 75 6.3.2.2 Quantitative Estimation of Fatty Acid Composition ...............................76 6.4 Analysis of Marine Nonsaponifiable Matter ......................................................................76 6.4.1 Lipid Saponification...............................................................................................76 6.4.2 Analysis of Sterols ..................................................................................................76 6.4.3 Analysis of Ether Lipids ........................................................................................ 77 6.5 Qualitative and Quantitative Analyses of Marine Lipid Classes ....................................... 77 6.5.1 Spectrophotometric Assessments of Total Lipid Extract ........................................ 78 6.5.2 Stereospecific Analysis of Lipid Classes ................................................................. 78 6.5.3 Column Chromatography..................................................................................... 79 69
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6.5.4 Thin-Layer Chromatography ................................................................................ 79 6.5.5 High-Performance Liquid Chromatography ......................................................... 79 6.5.6 Silver Ion Chromatography ................................................................................... 80 6.5.7 Nuclear Magnetic Resonance (NMR) Spectrometry ............................................ 80 6.5.8 Mass Spectrometry ................................................................................................81 6.5.9 Supercritical Fluid Chromatography ..................................................................... 82 References ................................................................................................................................. 82
6.1 Introduction 6.1.1 General Aspects of Lipid Compounds Lipids are found in all living organisms and have been shown to play two critical roles: (1) maintaining the integrity of plants and animals as structural compounds by forming a barrier separating the living cell from the outside world and (2) being a major source of cellular energy and function in living organisms where they can be stored. Different attempts have been carried out to define what is meant by the term lipid, although no satisfactory or widely accepted definition exists. Most textbooks describe lipids as a group of naturally occurring compounds, which have in common a ready solubility in organic solvents, such as hexane, toluene, chloroform, ethers, and alcohols. Such diverse compounds as hydrocarbons, carotenoids, steroids, soaps, triacylglycerols (TG), phospholipids (PL), gangliosides, and lipopolysaccharides would be included. Because of their structural and functional variety, a widely accepted division has been difficult. A simple physicochemical classification that empirically groups lipid molecules according to the hydrophilic–lipophilic balance has been proposed [1]. An alternative division into two broad classes has been shown to be convenient for lipid analysts [2]; in it, “simple lipids” (fatty acid and alcohol components) would be those that yield on hydrolysis at most two types of different products per mol, whereas “complex lipids” (glycerophospholipids, glycoglycerolipids, and sphingolipids) would yield three or more types of products per mol.
6.1.2 Marine Lipid Characteristics In many marine organisms, lipid is usually the second largest biochemical constituent after protein. Seafood lipids are known to provide high contents of important components for the human diet, such as nutritional lipid-soluble vitamins (namely A and D) and essential and ω3 polyunsaturated fatty acids (PUFA) that have shown a positive role in preventing certain human diseases, including cardiovascular ones [3]. Most animal and plant lipids from terrestrial and marine sources are similar in that they contain mainly even-numbered saturated and unsaturated fatty acids combined with glycerol (glycerides and glyceryl ethers), fatty alcohols (wax esters), sterols (sterol esters), phosphoric acid, and amines (PL). Marine lipids, however, differ from the other sources in that they contain a wider range of fatty acids, longer-chain fatty acids, and a larger proportion of highly unsaturated fatty acids, particularly C20:5ω3 (eicosapentaenoic acid, EPA) and C22:6ω3 (docosahexaenoic acid, DHA) [4]. Marine species have shown large variations in lipid content and composition as a result of endogenous and exogenous effects [5–7]. Related to exogenous effects, the catching season has been shown to play a key role regarding temperature and feeding availability; indeed, an inverse
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relationship between unsaturated fatty acid content and environmental temperature has been confirmed for many marine fish. With respect to endogenous effects, lipid matter has been described to exhibit a heterogeneous distribution throughout the body of marine species, probably affected by physiological and anatomical factors. Thus, differences according to the type of muscle and its location, age, sex, and sexual maturation have been pointed out. In all cases, content variations have specially been observed in fish locations to be employed as lipid depots.
6.1.3
Lipid Analysis in Marine Products
Researchers are required to analyze the lipid composition and its changes that arose during processing of food material from marine sources. The approach to the analysis of lipids in a given marine sample will depend on the amount of material in the sample, the equipment, and instrumentation available, but mainly the amount of information required. The present chapter is focused on describing the available traditional and advanced analytical methodology to assess the lipid composition of marine species on the basis of a food technologist and nutritionist requirements. A basic protocol procedure is exposed in Figure 6.1.
Marine products
Lipid isolation from tissues
Removal of nonlipid contaminants
Frozen storage
Fatty acid analysis
Lipid classes analysis
Traditional and advanced analytical methodology
Figure 6.1
Basic steps to be carried out for the lipid analysis of marine products.
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6.2 First Steps in Marine Lipid Analysis 6.2.1 Isolation of Lipids from Tissues Ideally, marine tissues should be extracted from the living organism as soon as possible after catching or slaughtering [2]. When this is not feasible, the tissue should be kept frozen (about −60°C or less) as rapidly as possible. Two main problems can arise with lipid fraction when employing inconvenient storage conditions. First, PUFA can autoxidize as a result of endogenous oxidant enzymes, although endogenous tissue antioxidants can provide some protection. The second problem is endogenous lipolytic enzymes that can lead to large amounts of unesterified fatty acids, diacylglycerides, phosphatidic acid, or lyso-phosphatidylglycerols in lipid extracts. Pure single lipid classes are soluble in a wide variety of organic solvents, but many of these are not suitable for extracting lipids from tissues as they are not sufficiently polar to overcome the strong forces of association between tissue lipids and the other cellular constituents, such as proteins. However, polar complex lipids, which do not normally dissolve readily in nonpolar solvents, may on occasion be extracted by these when they are in the presence of large amounts of simple lipids such as TG. The ideal solvent or solvent mixture for extracting lipids from tissues should be sufficiently polar to remove all lipids from their association with cell membranes or with lipoproteins but should not react chemically with those lipids. At the same time, it should not be so polar that TG and other nonpolar simple lipids do not dissolve and are left adhered to the tissues. Although there are limitations to its use and alternatives are frequently suggested, most workers in the field appear to accept two basic routines currently in general use, which yield essentially quantitative extractions of the major lipid classes when applied to homogenates of whole marine tissue extractions. The most popular is the method of Folch et al. [8], which employs chloroform–methanol (2:1) in a solvent–tissue ratio of 20:1. Where large amounts of tissue have to be extracted, the procedure of Bligh and Dyer [9] offers some advantages as it does not use large volumes of solvent; this method applies a single-phase solubilization of the lipids using chloroform–methanol (1:1) in a solvent–tissue ratio of 4:1. For all extraction methods, it is advisable to include an additional antioxidant at a level of 50–100 mg/L to the solvents. As an advanced alternative, supercritical fluid extraction shows an increasing demand, a major driving force being the environmental concern regarding the use of organic solvents. Its employment has recently been reviewed [10].
6.2.2
Removal of Nonlipid Contaminants
Most polar organic solvents used to extract lipids from tissues also extract significant amounts of nonlipid contaminants such as sugars, urea, amino acids, peptides, and salts. In addition, all solvents can contain contaminants, and as large volumes of solvents may be used to obtain small amounts of lipids, any such impurities can be troublesome. Most of the contaminating compounds can be removed from the lipid extract mixtures simply by shaking the combined solvents with one-quarter their total volume of a dilute salt solution (e.g., 0.88% KCl) [8]. A more elegant and complete, though more time-consuming, method of removing nonlipid contaminants is to carry out the washing procedure by liquid–liquid partition chromatography on a column of a dextran gel such as Sephadex G-25. This type of washing procedure was first developed by Wells and Dittmer [11] and simplified later by Wuthier [12] for large numbers of samples.
Lipid Compounds
6.2.3
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Lipid Manipulation and Storage
Wherever possible, lipids should be handled in an atmosphere of nitrogen, since PUFA will oxidize rapidly in air [2]. Autoxidation of double bonds in marine lipid fatty acids is particularly troublesome, and care must be taken at all steps in the analysis of lipids. When it is necessary to concentrate lipid extracts, large volumes of solvents are best removed by means of a rotatory film evaporator at a temperature that, in general, should not exceed about 40°C. Small volumes of solvent can be evaporated by carefully directing a stream of nitrogen onto the surface of the solvent. Lipids should not be left for any time in the dry state and should be stored in an inert nonalcoholic solvent such as chloroform from which air is excluded by flushing with a stream of nitrogen. Storage temperature should be −30°C as the highest temperature. Natural tissue antioxidants, such as tocopherols, afford some protection to lipid extracts, but it is usually advisable to add further synthetic antioxidants to storage solvents at the level of 50–100 mg/L [2]. As storage containers, glass is the best choice. Plastic ware of all kinds (other than that made from TeflonTM) can be specially troublesome and is best avoided, since plasticizers are very easily leached out. Conversely, it has been shown that lipids can themselves dissolve in some plastics, leading to selective losses of a proportion of the less polar constituents.
6.2.4
Lipid Quantification
For most common purposes, a known aliquot of the purified lipid extract is softly heated and the resulting dry lipid matter weighted, provided water absorption onto the dry extract lipid is avoided. For fast purposes, the Soxhlet method of extraction has been developed [13]. In it, a large diethyl ether volume is employed, and the resulting lipid extract can no more be employed for further analysis. This method proved to be accurate in the case of a high lipid content (low complex lipid content); if not, relatively important errors are obtained. According to the special relevance recently acquired by noninvasive technologies, application of nuclear magnetic resonance (NMR), near-infrared (NIR) spectrometry, and Fatmeter measurements is proving to be of increasing interest [14].
6.3 Marine Fatty Acid Analysis 6.3.1 Fatty Acid Methyl Esters Preparation Fatty acids are essential components of lipids. Their measurement by gas–liquid chromatography (GLC) is the most commonly used method for lipid analysis. Owing to the wide variety of fatty acid compounds in marine lipids (Table 6.1), this analysis is more complicated than that with other kinds of living organisms. Lipid extracts have to be converted into methyl ester derivatives. Two basic strategies can be applied [15,16]: acid catalysis and base catalysis. Then, fatty acid methyl esters (FAME) obtained are usually introduced in the GLC system without previous removal of contaminants.
6.3.1.1
Acid-Catalyzed Esterification and Transesterification
Free fatty acids (FFA) are methylated and O-acyl lipids transmethylated by heating them with a large excess of anhydrous methanol in the presence of an acidic catalyst. In addition, fatty acids
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Fatty Acids Commonly Present in Marine Speciesa
Abbreviated Name
Systematic Name
Trivial Name
Saturated Fatty Acids 14:0
Tetradecanoic
Myristic
15:0
Pentadecanoic
16:0
Hexadecanoic
Palmitic
17:0
Heptadecanoic
Margaric
18:0
Octadecanoic
Stearic
20:0
Eicosanoic
Arachidic
22:0
Docosanoic
Behenic
24:0
Tetracosanoic
Lignoceric
—
Monounsaturated Fatty Acids 16:1 ω7
9-Hexadecenoic
Palmitoleic
18:1 ω9
9-Octadecenoic
Oleic
18:1 ω7
11-Octadecenoic
Vaccenic
20:1 ω11
9-Eicosenoic
Gadoleic
20:1 ω9
11-Eicosenoic
Gondoic
22:1 ω11
11-Docosenoic
Cetoleic
22:1 ω9
13-Docosenoic
Erucic
24:1 ω9
15-Tetracosenoic
Nervonic
Polyunsaturated Fatty Acids
a
18:2 ω6
9,12-Octadecadienoic
Linoleic
18:3 ω3
9,12,15-Octadecatrienoic
Linolenic
18:4 ω3
6,9,12,15-Octadecatetraenoic
Stearidonic
20:4 ω6
5,8,11,14-Eicosatetraenoic
Araquidonic
20:5 ω3
5,8,11,14,17-Eicosapentaenoic
EPA
22:5 ω3
7,10,13,16,19-Docosapentaenoic
DPA or clupanodonic
22:6 ω3
4,7,10,13,16,19-Docosahexaenoic
DHA or cervonic
In all cases, the double-bond configuration is “cis.”
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from amide-bound lipids (sphingolipids) are also transesterified, whereas aldehydes are liberated from plasmalogens under acidic conditions. The commonest and mildest reagent used for the purpose is anhydrous hydrogen chloride in methanol. This is simply prepared by adding acetyl chloride slowly to cooled dry methanol. FAME are obtained by heating the reaction mixture in a stoppered tube at 50°C overnight. In order to guarantee complete solution of nonpolar lipid classes, a further solvent such as toluene should be employed. This reagent has been applied directly to fish muscle to obtain FAME without previous lipid extraction [17]. A different possibility consists of employing a solution of 1%–2% (v/v) concentrated sulfuric acid in methanol. Transesterification is carried out in the same manner and at much the same rate as with methanolic hydrogen chloride. Boron trifluoride in methanol is also used as a transmethylation catalyst and in particular as a rapid esterifying reagent for FFA. The reagent has a limited shelf life unless refrigerated, and the use of old or too concentrated solutions may result in the production of methoxy-substituted acids from unsaturated fatty acids and, accordingly, a PUFA loss.
6.3.1.2 Base-Catalyzed Transesterification O-acyl lipids are transesterified very rapidly in anhydrous methanol in the presence of a basic catalyst. However, FFA are not esterified. As with acid-catalyzed procedures, an additional solvent is necessary to solubilize nonpolar lipids such as cholesterol esters or TG. However, under base catalysis, aldehydes are not liberated from plasmalogens and amide-bound fatty acids are not affected. The commonest reagent used for this purpose has been sodium methoxide in anhydrous methanol, prepared simply by dissolving fresh clean sodium in dry methanol, although potassium methoxide or hydroxide have also been used as catalysts.
6.3.2
GLC Analysis of FAME
The advent of GLC revolutionized the analysis of the fatty acid components of lipids, so most performances have been carried out for qualitative and quantitative analysis [16]. Initially, glasspacked columns were widely employed [18]. Later on, the application of wall-coated open tubular (WCOT) columns to the analysis of fatty acids has provided a better knowledge of the complexity of marine fatty acids [19].
6.3.2.1
Qualitative Analysis of Fatty Acid Composition
During the previous decades, parameters known as equivalent chain lengths (ECLs) or carbon numbers have considerably been employed. ECL values can be calculated from an equation similar to that for Kovats’ indices or found by reference to the straight line obtained by plotting the logarithms of the retention times of a homologous series of straight-chain saturated FAME against the number of carbon atoms in the aliphatic chain of each acid. The retention times of the unknown acids should be measured under identical operating conditions, and the ECL values are read directly from the graph. Parallel to ECL value employment, known commercial FAME have been employed for the provisional identification of fatty acids by direct comparison of their retention times and those of the unknown esters on the same columns under identical conditions.
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In recent years, GLC/mass spectrometry (MS) has been widely accepted as one of the most valuable techniques for the identification of fatty acids and their derivatives [20]. A high proportion of the available data has been obtained for the methyl ester derivatives of fatty acids, as these are easily prepared and are widely used in chromatographic analysis. However, pyridinecontaining derivatives, such as picolinyl esters, have been shown to be suitable for direct mass spectrometric structural analysis of acids containing straight, branched, unsaturated, cyclic, or oxygenated chains. Finally, 4,4-dimethyloxazoline derivatives of fatty acids have been found to show several advantages and have been applied successfully to the structural determination of PUFA and cyclopropenoid fatty acids [21].
6.3.2.2
Quantitative Estimation of Fatty Acid Composition
With reliable modern gas chromatographs equipped with flame ionization detectors (FID), the areas under the peaks on the GLC traces are, within limits, linearly proportional to the amount (by weight) of material eluting from the columns [15,16]. Problems of measuring this area arise mainly when components are not completely separated, and there is no way of overcoming this difficulty entirely. A known quantity of an internal standard should be added to the lipid sample. In most cases, nonadecanoic acid is employed and added before the methylation step; quantitative results would first be calculated on its basis. On the other hand, commercially available standard mixtures containing accurately known amounts of methyl esters of saturated, monoenoic, and polyenoic fatty acids should be analyzed under the same GLC conditions for checking the quantification results. If necessary, calibration factors may have to be calculated for each fatty acid component to correct the areas of the relevant peaks in the mixtures analyzed; this is specially relevant for PUFA. Results can be expressed as weight percentages of the fatty acids present or as molar amounts of each fatty acid.
6.4 Analysis of Marine Nonsaponifiable Matter 6.4.1
Lipid Saponification
Lipids may be hydrolyzed (saponified) by heating them under reflux with an excess of dilute aqueous ethanolic alkali; the fatty acids on one side and diethyl-ether-soluble nonsaponifiable materials on the other side are separately recovered for further analysis [2]. According to the previous section, the resulting FFA have to be transformed into their corresponding FAME for further analysis by an acid-catalyzed method. On the other hand, the nonsaponifiable layer will contain any long-chain alcohols and sterols originally present in the lipid sample in the esterified form, as well as the deacylated residues of ether lipid compound. Such compounds can be divided into sterols and “ether lipids.”
6.4.2
Analysis of Sterols
Sterols are biological compounds, the basic structure of which includes the cyclopentanophenanthrene ring. Total sterol content can be determined directly and spectrophotometrically from the lipid extract by using the method of Huang et al. [22], based on the Liebermann–Buchardt reaction. For a complete analysis, sterols can be fractionated and analyzed by means of different
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chromatographic techniques [23,24]. For GLC analysis, marine sterols have to be converted into more volatile compounds such as acetate [25] and trimethylsilyl (TMS) [26] derivatives. Cholesterol has been shown to be the most abundant sterol in all marine living beings, although invertebrates have shown a significant presence of other sterols [27]. Chromatographic methods for cholesterol analysis [28] are of relevant importance in foods in relation to human health concerns. Although the GLC is normally carried out with cholestane as internal standard, high-performance liquid chromatography (HPLC) methods can offer a nondestructive alternative, but they suffer from the limitation of the lack of a distinctive chromophore in the analyte. Great attention has been accorded to the assessment of cholesterol oxide formation in marine products [29].
6.4.3 Analysis of Ether Lipids Marine lipids may contain fatty acid residues as the only radicals, or they may include alkyl and alkenyl radicals. The first type is the major one in marine lipids, and its analysis has already been discussed in Section 6.3. The others are often united into a group called “ether lipids.” Such compounds are basically found as PL classes (specially in phosphatidylethanolamine), being normally placed as the radical in position 1 and specially abundant in marine invertebrates [5,27]. Unlike fatty acids, information on ether lipid composition in marine PL is less abundant, although a great interest has been accorded to their isolation because of their medical and cosmetic applications [30]. Methods for separating and quantifying ether-linked glycerides have been reviewed [31,32]. The alkyl groups of 1-alkyl-2,3-diacyl-sn-glycerols are generally saturated or cis-monoenoic even– numbered components (16:0, 18:0, and 18:1, mostly). The alkyl moieties are usually analyzed in the form of 1-alkylglycerol or as volatile nonpolar derivatives of this compound, such as acetate, trifluoroacetate, TMS, or isopropylidene derivatives by GLC. The determination of double-bond positions in long alkyl chains has been carried out by means of picolinyl and nicotinylidene derivatives by GLC-MS [33]. Further, supercritical fluid chromatography (SFC) has been employed for the glycerol ether analysis of liver oils of shark species [34]; thus, chimyl, batyl, and selachyl alcohols were found to be the most abundant. Concerning alkenylglycerols, such compounds tend to be decomposed during GLC analysis and are best reduced by catalytic hydrogenation to alkylglycerols. Although the vinyl ether linkage is unaffected by basic hydrolysis conditions, it can be cleaved by acid-catalyzed transmethylation, which generates dimethyl acetals from the liberated fatty aldehydes. Accordingly, alkenyl compounds have been directly identified and quantified by GLC together with FAME [35]. Adsorption thin-layer chromatography (TLC) on silica gel layers can be used to separate simple alkyl and alkenyl lipids; neutral plasmalogens tend to migrate ahead of alkyldiacylglycerols, which in turn migrate just in front of TG. They can be separated by a double development in a single direction with hexane–diethyl ether (95:5, v/v) as a solvent system. Neutral plasmalogens may be detected by spraying the TLC plates with 2,4-dinitrophenyl-hydrazine (0.4%) in 2M HCl, whereas no simple spot test is available for the identification of alkyldiacylglycerols.
6.5
Qualitative and Quantitative Analyses of Marine Lipid Classes
Lipid samples obtained from extraction of biological material are complex mixtures of individual lipid classes [16]. Often, no single procedure will achieve the desired analysis, and combinations of techniques must be used until the required purposes are served. In this section, different analytical
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approaches will be discussed, focused on the qualitative and quantitative analyses of marine lipid classes. The fatty acid composition of each lipid class can be determined by GLC of the methyl ether derivatives, prepared by esterification or transesterification of the purified lipid class, according to details explained in Section 6.3.
6.5.1 Spectrophotometric Assessments of Total Lipid Extract Some classical methods are available for the analysis of lipid classes or lipid class groups when applied directly onto the lipid extract without prior separation. For FFA assessment, titrimetric methods were used for many years, although some interference of polar lipids was found. Procedures that involve spectrophotometric measurement of highly colored copper complexes are now favored. A very popular one is that proposed by Lowry and Tinsley [36], where an FFA-cupric acetate-pyridine complex is involved. More recently, a rapid NIR spectrometry has been applied for the direct FFA determination in fish oil [37]. Traditional determination of PL content in lipid extracts has involved the digestion of PL with the release of inorganic phosphate; then, this is made to react with ammonium molybdate to form phosphomolybdic acid, which is reduced and determined spectrophotometrically [38]. An alternative and successful method has been proposed by Raheja et al. [39] without previous digestion; in it, PL present in the lipid extract are made to react with ammonium molybdate in an organic phase and then measured spectrophotometrically. Ester linkages can be quantified by the method of Vioque and Holman [40]. In it, such functional groups are made to react with hydroxamic acid and further complexed with Fe (III). This method can be applied to total lipid extract or to any lipid class after previous isolation [7,41]. Finally, a method for the quantification of esterified and unesterified total sterols is mentioned in Section 6.4.2 [22].
6.5.2 Stereospecific Analysis of Lipid Classes The determination of fatty acid composition at each location in lipid classes has ever since attracted great attention. Compared with the data compiled for plant oils and for fats from land animals, the results so far reported for aquatic animals are few. This is due to the great complexity of fatty acids present in these oils and fats, giving rise to a tremendous number of species. The advent of new NMR, HPLC, and GLC technologies combined with MS in the last decades has provided quick and useful procedures for the stereospecific lipid analysis, according to the information provided in following sections. However, traditional methodologies are still employed in cases where such advanced technologies are not available and are reviewed in this section. Most living organisms have developed lipolytic enzyme systems that are able to distinguish between bonds to the various positions of glycerol or between certain types of bonds in specific lipids. In many cases, these enzymes can be isolated and used in simple incubations in vitro as an aid in structural analyses of lipids, this including chromatographic separation and further analysis of fatty acids after previous methylation and transmethylation. A wide use was found for lipase hydrolysis, although it turned out not to be accurate enough for marine lipids, since the presence of double bonds in the proximity of a carbonyl group of fish PUFA reduces the rate of deacylation of glycerides. Accordingly, the Grignard reagent has widely been employed in the case of marine substrates [42]. Additionally, preparative TLC on silicic acid impregnated with 5% (w/w) boric acid has been applied to prevent acyl migration during chromatographic separation.
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Traditional research accounts for consecutive series of methods combining chemical reactions and enzymatic releases of fatty acids in different positions for resolution of the molecular species. Such stereospecific studies have widely been focused onto TG [43,44] and PL [45,46] classes.
6.5.3 Column Chromatography Normal-pressure or low-pressure column chromatography (CC) was widely employed in the past and is now mostly used as a way of preliminary fractionation of lipid classes. Separation can be carried out on silicic acid, acid-washed florisil, or florisil as adsorbents, being simple lipids eluted in a stepwise sequence with hexane containing increasing proportions of diethyl ether, whereas complex lipids are recovered by elution with methanol [41,47]. The principal advantages of the method are the ease of preparation of a column and the comparatively large amount of lipid that can be separated. Column chromatography on diethylaminoethyl (DEAE)-cellulose has shown to be a valuable method for the isolation of particular groups of complex lipids in comparatively large amounts, although lengthy conditioning may be necessary before columns can be employed [2,47]. Aminopropyl-bonded phase cartridges have been much used for the isolation of simple and complex lipid fractions, although particular care is required to recover the acidic lipids quantitatively.
6.5.4 Thin-Layer Chromatography Many text books and reviews report TLC application on lipids for routine separations, identification, and quantification [48,49]. A variety of solvent systems have been used to separate simple lipids on an analytical or semipreparative scale by single or two-dimensional TLC. Those used most frequently contain hexane, diethyl ether, and acetic (or formic) acid in various proportions. For preparative purposes, 20–50 mg of marine lipid may be applied with ease as a band on a 20×20 cm plate coated with a layer of silica gel of 0.5 mm thickness [7,41]. However, precoated plates are much more convenient than laboratory-made plates, in spite of the relatively higher costs [50]. In all cases, lipid classes can be detected by any of the nonspecific available reagents and identified by their migration characteristics relative to authentic standards chromatographed simultaneously alongside the samples under investigation. The improvement and versatility of TLC enable it to be used for several modern applications, which include highly automated techniques right from sample application and development to detection and quantification. Such techniques would include high-pressure TLC (HPTLC), overpressure TLC (OPTLC), and tubular TLC (TTLC). In addition, coupling of TLC with other techniques such as HPLC, infrared (IR) spectrometry, MS, and NMR has increased its analytical power in several applications. The perceived weakness of TLC has been recognized as the quantification aspect, and this has led to the evolution of the TLC/FID Iatroscan system, which has been used routinely for lipid analysis in the last decades. It combines the separation capabilities of conventional TLC with the quantification power of the FID and has application in the quantitative analysis of all substances separable by conventional TLC. The system has been successfully used for marine lipid class analysis [51].
6.5.5 High-Performance Liquid Chromatography In recent years, HPLC has undoubtedly been the most widely applied separation technique in the analysis of most simple and complex lipid classes [48,52]. HPLC is much more expensive than
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TLC in terms of both equipment and running costs, but it can be automated to a considerable degree and gives much cleaner fractions in micropreparative applications. It can give better and more consistent separations of minor components, while no oxidation of the unsaturated fatty acid constituents needs to occur during fractionation on an HPLC column. An isocratic and gradient elution procedure with ultraviolet (UV) detection has been employed for marine PL analysis. In the detection, some of the more impressive separations have made use of FID systems, but others have obtained satisfactory results, with UV detection at 206 nm both on an analytical and on a preparative scale. Finally, evaporative light-scattering detection has successfully been applied [16]. HPLC has specially been applied to the most abundant lipid classes; therefore, quantification and stereospecific analyses have been carried out. Thus, TG separation according to the carbon number or partition number has been achieved [53]. For PL classes, HPLC analysis has been accepted as the most accurate one, by employing both gradients of polar solvents and microparticulate silicic acid [6,54].
6.5.6 Silver Ion Chromatography Silver ions, like the ions of other transition metals, interact specifically with the olefinic double bonds of unsaturated compounds to form weak charge transfer complexes. The complexes are usually unstable and exist in equilibrium with the free form of the olefin. However, such complexation is favorable for use in chromatography and enables the performance of the various Ag+-chromatographic techniques developed so far. Thus, Ag+-chromatography has been performed in conjunction with CC, TLC, and HPLC, being successfully applied to all lipid classes in marine species by separating molecules according to unsaturation degree [55]. Ag+-TLC is used mostly in the preparative mode, as a complementary separation method to GLC or GLC-MS. Both homemade and precoated glass plates are used in Ag+-TLC. The usual supporting materials are silica gel G for FAME and TG and silica gel H for complex lipids. Thus, the complementary employment of GLC or GLC-MS together with Ag+-TLC is considered one of the most powerful tools for elucidation of fatty acid composition in complex lipid samples [56]. On the other hand, Ag+-HPLC and reverse phase (RP)-HPLC applied in complementary ways were effective in the analysis of TG in fish oils [57]. Perona and Ruiz-Gutiérrez [53] were able to resolve a large number of sardine TG molecular species by RP-HPLC; further identification of most peaks was carried out by using preparative Ag+-TLC followed by fatty acid analysis by GLC.
6.5.7 Nuclear Magnetic Resonance (NMR) Spectrometry In recent years, high-resolution NMR spectrometry (1H-NMR, 13C-NMR, 31P-NMR) has increasingly been applied to the identification of lipid structures to determine patterns of branching, or substitution, and in particular to the detection, and often the location, of the double-bond systems in fatty acid chains. In the past 20 years, some important articles and reviews have been published [58], so it has become an extremely powerful technique for obtaining qualitative and quantitative information of the lipid class profile of a marine tissue extract. The procedure is rapid and nondegradative.
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Based on 1H-NMR spectrometry [59], a rapid and structure-specific method for the determination of ω3-PUFA in fish lipids was presented. The different chemical shifts observed for the methyl resonance of ω3-PUFA (δ = 0.95 ppm) with respect to the methyl resonance of all other fatty acids (δ = 0.86 ppm) provided the possibility of proposing this new analytical tool. In a first attempt for 13C-NMR application [60], the high-resolution NMR spectra of four fish oils were recorded. Signals in the spectra were assigned, and attention was focused on the identification of specific signals for ω3 fatty acid group and also individually for DHA, EPA, and stearidonic acid. Later on [61], 13C-NMR spectrometry was successfully used to determine the proportions of saturated, mono- and diene-, ω6, ω3, and highly unsaturated fatty acids of lipid extract of Atlantic salmon muscle. A good agreement could be observed between NMR values and those from the GLC analysis. Quantitative analysis of fatty acid composition and alpha-beta distribution in TG tuna fish was achieved [62]; results obtained using high-resolution 13C-NMR were in good agreement with those obtained by GLC. FFA carbonyl resonances were detected at the lower field of the carbonyl region, thus providing a suitable tool for lipolysis analysis. The positional distribution (1, 2, and 3 locations) of ω3 fatty acids in depot fat of several fish species was examined by 13C-NMR [63]. It could be observed that DHA was concentrated in the 2-location of TG in depot fats. Finally, 13C-NMR was employed for the plasmalogen analysis in fish lipid samples showing a good agreement with the data obtained by GLC [64]. Application of 31P-NMR has shown to be far shorter than with 1H and 13C, so little application is specially available for marine lipids [58]. This NMR technique can provide a single signal for each PL class, according to each corresponding resonance; its intensity should be proportional to the quantity. The 31P-NMR application has also shown the possibility of analyzing the ether structures within the glycerol backbone of phosphatidylethanolamine and phosphatidylcholine.
6.5.8 Mass Spectrometry MS has long been used as a powerful tool for the analysis of the molecular weight, empirical formula, and complete structure of an unknown compound, although an increasing importance has been obtained lately for quantitative analysis [20,65,66]. The first step for any MS method is ionization of the sample molecules in the gas phase. Following ionization to a negatively or positively charged species (most commonly the later), the molecules or their fragments can be separated and identified on the basis of their mass-to-charge ratio (m/z). Over the years, many of the advances in MS have involved new ionization techniques. The information-rich nature of MS makes it the most desirable detector for many explanations, but, although GLC is conveniently coupled to electron impact ionization (EI) and chemical ionization (CI) sources, the condensed mobile phase used for liquid separations is not readily compatible with high vacuum ionization sources. After different approaches, Arpino [67] likened the HPLC-MS union. This development paralleled the development of atmospheric pressure chemical ionization (APCI). Recent developments in MS have been very interesting for complex lipid molecules. Thus, soft ionization MS techniques such as fast atom bombardment, thermospray, and electrospray have the ability to ionize lipid molecules without causing extensive fragmentation. Thus, fragmentation of the molecular ion species produced by soft ionization processes can further be achieved in a second mass spectrometer (MS/MS) by collision-induced dissociation. Among the different food lipids, marine lipids have received lesser attention, probably due to their more complicated structure. Some applications concerning the marine lipids’ study will now be mentioned.
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The oxidative decomposition of cholesterol in different fish products was investigated by means of MS analysis of cholesterol oxide TMS derivatives with a quadrupole mass spectrometer fitted with an EI source [68]. A wide range of cholesterol oxides were identified and quantified. The qualitative and quantitative compositions of 1-O-alk-1-enylglycerolipids of albacore tuna (Thunnus alalunga) were studied along the canning process [35]; analysis was carried out in conjunction with FAME by means of their dimethyl acetal derivatives resulting from the acid transmethylation of lipid extracts. Minor fatty acids from mussels (Mytilus galloprovincialis) were enriched by Ag+-TLC and then analyzed by GLC-MS as 2-alkenyl-4,4,-dimethyloxazoline derivatives [69]. The mass spectrometer was operated in the EI mode (70 eV), and several nonmethylene interrupted fatty acids were singled out. Lately, Rezanka [70] described a method for the enrichment of long-chain fatty acids from fatty acids of a green freshwater alga and their identification as picolinyl esters by means of HPLCMS with APCI; the method was based on the use of preparative reversed-phase HPLC followed by subsequent identification by APCI HPLC-MS.
6.5.9 Supercritical Fluid Chromatography In this advanced technique [10,71], analytes are eluted from a capillary chromatographic column, which uses a highly compressed gas above its critical temperature and critical pressure. Carbon dioxide is by far the most commonly used SFC mobile phase because of its low critical temperature, whereas its critical pressure and critical density are high enough for good solvation of many potential analytes. An important advantage is that it is compatible with FID, which has great sensitivity and linearity. In addition, the use of SFC can substantially reduce the dependence on organic solvents in solvent extraction or HPLC analysis. Analytical SFC has been shown to be particularly applicable to the analysis of higher molecular weight lipid moieties, such as mixed glyceride compositions ranging from 200 to 900 in molecular weight. Concerning marine species analysis, in a first attempt Baltic herring flesh TG were separated in eight fractions by Ag+-TLC, and the four most unsaturated fractions were analyzed by capillary SFC according to their acyl carbon numbers [72]. Later on, simple classes from marine oils of different species were separated and quantified by capillary SFC [73]; carbon dioxide as the mobile phase, a nonpolar capillary column, and a FID were employed in it. The liver oils of several shark species were analyzed by SFC [34]; thus, the method was capable of direct quantification of squalene and cholesterol, whereas quantification of TG, cholesterol esters, and diacylglycerol ethers required TLC fractionation before SFC analysis. Purification of PUFA (DHA and EPA) ethyl esters from tuna oil was carried out by SFC [74]; an optimization of process parameters was achieved to obtain a maximal production rate.
References 1. Small, D., Handbook of Lipid Research, The Physical Chemistry of Lipids, Plenum Press, New York, 1986, Vol. 4, p. 89. 2. Christie, W., Lipid Analysis, 2nd edn., Pergamon Press, Oxford, U.K., 1982, p. 17. 3. Simopoulos, A., Nutritional aspects of fish, in Seafood From Producer to Consumer, Integrated Approach to Quality, Luten, J., Börrensen, T., and Oehlenschläger, J., eds., Elsevier Science, London, U.K., 1997, p. 589.
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4. Ackman, R., Fatty Acids, in Marine Biogenic Lipids, Fats, and Oils, Ackman, R., ed., CRC Press, Boca Raton, FL, 1989, Vol. 1, p. 103. 5. Pearson, A., Love, J., and Shorland, F., “Warmed-over” flavor in meat, poultry and fish. Adv. Food Res., 23, 2, 1977. 6. Vaskowski, V., Phospholipids, in Marine Biogenic Lipids, Fats, and Oils, Ackman, R., ed., CRC Press, Boca Raton, FL, 1989, p. 199. 7. Sieiro, P., Aubourg, S. and Rocha, F., Seasonal study of the lipid composition in different tissues of the common octopus (Octopus vulgaris). Eur. J. Lipid Sci. Technol., 108, 479, 2006. 8. Folch, J., Lees, M., and Stanley, G., A simple method for the isolation and purification of total lipids from animal tissue, J. Biol. Chem., 226, 497, 1957. 9. Bligh, E. and Dyer, W., A rapid method of total extraction and purification. Can. J. Biochem. Physiol., 37, 911, 1959. 10. King, J., Supercritical fluid chromatography (SFC)-Global perspective and applications in lipid technology, in Advances in Lipid Methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 301. 11. Wells, M. and Dittmer, J., The use of sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry, 2, 1259, 1963. 12. Wuthier, R., Purification of lipids from nonlipid contaminants on sephadex bead columns, J. Lipid Res., 7, 558, 1966. 13. Prost, E. and Wrebiakowski, H., Evaluation of soxhlet’s and Bligh and Dyer’s methods in the determination of fat in meat. Z. Lebensm. Unters. Forsch., 149, 193, 1972. 14. Nielsen, D., Hyldig, G., Nielsen, J., and Nielsen H., Lipid content in herring (Clupea harengus L.). Influence of biological factors and comparison of different methods of analyses: Solvent extraction, Fatmeter, NIR and NMR. Food Sci. Technol., 38, 537, 2005. 15. Kuksis, A., Separation and determination of structure of fatty acids, in Handbook of Lipid Research, Fatty Acids and Glycerides, Kuksis, A., ed., Plenum Press, New York, 1978, Vol. 1, p. 1. 16. Christie, W., Lipid Analysis, 3rd edn., The Oily Press, Bridgwater, U.K., 2003, p. 37. 17. Lepage, G. and Roy, C., Direct transesterification of all classes of lipids in a one step reaction. J. Lipid Res., 27, 114, 1986. 18. Hammond, E., Packed-column gas chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K. and New York, 1986, p. 113. 19. Ackman, R., WCOT (capillary) Gas–liquid chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K. and New York, 1986, p. 137. 20. Le Quéré, J., Gas chromatography-mass spectrometry and tandem mass spectrometry in the analysis of fatty acids, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Perkins, E., eds., AOCS Press, Champaign, IL, 1995, p. 191. 21. Medina, I. and Garrido, J., One-step conversion of fatty acids into their 2-alkenyl-4,4,-dimethyloxazoline derivatives directly from total lipids. J. Chrom. A, 673, 101, 1994. 22. Huang, T., Chen, C., Wefler, V., and Raftery, A., A stable reagent for the Liebermann-Buchardt reaction. Anal. Chem., 33, 1405, 1961. 23. Teshima, S., Sterols and crustaceans, mollusks and fish, in Physiology and Biochemistry of Sterols, Patterson, G. and Nes, W., eds., American Oil Chemists’ Society Press, Champaign, IL, 1990, p. 229. 24. Hoving, E., Chromatographic methods in the analysis of cholesterol and related lipids. J. Chrom. B, 671, 341, 1995. 25. Aubourg, S., Pérez-Martín, R., and Gallardo, J., Stability of lipids of frozen albacore (Thunnus alalunga) during steam cooking. Int. J. Food Sci. Technol., 24, 341, 1989. 26. Krzynowek, J. and Panunzio, L., Cholesterol and fatty acids in several species of shrimp. J. Food Sci., 54, 237, 1989. 27. Joseph, J., Distribution and composition of lipids in marine invertebrates, in Marine Biogenic Lipids, Fats and Oils, Ackman, R., ed., CRC Press Inc., Boca Raton, FL, 1989, Vol. 2, p. 49. 28. Fenton, M., Chromatographic separation of cholesterol in foods. J. Chrom., 624, 369, 1992.
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29. Ohshima, T., Formation and content of cholesterol oxidation products in seafood and seafood products, in Cholesterol and Phytosterol Oxidation Products, Guardiola, F., Dutta, P., Codony, R. and Savage, G., eds., American Oil Chemists’ Society Press, Champaign, IL, 2002, p. 186. 30. Hayashi, K., Occurrence of diacyl glycerol ethers in liver lipids of gonatid squid Gonatopsis borealis. Nippon Suisan Gakkaishi, 55, 1383, 1989. 31. Sargent, J., Ether-linked glycerides in marine animals, in Marine Biogenic Lipids, Fats and Oils, Ackman, R., ed., CRC Press Inc., Boca Raton, FL, 1989, Vol. 1, p. 175. 32. Urata, K. and Takaishi, N., Ether lipids based on the glyceryl ether skeleton: Present state, future potential, J. Am. Oil Chem. Soc., 73, 819, 1996. 33. Harvey, D., Nicotinylidene derivatives for the structural elucidation of glycerol mono-ethers and mono-esters by gas chromatography/mass spectrometry. Biol. Mass Spectrom., 20, 87, 1991. 34. Borch-Jensen, C., Magnussen, M., and Mollerup, J., Capillary supercritical fluid chromatographic analysis of shark liver oils. J. Am. Oil Chem. Soc., 74, 497, 1997. 35. Medina, I., Aubourg, S., and Pérez-Martín, R., Analysis of 1-O-alk-1-enyl glycerophospholipids of albacore tuna (Thunnus alalunga) and their alterations during thermal processing. J. Agric. Food Chem., 41, 2395, 1993. 36. Lowry, R. and Tinsley, I., Rapid colorimetric determination of free fatty acids. J. Am. Oil Chem. Soc., 53, 470, 1976. 37. Zhang, H. and Lee, T., Rapid near-infrared spectroscopic method for the determination of free fatty acid in fish and its application in fish quality assessment. J. Agric. Food Chem., 45, 3515, 1997. 38. Barlett, G., Phosphorus assay in column chromatography. J. Biol. Chem., 234, 466, 1959. 39. Raheja, R., Kaur, C., Singh, A., and Bhatia, I., New colorimetric method for the quantitative determination of phospholipids without digestion. J. Lipid Res., 14, 695, 1973. 40. Vioque, E. and Holman, R., Quantitative estimation of esters by thin-layer chromatography. J. Am. Oil Chem. Soc., 39, 63, 1962. 41. Gallardo, J., Aubourg, S., and Pérez-Martín, R., Lipid classes and their fatty acids at different loci of albacore (Thunnus alalunga): Effects of the pre-cooking, J. Agric. Food Chem., 37, 1060, 1989. 42. Aubourg, S., Sotelo, C., and Gallardo, J., Zonal distribution of fatty acids in albacore (Thunnus alalunga) triglycerides and their changes during cooking. J. Agric. Food Chem., 38, 255, 1990. 43. Christie, W., Stereospecific analysis of triacyl-sn-glycerols, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Perkins, E., eds., AOAC Press, Champaign, IL, 1995, p. 93. 44. Myher, J., Kuksis, A., Geher, K., Park, P., and Diersen-Schade, D., Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids. Lipids, 31, 207, 1996. 45. Brockerhoff, H., Determination of the positional distribution of fatty acids in glycerolipids. Methods Enzymol., 35, 315, 1975. 46. Aubourg, S., Medina, I., and Pérez-Martín, R., Polyunsaturated fatty acids in tuna phospholipids: Distribution in the sn-2 location and changes during cooking. J. Agric. Food Chem., 44, 585, 1996. 47. Hemming, F. and Hawthorne, J., Lipid Analysis. Editorial Acribia, S. A., Zaragoza (Spain), 2001, p. 5. 48. Hamilton, R., Thin layer chromatography and high-performance liquid chromatography, in Analysis of Oils and Fats, Hamilton, R. and Rossell, J., eds., Elsevier Applied Science, London, U.K., 1986, p. 243. 49. Shukla, V., Thin-layer chromatography of lipids, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Ackman, R., eds., AOCS Press, Champaign, IL, 1995, p. 17. 50. Nakamura, T., Fukuda, M., and Tanaka, R., Estimation of polyunsaturated fatty acid content in lipids of aquatic organisms using thin-layer chromatography on a plain silica gel plate. Lipids, 31, 427, 1996. 51. Shantha, N. and Napolitano, G., Lipid analysis using thin-layer chromatography and the Iatroscan, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 1.
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52. Shukla, V., High-performance liquid chromatography: Normal-phase, reverse-phase detection methodology, in New Trends in Lipid and Lipoprotein Analyses, Sebedio, J. and Ackman, R., eds., AOCS Press, Champaign, IL, 1995, p. 38. 53. Perona, J. and Ruiz-Gutiérrez, V., Characterization of the triacylglycerol molecular species of fish oil by reversed-phase high performance liquid chromatography. J. Liq. Chrom. Rel. Technol., 22, 1699, 1999. 54. Medina, I., Aubourg, S., and Pérez-Martín, R., Composition of phospholipids of white muscle of six tuna species. Lipids, 30, 1127, 1995. 55. Nikolova-Damyanova, B., Lipid analysis by silver ion chromatography, in Advances in Lipid methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 43. 56. Joh, Y., Elenkov, I., Stefanov, K., Popov, S., Dobson, G., and Christie, W., Novel di-, tri- and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea. Lipids, 32, 13, 1997. 57. Laakso, P. and Christie, W., Combination of silver ion and reversed-phase high-performance liquid chromatography in the fractionation of herring oil triacylglycerols. J. Am. Oil Chem. Soc., 68, 213, 1991. 58. Diehl, B., Multinuclear high-resolution nuclear magnetic resonance, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 87. 59. Sacchi, R., Medina, I., Aubourg, S., Addeo, R., and Paolillo, L., Proton nuclear magnetic resonance rapid and structure-specific determination of ω-3 polyunsaturated fatty acids in fish lipids. J. Am. Oil Chem. Soc., 70, 225, 1993. 60. Gunstone, F., High resolution NMR studies of fish oils. Chem. Phys. Lipids, 59, 83, 1991. 61. Aursand, M., Rainuzzo, J., and Grasdalen, H., Studies of fatty acids in Atlantic salmon (Salmo salar) by 13C and 1H nuclear magnetic resonance (NMR) spectroscopy, in Quality Assurance in the Fish Industry, Huss, H., ed., Elsevier Science Publishers B. V., Amsterdam (Holland), 1992, p. 407. 62. Sacchi, R., Medina, I., Aubourg, S., Giudicianni, I., Addeo, F., and Paolillo, L., Quantitative high resolution 13C-NMR analysis of lipids extracted from the white muscle of Atlantic tuna. J. Agric. Food Chem., 41, 1247, 1993. 63. Aursand, M., Jørgensen, L., and Grasdalen, H., Positional distribution of ω3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy. J. Am. Oil Chem. Soc., 72, 293, 1995. 64. Sacchi, R., Medina, I., and Paolillo, L., One and two-dimensional NMR study of plasmalogens (alk-1-enyl-phosphatidylethanolamine). Chem. Phys. Lipids, 76, 201, 1995. 65. Kuksis, A., Mass spectrometry of complex lipids, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, England, U.K., 1998, p. 181. 66. Byrdwell, W., APCI-MS in lipid analysis, in Advances in Lipid Methodology—Five, Adlof, R., ed., The Oily Press, Bridgwater, England, U.K., 2003, p. 171. 67. Arpino, P., On-line liquid chromatography/mass spectrometry? An odd couple! Trends Anal. Chem., 1, 154, 1982. 68. Oshima, T., Li, N., and Koizumi, C., Oxidative decomposition of cholesterol in fish products. J. Am. Oil Chem. Soc., 70, 595, 1993. 69. Garrido, J. and Medina, I., Identification of minor fatty acids in mussels (Mytillus galloprovincialis) by GC-MS of their 2-alkenyl-4,4-dimethyloxazoline derivatives. Anal. Chim. Acta, 465, 409, 2002. 70. Rezanka, T., Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry from green alga Chlorella kesslerri. J. Sep. Sci., 25, 1332, 2002. 71. Blomberg, L., Demirbuker, M., and Andersson, M., Characterization of lipids by supercritical fluid chromatography and supercritical fluid extraction, in Lipid Analysis in Oils and Fats, Hamilton, R., ed., Blackie Academic and Professional, London, U.K., 1998, p. 34.
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72. Kallio, H., Vauhkonen, T., and Linko, R., Thin-layer silver ion chromatography and supercritical fluid chromatography of Baltic herring (Clupea harengus membras) triacylglycerols. J. Agric. Food Chem., 39, 1573, 1991. 73. Staby, A., Borch-Jensen, C., Balchen, S., and Mollerup, J., Quantitative analysis of marine oils by capillary supercritical fluid chromatography. Chromatographia, 39, 697, 1994. 74. Alkio, M., Gonzales, C., Jäntti, K., and Aaltonen, O., Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography. J. Am. Oil Chem. Soc., 77, 315, 2000.
Chapter 7
Lipid Oxidation Turid Rustad Contents 7.1 Introduction ..................................................................................................................... 87 7.2 Analysis of Lipid Oxidation ............................................................................................. 88 7.2.1 Primary Oxidation Products ................................................................................. 88 7.2.2 Secondary Oxidation Products ............................................................................. 89 7.2.3 Stability Methods ................................................................................................. 92 7.2.4 Instrumental Methods .......................................................................................... 92 7.2.5 Sensory Analysis of Rancidity ............................................................................... 93 7.3 Summary ......................................................................................................................... 93 References ................................................................................................................................. 93
7.1
Introduction
Marine lipids are good and natural sources of polyunsaturated n-3 fatty acids (PUFA) such as docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3). These fatty acids have beneficial health effects and are reported to prevent coronary heart diseases and have a positive effect on the brain and nervous system as well as stimulating the immune system [1,2]. However, due to the high content of long-chain PUFAs, marine lipids are highly susceptible to oxidation. Lipid oxidation is the most important factor limiting the shelf life of marine oils and is also an important factor determining the shelf life of seafood products, except when microbial processes limit the shelf life. Reaction products from lipid oxidation have a negative effect on the sensory properties of fish products. The volatile, secondary oxidation products, especially those that originate from n-3 PUFAs are components that have a low threshold and therefore have a negative impact on the sensory quality of the food even in low concentrations [3]. This can lead to 87
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loss of products, complaints from the consumers, and reduced sales. Some of the reaction products from lipid oxidation may also have negative health effects. Lipid oxidation can be divided into three types, autoxidation, photooxidation, and enzymatic oxidation. Autooxidation of lipids takes place when the unsaturated fatty acids are exposed to oxygen and proceeds through an autocatalytic chain reaction [3]. Free radicals are formed when hydrogen ions are extracted from the fatty acids. The radicals react with oxygen forming peroxy radicals and hydroperoxides. The peroxides are easily broken down to alkoxy radicals, leading to a wide variety of reaction products. These include nonradical species such as aldehydes, ketones, acids, and alcohols, and also more complex reaction products such as epoxy and polymeric compounds are formed during the propagation and termination steps [4]. The secondary oxidation products include both low molecular weight, volatile compounds and nonvolatile components with a relatively high molecular weight. The secondary oxidation products can also react further, resulting in a wide variety of degradation products, which makes it difficult to find where the components originated. This also makes the determination of the degree of oxidation a challenging task. When the decomposition of a hydroperoxide has resulted in the formation of a low-molecular weight volatile compound, the parent triglyceride is left with a shorter fatty acid. If this contains a terminal carbonyl group, the molecule is called a core aldehyde. However, the influence of these compounds has been little studied [3]. The fatty acids and the lipid oxidation products in foods can also react with other components in the food such as proteins, carbohydrates, and water, making it even more difficult to determine the degree of rancidity.
7.2 Analysis of Lipid Oxidation Many different methods have been implemented both by the industry and in research to determine the degree of lipid oxidation both in marine oils and in seafood. Methods to determine the degree of lipid oxidation can be divided into two main groups, methods that determine the primary oxidation products and methods that measure the secondary oxidation products.
7.2.1
Primary Oxidation Products
The most common methods to determine primary oxidation products are peroxide value (PV) and conjugated dienes. PV is one of the classical methods for determination of oxidative status; it is both one of the oldest and one of the most used methods. For determination of PVs in foods, the lipid can be extracted using the methods of Ref. [5] or [6] before analysis. Several analytical procedures are available, but it is important to keep in mind that the results for PV measurements will vary both according to the method used and how the procedure is performed [3]. A simple titration method where the sample is dissolved in chloroform–acetic acid (or isooctane–acetic acid) is often used for fats and oils. Potassium iodide is added, this is oxidized by the hydroperoxides or other components present in the sample, and the liberated iodine is titrated with sodium thiosulfate with starch as an indicator. The PV is expressed in milliequivalent of iodine per kilogram of lipid or as millimolar of peroxide per kilogram of lipid [7]. This method requires a sample of 5 g if the PV is below 10 and about 1 g if the PV is higher [3]. The sensitivity is about 0.5 meq/kg, but this can be improved by determining the endpoint colorimetrically or by
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determining the liberated iodine electrometrically using a platinum electrode. Oxygen in the air, light, and absorption of iodine by the unsaturated fatty acids in the oil may interfere and cause variations in the results. Care should therefore be taken in standardizing how the procedure is performed. Several colorimetric methods for determination of PV values are used. One of these is the colorimetric ferric thiocyanate method. In this procedure ferrous ions are oxidized to ferric ions, which react with ammonium thiocyanate forming ferric thiocyanate, which is a red complex with an absorption maximum of 500 nm [3]. This method is more sensitive and requires smaller samples. The method of The International Dairy Federation—often called the IDF method [8] as modified by Ueda et al. [9] and Undeland et al. [10]—requires a low amount of sample (less than 10 mg). Nielsen et al. compared five different methods for determination of PVs [11]—the titration method, the colorimetric ferro method, the micromethod determining oxidation of iodide to free iodine, the FOX2 method determining oxidation of ferrous salts to ferric ions and reaction with xylenol orange, and the modified IDF method. Even if new instrumental methods now have been developed for determination of PVs, it is often desirable to use a method that either does not require instruments or requires only a spectrophotometer. The different methods gave different PVs for the same sample, and there was no consistency in the levels of PV determined by the different methods. Based on the fact that the methods chosen should have a large linear range, a high reproducibility, and use a low amount of solvent, the IDF method was chosen as the best of these methods. However, also for this method care should be taken in standardizing the procedure, with regard to chemicals used, how these are stored, and how the procedure is performed. Small changes in quality of ethanol can give widely different standard curves and thereby influence the results. In order to determine individual peroxides, high-performance liquid chromatography (HPLC) methods can be used [3]. After the initiation phase, the level of primary oxidation products increases and passes through a maximum. Using PV as a sole determination of oxidation level can therefore be misleading, and it is important to know the history of the oil or the seafood to interpret the measurement of PV. Due to rapid polymerization of EPA and DHA compared with the formation of stable peroxides of these fatty acids, PV is reported to be an unreliable indicator of lipid peroxidation in fish [4]. Conjugated diene hydroperoxides are formed when polyunsaturated fatty acids oxidize. The fatty acid chain then contains a structure with alternating simple and double bonds. Conjugated dienes have a strong absorption maximum at 230–235 nm [12]. Frankel [3] suggests measuring the absorbance of conjugated dienes at 243 nm. A known amount of sample is diluted in methanol (esters), isooctane, or hexane [13]. Conjugated dienes are useful for bulk lipids, and the AOCS method requires a sample size of around 10 mg. For use on tissue extracts, extraction and separation techniques are necessary. The sensitivity and specificity can be increased by using second derivative spectra [12].
7.2.2
Secondary Oxidation Products
Development of peroxides and conjugated dienes follows the same process and can be reduced after a certain oxidation level. These methods are therefore most useful as a measure of lipid oxidation for lipids with a low level of oxidation. Peroxides are unstable and are rapidly transformed into secondary [14] oxidation products, and determinations of PV have to be combined with the determination of secondary products such as thiobarbituric acid-reactive substances (TBARS) and
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anisidine value (AnV). These methods determine the presence of aldehydes, which are secondary oxidation products. For determination of secondary oxidation products, the AnV is a common method. This determines the amount of aldehydes (mainly 2-alkenals and 2,4-dienals). The sample is dissolved in isooctane, p-anisidine dissolved in acetic acid is added, and the absorbance at 350 nm is determined after 10 min [15]. The AnV of freshly deodorized oils is caused by core aldehydes. AnV can also be determined using Fourier transform infrared (FT-IR) [16]. The Totox value is still one of the most commonly used oxidation parameters used in commercial laboratories and laboratories in the edible oil industry. This value is a combination of the PV and the AV. The Totox value is given as 2*PV + AnV. The determination of TBARS (or TBA) is a common method to determine secondary oxidation products. There are many published methods to determine TBARS, but as for the determination of PV, different methods give different results. All the methods are based on the pink color absorbance formed by reaction between TBA and oxidation products of polyunsaturated lipids. Originally, the colored complex was ascribed to the condensation of two moles of TBA and one mole of malonaldehyde (MDA), which is formed as a decomposition product from lipid hydroperoxides under the acidic test conditions [3]. However, the reaction is not specific, and the color is formed by many different secondary oxidation products, hence the name TBARS. Many factors influence the color in the TBA test—temperature, time of heating, pH, metal ions, and antioxidants. Some of the MDA detected in this test is formed during the peroxidation of the lipids, but most of it is formed during the decomposition of the lipid peroxides during the acid heating stage. This process is accelerated by metal ions [12]; in addition H2O2, antioxidants, and chelating agents may also influence the peroxide decomposition during the assay. Many variations of this test are being used. In the AOCS method [13], the lipids are dissolved in a solution of thiobarbituric acid in butanol, the sample is incubated at 95°C for 2 h, and the absorbance of the solution is read at 530 nm. In another method, the oxidation products are extracted in trichloroacetic acid (TCA) before the reaction with TBA. In the micromethod of Ke and Woyewoda [17], the lipids are boiled for 45 min with a mixture of TBA, sulfite, and chloroform before adding TCA, and the optical density of the water phase is determined at 538 nm. In other variations, the TBARS are separated by steam distillation or HPLC to increase selectivity. The TBA test can be standardized using MDA, which is generated by acid hydrolysis of 1,1,3,3-tetraethoxypropane [3]. However, alkanals, alkenals, and 2,4-dienals also react with TBA, forming a yellow pigment absorbing light at 450 nm. Dienals also give a red pigment absorbing at 530 nm. In addition, many other components in foods can react with TBA or interfere with the measurements. Protein, amino acids, nucleic acids, nitrite, sucrose and other sugars, reaction products from browning reactions, antioxidants, and trace metals can influence the result [3,13]. The TBARS values for different foods with the same level of oxidation (based on flavor scores) can vary significantly [3,13,18]. However, TBARS values have been found to correlate with sensory scores within the same materials [19]. The volatile compounds formed as a result of lipid oxidation can be analyzed using electronic noses/gas-sensor array systems [20]. Different types of headspace analyses can be used, where the headspace volatiles over the samples are sampled, separated, and identified using different gas sensors. Of these methods, static headspace and solid-phase microextraction (SPME) are the least sensitive. Purge and trap techniques, where the samples are flushed or purged with nitrogen and the volatiles in the gas flow are trapped on a solid absorber, are highly sensitive. After sampling, the volatiles can be thermally desorbed into a gas chromatograph for separation. The mass spectra of the compounds can also be compared with spectra of pure standard compounds and
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identified [21]. The advantages of this method are that it is flexible, and the amount of sample and sampling conditions can be varied according to the needs. However, quantification of headspace data, especially from solid matrixes, is complicated, and the results are dependant on the sample material. Small variations in sampling procedures can give large variations in the data; the data handling is also difficult. Analysis of volatiles is discussed by Ólafsdóttir and Jónsdóttir in Chapter 8. Lipid oxidation products can interact with other components in food, such as amino acids, peptides, proteins, nucleic acids, deoxyribonucleic acid (DNA), phospholipids, and so on, and form fluorescent products. Reactions between lipid oxidation products and other components in seafood or seafood products may lead to underestimation of the degree of lipid oxidation, as measured by methods such as PV and TBARS. Hydroperoxides (primary lipid oxidation products) and aldehydes (secondary oxidation products) can react with amino groups in proteins, forming Schiff bases. This reaction can lead to formation of brown-colored compounds [22,23]. The fluorescent compounds formed from lipids are the result of oxidation of phospholipids or are formed from oxidized fatty acids in the presence of phospholipids. Fluorescence techniques are highly sensitive and 10–100 times more sensitive for detection of MDA than TBARS [3]. The different chromophores formed as a result of oxidized lipids, reactions between oxidized lipids and proteins/ peptides, or reactions between oxidized lipids and DNA have different excitation and emission maxima as shown in Table 7.1. Fluorescence has traditionally been applied to samples in solution, for example, for assessment of lipid oxidation during fish processing [24–26]. Aubourg and Medina [26] extracted fish muscle with a 2/2/1.8 chloroform/methanol/water mixture and measured fluorescence both in the water and in the organic phase. They measured the fluorescence intensity both at 393/463 nm and 327/415 nm. The fluorescence intensities were divided by the fluorescence intensity of quinine sulfate and the fluorescence shift calculated. The fluorescence shift was found to be a more effective index of changes in fish quality than other commonly used methods. When fluorescence measurements are done on samples in solution, and the concentration is below a certain level, the measured intensity follows the Beer–Lambert law. When the samples are turbid or solid or the concentration is high, scatter, quenching, and so on, destroy this relationship. Instead, front-face fluorescence
Table 7.1 Excitation and Emission Maxima for Chromophores Formed as a Result of Oxidized Lipids, Reactions between Oxidized Lipids and Proteins/Peptides or Reactions between Oxidized Lipids and DNA Chromophore
Excitation Maxima (nm)
Emission Maxima (nm)
Oxidized phospholipids/oxidized fatty acids + phospholipids
365
435–440
MDA + phospholipids
400
475
Oxidized arachidonic acid + dipalmityl phosphatidylethanolamine
360–390
430–460
Oxidized arachidonic acid + DNA
315
325
Peroxides/secondary oxidation products + DNA in the presence of metal ions or reducing agents
320
420
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spectroscopy can be used. Using solid-phase fluorescence is a relatively new approach, but studies on the use of this technique in dried fish were published in 1992 [27]. Fluorescence spectroscopy on intact samples has been shown to be a sensitive technique, comparable to sensory analysis and gas chromatography, for measuring lipid oxidation [28]. One challenge is that fluorescence spectra can be very complex and that not only the oxidation products but also connective tissue, adipose tissue, porphyrins, and additives may contribute to the spectra. So far, little has been done to study the fluorescence spectra of the different oxidation products that are formed in foods. In a study of different model systems including fish and meat, Veberg et al. [28] concluded that fluorescence spectroscopy may be able distinguish between different oxidation products formed but that this would require using the whole spectrum and not only the intensity at the maximum wavelength. Fluorescence spectroscopy has a great potential for on-line or at-line applications.
7.2.3
Stability Methods
Several techniques based on accelerated oxidation are used for evaluation of oxidation; these include the oil stability index method [29], the Rancimat test [30], and oxidative stability measurement by Oxidograph [31] and they are all suitable for analyzing oil systems. The Rancimat, oil stability index (OSI), active oxygen method (AOM), and Oxidograph are techniques for measuring the stability of oils toward oxidation. In Rancimat and OSI instruments, the oil can be heated to 80°C or more while air is bubbled through it. This results in the formation of low molecular weight acids that are flushed out with the air and collected in vessels containing distilled water. The change in conductivity is measured, and the point where it changes most is called the induction time. The AOM method is performed in a somewhat similar way, but it measures the time taken to reach a certain PV. The Oxidograph instrument finds the induction time based on measurement of the decline in pressure caused by the absorption of oxygen in a closed vessel.
7.2.4
Instrumental Methods
Many instrumental methods have been developed for the determination of oxidation parameters in oils and foods, including near-infrared spectroscopy (NIR), Fourier-transform near-infrared (FT-NIR), and FT-IR spectroscopy methods [16,32,33]. Lipid oxidation products can produce very weak chemiluminescence (CL). It has been shown that sodium hypochlorite-induced decomposition of hydroperoxides gives strong CL [34,35]. The level of hydroperoxides in fish oil can be determined using a rapid CL method [14]. In recent years, new methods have been developed, and these include assessment of free radicals using electron spin resonance (ESR) spectroscopy and use of different chromatographic methods to determine both primary and secondary oxidation products. The gas chromatography–mass spectrometry (GC–MS) techniques can be used to determine a wide range of volatile secondary lipid oxidation products [36]. The liquid chromatography–mass spectrometry (LC–MS) techniques can also determine nonvolatile products—of special interest are the core aldehydes [3,37]. Free radical assessments by the ESR spin-trapping technique detected the very early stages of lipid oxidation, and a few minutes of oxidation of docosahexaenoate (DHA) resulted in significant changes in the ESR spectra. The levels of free radicals trapped in cod liver oil and salmon oil during the first hours of oxidation were in accordance with the oxidative stability measured by conventional methods [4]. 1H NMR spectra can be used to study specific lipid oxidation products, such as different hydroperoxides, aldehydes, and also cyclic compounds, obtaining information
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that cannot usually be obtained by single conventional analytical methods [4]. Multivariate data analysis is a valuable tool in elucidating changes in spectra during storage and showed the resonances that came from n-3 fatty acids during oxidation. However, the sensitivity was low (detection levels ∼0.01 nM). The sensitivity could be improved by the use of CryoProbe technology.
7.2.5
Sensory Analysis of Rancidity
The ultimate measurement of rancid odor and taste is sensory analysis by a trained panel. A trained panel can be a very valuable tool for detection of early lipid oxidation of foods containing n-3 fatty acids. Some of the degradation products from long-chain n-3 PUFAS have a profound effect on odor and flavor in concentrations as low as in the parts per billion range [3]. In general, the oxidation products from n-3 fatty acids have a lower sensory threshold than those of oxidation products from other fatty acids. The detection of these low levels is not straightforward with classical lipid oxidation measurement methods. Odor threshold values vary both with the chemical structure of the carbonyl compounds and with the food matrix and based on how the sensory detection is performed, through the nose (nasal) or through the mouth (retronasal). Even if sensory methods can give sufficient information, their use is limited by the cost of employing a trained panel. It can also be difficult to compare data from different panels using different vocabularies or data from the same panel analyzed at different times. In addition, sensory analysis requires relatively large amounts of samples, and the use of chemical and instrumental analyses is recommended to support and complement the sensory analysis [3].
7.3 Summary Many different methods for the analysis of lipid oxidation exist. However, for many of these methods the results obtained vary not only with the method used but also with the analytical procedure that is performed, so care should be taken in standardizing the procedures. The ultimate wish from the food industry would be a rapid nondestructive method that can be applied on-line to analyze the oxidative or sensory quality in raw materials, intermediary goods, and finished products during seafood processing. However, even if there are many different methods that are used to determine lipid oxidation, today it is not possible to use only one method to determine lipid oxidation. There is, however, a rapid development in analytical methods to determine lipid oxidation, but for many of these methods calibration and verification are needed before they can be used for routine analysis.
References 1. Boissonneault, G.A., Dietary fat, immunity, and inflammatory disease, in Fatty Acids in Foods and Their Health Implications, C.K. Chow, Ed., Marcel Dekker: New York, 2000, pp. 777–795. 2. Narayan, B., K. Miayshita, and M. Hosakawa, Physiological effects of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA)—A review. Food Rev. Int., 2006. 22: 291–306. 3. Frankel, E.N., Lipid Oxidation, 2nd ed., The Oily Press: Bridgewater, U.K., 2005. 4. Falch, E., Lipids from residual fish raw material, in Department of Biotechnology. 2005, Norwegian University of Science and Technology: Trondheim, 2005, 206. 5. Folch, J., M. Lees, and G.H. Sloan Stanley, A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 1957. 226: 497–509.
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6. Bligh, E.G. and W.J. Dyer, A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 1959. 37: 911–917. 7. AOCS, Method Cd 8-53, in Official Methods and Recommended Practices of the American Oil Chemists’ Society, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 8. Sato, K. et al., Type V collagen in trout (Salmo gairdneri) muscle and its solubility change during chilled storage of muscle. J. Agric. Food Chem., 1991. 39: 1222–1225. 9. Ueda, S., M. Hayahashi, and M. Namiki, Effect of ascorbic acid in a model food system. Agric. Biol. Chem., 1986. 50: 1–7. 10. Undeland, I., M. Stading, and H. Lignert, Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage. J. Sci. Food Agric., 1998. 78: 441–450. 11. Nielsen, N.S., M. Timm-Heinrich, and C. Jacobsen, Comparison of wet-chemical methods for determination of lipid hydroperoxides. J. Food Lipids, 2003. 10: 35–50. 12. Halliwell, B. and J.M.C. Gutteridge, The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem. Sci., 1990. 15: 129–135. 13. AOCS, AOCS Official Method Ti 1a-64, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 14. Pettersen, J., Chemiluminescence of fish oils and its flavour quality. J. Sci. Food Agric., 1994. 65: 307–313. 15. AOCS, Method Cd 18–90, in Official Methods and Recommended Practices of the American Oil Chemists’ Society, D. Firestone, Ed., AOCS: Champaign, IL, 1995. 16. Guillen, M.D. and N. Cabo, Fourier transform infrared spectra data versus peroxide and anisidine values to determine oxidative stability of edible oils. Food Chem., 2002. 77: 503–510. 17. Ke, P.J. and A.D. Woyewoda, Microdetermination of thiobarbituric acid values in marine lipids by a direct spectrophotometric method with a monophasic reaction system. Anal. Chim. Acta, 1979. 106: 279–284. 18. Nawar, W.W., Lipids, in Food Chemistry, O.R. Fennema, Ed., Marcel Dekker Inc.: New York, 1996, pp. 225–319. 19. Wold, J.P. et al., Rapid assessment of rancidity in complex meat products by front face fluorescence spectroscopy. J. Food Sci., 2002. 67: 2397–2404. 20. Olsen, E., Analysis of early lipid oxidation in foods with n-3 fatty acids, in Dept. of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences: Ås., Norway, 2005, 160. 21. Hübschmann, H.-J., Basics, in Handbook of GC/MS-Fundamentals and Applications. Wiley-VCH Verlag GmbH: Weinheim, 2001, pp. 7–212. 22. Tironi, V.A., M.C. Tomas, and M.C. Anon, Structural and functional changes in myofibrillar proteins of sea salmon (Pseudopercis semifascata) by interaction with malonaldehyde (RI). J. Food Sci., 2002. 67: 930–935. 23. Pokorny, J., Interaction of oxidised lipids with protein. La Rivista Italiana Delle Sostanze Grasse, 1977. 27: 389–393. 24. Aubourg, S., I. Medina, and J.M. Gallardo, Quality assessment of blue whiting (Micrometistius poutassou) during chilled storage by monitoring lipid damages. J. Agric. Food Chem., 1998. 46: 3662–3666. 25. Aubourg, S., Lipid damage detection during the frozen storage of an underutilized fish species. Food Res. Int., 1999. 32: 497–502. 26. Aubourg, S.P. and I. Medina, Influence of storage time and temperature on lipid deterioration during cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) frozen storage. J. Sci. Food Agric., 1999. 79: 1943–1948. 27. Hasegawa, K., Y. Endo, and K. Fujimoto, Oxidative deterioration in dried fi sh model systems assessed by solid sample fluorescence spectrophotometry. J. Food Sci., 1992. 57: 1123–1126. 28. Veberg, A., G. Vogt, and J.P. Wold, Fluorescence in aldehyde model systems related to lipid oxidation. LWT-Food Sci. Technol., 2006. 39: 562–570.
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29. Jebe, T.A., M.-G. Matlock, and R.T. Sleeter, Collaborative study of the oil stability index analysis. J. Am. Oil Chem. Soc., 1993. 70: 1055–1061. 30. Mendez, E. et al., Comparison of Rancimat evaluation modes to assess oxidative stability in fi sh oils. J. Am. Oil Chem. Soc., 1997. 74: 331–332. 31. Vinter, H. The Oxidograph. A development within accelerated measurement of stability, in Scandinavian Symposium of Lipids (Lipidforum) 16th, 1991. pp. 160–162. 32. Moh, M.H. et al., Determination of peroxide value in thermally oxidized crude palm oil by near infrared spectroscopy. J. Am. Oil Chem. Soc., 1999. 76: 19–23. 33. Li, H. et al., Determination of peroxide value by Fourier transform near-infrared spectroscopy. J. Am. Oil Chem. Soc., 2000. 77: 137–142. 34. Yamamoto, Y. et al., Study of oxidation by chemiluminescence. IV. Detection of low levels of lipid hydroperoxides by chemiluminescence. J. Am. Oil Chem. Soc., 1985. 62: 1248–1250. 35. Matthäus, B., C. Wiezorek, and K. Eichner, Fast chemiluminescence method for detection of oxidized lipids. Fat Sci. Technol., 1994. 96: 95–99. 36. Jonsdottir, R., M. Bragadottir, and G. Olafsdottir, The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. J. Aquat. Food Prod. Technol., 2007. 16: 67–86. 37. Kuksis, A., H. Kamido, and A. Ravandi, Glycerophospholipid core aldehydes: Mechanism of formation, methods of detection, natural occurrence, and biological significance, in Lipid Oxidation Pathways, A. Kamal-Eldin, Ed., AOCS Press: Champaign, IL, 2003, pp. 138–189.
Chapter 8
Volatile Aroma Compounds in Fish Guðrún Ólafsdóttir and Rósa Jónsdóttir Contents 8.1 8.2 8.3 8.4
Introduction ..................................................................................................................... 98 Development of Fish Aroma............................................................................................. 98 Fresh Fish Odors .............................................................................................................. 99 Identification of Quality Indicators ................................................................................ 100 8.4.1 Microbial Spoilage Odors ....................................................................................103 8.4.1.1 Sweet, Sour, and Malty Odors ...............................................................103 8.4.1.2 Dried Fish, Ammonia-Like, and Stale Odors .........................................105 8.4.1.3 Putrid, Onion, and Cabbage-Like Odors ...............................................105 8.4.1.4 Miscellaneous ........................................................................................105 8.4.2 Oxidatively Derived Odors ..................................................................................106 8.4.2.1 Cooked Odor—Boiled Potato and Rancid Odors .................................106 8.4.2.2 Washed Cod Muscle System ..................................................................108 8.4.3 Processing Odors ................................................................................................. 111 8.4.3.1 Smoked Fish Odors ............................................................................... 111 8.4.3.2 Ripening Odor—Salted and Dried Fish Odor.......................................112 8.5 Conclusions .....................................................................................................................113 References ................................................................................................................................113
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8.1 Introduction Health and wellness are the main drivers in new product development. Fish being a valuable source of polyunsaturated fatty acids (PUFA) and other nutrients is a prominent candidate as the healthy choice for consumers. Research has aimed at strengthening the marine-based food industry in the development of fish products of acceptable quality to meet new trends in lifestyles. A prerequisite for increased consumption of fish products is their availability on the market as fresh and high-quality products of delicate flavor. Volatile compounds play an important role in the odor quality characteristics and consumer acceptance of fish. The understanding of odor development by chemical, biochemical, and microbiological processes in fish postharvest is of importance to be able to control the various extrinsic factors that influence the formation of volatile degradation products and consequently the quality of fish products. Research over the years has led to improved chilling and packaging technologies aimed at reducing microbial growth. As a result, extension in shelf life of fresh chilled fish has been achieved. However, oxidative processes causing odors and texture changes become noticeable during extended storage and limit the shelf life. Enhanced oxidation during cooking resulting in off odor development is of concern and an obstacle for application of fish in convenience food. Improved understanding of the role of oxidation of polyunsaturated fatty acids in the development of off odors in fish products has directed research efforts to search for effective means to control oxidative processes. Consequently, studies on application of natural antioxidants are of prime interest to underpin further utilization of fish in innovative product development as fresh, cooked, processed, or hydrolyzed products and as ingredients in functional foods.
8.2 Development of Fish Aroma An overview of changes during handling and processing influencing the development of aroma in fish is generalized in Figure 8.1. Initially, the changes are dominated by autolytic activity, including degradation of nucleotides, formation of taste, active inosine, and accumulation of hypoxanthine (Hx), lowering of pH and endogenous enzyme activity, followed by oxidation processes. Finally, the proliferation of the specific spoilage organisms (SSO) results in the development of volatile compounds, contributing to spoilage changes and thus influencing the freshness and quality of the end product of chilled fish [1–3]. It is well established that enzyme lipoxygenase (LOX)-mediated conversions of polyunsaturated fatty acids (PUFA) to volatile aroma compounds initiates the development of plant-like aroma of fresh fish [4–6]. Other prooxidants like hemeproteins (hemoglobin and myoglobin) are also involved in the initiation of the oxidative processes in fish muscle [7], leading to the formation of secondary oxidation products and off flavors [8]. Degradation of soluble muscle constituents such as sarcoplasmic proteins and microbial metabolism contributes to changes in the aroma profile of fish during storage. The pool of components that are degraded and cause off flavors because of microbial growth are mainly soluble substances in the muscle. They are composed of the various nonprotein nitrogenous components (NPN), including small peptides such as carnosine and anserine, amino acids, guanidine compounds like creatine, TMAO, and nucleotides. Some of these compounds influence the taste of fish-like peptides (i.e., anserine), and the individual amino acids glycine, valine, alanine, and glutamic acid are known to contribute to taste together with the degradation components of the nucleotides such as inosine. Proteolysis plays a critical role in postmortem changes, resulting in undesirable texture changes in fish. Endogenous enzyme
Volatile Aroma Compounds in Fish
Handling chilling, freezing, and cooking
99
Processing smoking, salting, drying, and hydrolysis
Endogenous enzymes
Microbial metabolism
i.e., LOX proteases, hydrolases, phospholipases TMAOase
Specific spoilage organisms (SSO)
Fresh fish aroma seaweedy, cucumber, metallic, neutral
◾
Spoilage aroma sweet, malty, sour, putrid, dried fish, ammonia-like
Oxidation Prooxidants: metals (Fe,Cu) Hb, Mb Antioxidants: α-tocopherol, ascorbic acid, polyphenols
Lipids phosholipids/PUFA Proteins sarcoplasmic, peptides Soluble substances, NPN, nucleotides, amino acids
Oxidized aroma Processed aroma green-like, boiled potato, popcorn, caramel, malty, stockfish, stale, rancid potato, mushroom, cucumber
Figure 8.1 Overview of changes in fish influencing the development of characteristic aroma of fresh, oxidized, spoiled, and processed fish. LOX, lipoxygenase; SSO, specific spoilage organisms; Hb, hemoglobin; Mb, myoglobin; PUFA, polyunsaturated fatty acids; NPN, nonprotein nitrogen-containing compounds; TMAO, trimethylamineoxide.
activity influences the deterioration of fish muscle, including calpains (neutral calcium-dependent proteases) and cathepsins (lysosomal proteases), but the mechanism of this activity is not fully elucidated [9].
8.3 Fresh Fish Odors The delicate flavor of fish is mostly contributed by volatile compounds and taste active substances in the aqueous phase, whereas volatiles generated from fat result in variation in the specific flavor character of different fish species. Newly caught marine fish contains low levels of volatile compounds and is nearly odorless. Soon after harvest, LOX activity on the skin and gills of both freshwater and marine species (rainbow trout, river trout, and sardines) plays a role in the formation of odorous volatiles, contributing to green, pleasant aromas of fish [6,10–13]. The compounds that contribute to the characteristic plant-, cucumber-, melon-, and mushroom-like odors are unsaturated carbonyl compounds and alcohols with six, eight, or nine carbon atoms [4,5,14,15]. Josephson et al. [5] summarized the occurrences of volatile compounds in freshwater and saltwater species and concluded that the four common compounds found in saltwater species, hexanal, 1-octen-3-ol, 1,5-octadien-3-ol, and 2,5-ocatadien-1-ol, were responsible for the moderate, faint odor of saltwater species. On the other hand, the unsaturated C9 carbonyl compounds such as 2,6-nonadienal, which have potent green, plant-, cucumber-, and melon-like odors, were characteristic for freshwater and euryhaline fish. The overall perceived odor is dependent on the level of influential compounds and their odor thresholds along with possible synergistic effects. Some components are desirable at low levels, but
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if their concentration increases, they may contribute to off odors. An example is the enzymically derived long-chain alcohols and carbonyls that exhibit characteristic fresh, plant-like notes in fresh fish, but when accumulated in higher levels because of autooxidation, they contribute to oxidized and fishy odors in stale fish [16]. Another example is iodine-like off flavor in prawns associated with bromophenols originating from the feed chain [17]. However, in nominal levels the bromophenols appear to contribute to natural sea-, iodine-, and marine-like flavors of seafood [18]. Environmental conditions and seasonal effects like spawning can influence the odor quality of fish. The volatile pattern changes in mature salmon when migrating from the sea for spawning. C9 LOX-derived compounds have been found in higher levels in spawning euryhaline and freshwater fish [5]. Seasonal effects have also been reported for capelin, a saltwater species, which has a very characteristic cucumber odor during spawning. 2,6-Nonadienal was identified to be the most characteristic compound for the cucumber-like capelin odor [19]. Studies performed in Japan, on accumulation of hydroperoxides in fish tissues, indicate their involvement in the development of fresh fish aroma associated with seasonal variation. Accumulation of certain hydroperoxide isomers coincided with the period of enhancement of characteristic aroma in sweet smelt. They were suggested as the possible precursors of nine-carbon volatile compounds, including (E)-2-nonenal, (E,Z)-2,6-nonadienal, and 3,6-nonadien-1-ol in sweet smelt tissues [20].
8.4 Identification of Quality Indicators Different characteristic odors develop in various fish species during storage. Fatty species develop rancid odors and taste; lean species typically develop sweet, boiled potato-, and amine-like odors; and species of the salmonidae family develop earthy, muddy, and sweet odors. Volatile compounds formed by microbial metabolism and oxidation contributing to these odors have been identified by gas chromatography methods and suggested as indicators of quality. Some of the influential odor compounds that have very low odor thresholds are often present in low levels, and these are difficult to detect by analytical techniques. Therefore, it is useful to monitor the overall pattern of volatile compounds and select indicator compounds, which are present in higher levels and can be quantified. Rapid methods can then be applied to detect indicators or alternatively classes of compounds if the pattern of the volatile compounds is known and a connection has been verified between the indicator compounds and the compounds that are responsible for the odors and quality changes. This has been the approach in our studies, where it has been demonstrated by monitoring key volatiles to study changes in different fish products during storage, odor, and quality changes can be explained in, for example, cod during storage [21,22] and in smoked salmon [23]. Both single compounds such as TMA and ethanol and multicompound indices based on combination of alcohols, amines, and sulfur compounds representing the different changes occurring during storage have been suggested by numerous researchers as indicators for freshness and spoilage [22,24–29]. Volatile degradation compounds as quality indicators can be detected by rapid techniques such as electronic nose to monitor and predict quality changes in various fish species and in smoked salmon [19,21–22,30–36]. Table 8.1 summarizes the occurrence of volatile compounds detected in our studies on cod [22] and haddock fillets [31] and smoked salmon [23]. Purge and trap on Tenax and SPME methods were applied for sampling, and identification was based on GC–FID, GC–MS, and GC–O. The main classes of compounds detected during storage are alcohols, aldehydes, ketones, amines, acids, esters, and sulfur compounds. The aldehydes contribute most to the spoilage odors because of their low flavor thresholds, as seen by the detected odors listed in Table 8.1.
Volatile Aroma Compounds in Fish
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Table 8.1 Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Raw Cod
Boiled Cod
Raw Haddock
Smoked Salmon
Odor Description (GC–O)
Ethanol
×
×
×
×
—
2-Methyl-1-propanol/pentane
×
×
×
1-Penten-3-ol
×
×
×
×
—
3-Methyl-1-butanol
×
×
×
×
—
2-Methyl-1-butanol
×
×
—
2,3-Butandiol
×
×
—
Compound
Alcohols
1-Octen-3-ol 2-Ethyl-1-hexanol
× ×
1-Octanol
—
×
Mushroom
×
—
×
—
Aldehydes Acetaldehyde
×
×
×
—
2-Methyl-propanal
×
—
2-Methyl-butanal
×
Sweet, caramel, fish fillet
3-Methyl-butanal
×
×
×
×
Hexanal
×
×
×
×
cis-4-Heptenal Heptanal
×
2,4-Heptadienal, (E,E)-
×
×
×
Sweet, caramel, flowery —
×
Rancid
×
Boiled potato, earthy
×
Sweet, fatty
Nonanal
×
×
×
×
—
Decanal
×
×
×
×
Fresh, floral
Undecanal
×
×
×
Sweet, candy
×
×
×
Ketones 2-Butanone 2,3-Butandione
×
— N/A (continued)
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Table 8.1 (continued) Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Compound
Raw Cod
Boiled Cod
Raw Haddock
2-Pentanone 3-Pentanone
×
×
Smoked Salmon
Odor Description (GC–O)
×
—
×
Sweet, caramel
2,3-Pentanedione
×
—
3-Hexanone
×
—
3-Methyl-2-butanone
×
3-Hydroxy-2-butanone
×
6-Methyl-5-hepten-2-one
×
×
— ×
×
Sweet, sour Flowery, sweet, heavy, spicy
Amine Trimethylamine
×
×
×
×
×
×
×
TMA-like, dried fish
Acid Acetic acid
×
—
×
—
Esters Ethyl acetate Ethanthiocacid, S-methylester
× ×
Propanoic acid, ethyl ester
×
N/A —
Propanoicacid-2-methyl, ethylester
×
N/A
Acetic acid, 2-methylpropyl ester
×
N/A
Butanoic acid, ethyl ester
×
×
×
Sickenly sweet, vomit
2-Butenoic acid, ethyl ester
×
N/A
Butanoic acid, 2-methyl, ethylester
×
N/A
Butanoic acid, 3-methyl, ethylester
×
N/A
Hexanoic acid, ethyl ester
×
N/A
Sulfur Compounds Methanethiol Dimethyl sulfide
× ×
×
— ×
—
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Table 8.1 (continued) Volatile Compounds Detected in Cod [22], Haddock Fillets [31], and Smoked Salmon [23] during Chilled Storagea Raw Cod
Boiled Cod
Raw Haddock
Dimethyl disulfide
×
×
×
Onion like
Dimethyl trisulfide
×
×
×
Rotten, sulfur, cabbage
Compound
Smoked Salmon
Odor Description (GC–O)
a
Volatiles in boiled cod were analyzed in samples of raw chilled cod fillets [22] by heating corresponding samples at 80°C for 60 min. —, not detected by GC–O; N/A, data not available for haddock.
Seaweedy and marine-like odors, as well as green plant-, cucumber-, mushroom-, or geraniumlike odors are characteristic sensory odor descriptors for fresh whole fish. In general when fish is cooked, the aroma of the fillet is described as sweet and reminiscent of shellfish, meat-like, and sometimes metallic. After several days of storage, the freshness notes disappear and the odor of the uncooked fish becomes neutral. Sweet-milky and vanilla/caramel-like odors are typical in cooked fish. During prolonged storage boiled potato odor develops, and when combined with frozen storage odor, dried fish/stockfish, and TMA-like smell, and finally sour and dirty tablecloth odor, the fish is no longer fit for consumption. The odor descriptors in Table 8.1 based on GC–O analysis of cod and smoked salmon represent most of these overall changes.
8.4.1 Microbial Spoilage Odors The spoilage odors in chilled fish vary depending on the dominant microflora in the products, which is mostly affected by handling, cooling, packaging, and temperature conditions during storage [33,34]. An example of the spoilage pattern of volatile compounds in chilled fish is illustrated in Figure 8.2, showing results from a storage study of cod fillets packed in styrofoam boxes during chilled storage (0.5°C) [22]. The aim was to screen for potential quality indicators and determine which compounds and classes of compounds were most abundant in the headspace and also to identify the most influential spoilage odors contributing to sensory rejection. Identification of volatile compounds was based on GC–MS analysis (see Table 8.1), and quantification of the main classes of compounds was based on the sum of the PAR for respective compounds in each class. The loss of freshness of cod fillets and early spoilage changes were related to the formation of ketones, alcohols, and aldehydes, contributing to sweet, sour, and malty odors. Late spoilage changes, development of spoilage odors, and the end of shelf life of cod fillets on day 12 of storage are explained by the presence of TMA, esters, acids, and sulfur compounds produced by microbial degradation of fish components, mainly amino acids.
8.4.1.1
Sweet, Sour, and Malty Odors
Ketones, alcohols, and aldehydes detected on day 4 of storage and their increasing levels on days 7 and 10 (Figure 8.2) were associated with the development of sweet, sour, caramel-like, and malty spoilage odors. The microbially derived alcohols 2-methyl-1-propanol, 3-methyl-1-butanol, and 2,3-butandiol were found in the highest levels on day 12 at sensory rejection. The flavor thresholds
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Peak area ratio (PAR)
100 80
Alcohols Aldehydes
60
Ketones TMA
40
Aceticacid Esters
20 0 0
2
4 6 8 10 Days of storage
12
14
Figure 8.2 GC–MS analysis of volatile compounds showing changes in the levels (PAR, peak area ratio) of the main classes of compounds contributing to spoilage in cod fillets packed in styrofoam boxes during storage at 0.5°C until sensory rejection on day 12. (Modified from Ólafsdóttir, G., Volatile compounds as quality indicators in fish during chilled storage: Evaluation of microbial metabolites by an electronic nose, PhD thesis, University of Iceland, Reykjavík, 2005.)
of alcohols are higher than those of carbonyls, and they did not contribute to the odor of the fillets as evaluated by GC–O (Table 8.1) [22]. Ethanol was detected in high levels initially (on days 4 and 7) and then declined. The initial high levels of ethanol in spoilage of fish has been related to the utilization of carbohydrate sources, whereas the formation of branched-chain alcohols and aldehydes such as 2-methyl-1-propanol, 3-methyl-1-butanol, and 3-methyl-butanal probably originate from degradation of valine and leucine, respectively. The branched chain aldehyde, 3-methyl-butanal, was characterized by sweet, caramel, and fish-fillet-like odors by GC–O in our study. Lindsay [8] suggested using short-chain alcohols such as ethanol, butanol, and 3-methyl-1-butanol as potential indices of refrigerated fish spoilage based on studies of freshwater whitefish. Propanol was suggested as a potential indicator when using modified atmosphere packaging techniques. In chilled haddock fillets stored in styrofoam boxes, TMA, 2-methyl-1-propanol, 3-methyl-1-butanol, 3-hydroxy-butanone, ethyl acetate, and butanoic acid ethyl ester were found in the highest amounts and increased with storage. Dimethyl disulfide and dimethyl trisulfide were detected at the end of storage time when samples were spoiled, whereas dimethyl sulfide was detected initially and throughout storage [31]. In cultured and wild sea bream stored in ice for 23 days, TMA, 3-methyl-1-butanol, 1-penten-3-ol, piperidine, methanethiol, dimethyl disulfide, dimethyl trisulfide, and acetic acid were identified as spoilage indicators [29]. The formation of acetoin (3-hydroxy-2-butanone) was characteristic for the spoilage of chilled cod fillets packed in styrofoam boxes and was attributed to the growth of Photobacterium phosphoreum [22]. Levels of acetoin increased earlier than those of TMA, and, therefore, it is more useful to monitor the loss of freshness as an early indicator of spoilage. The concentration of acetoin was much higher than the lipid derived ketones detected, such as 2-butanone, 3-pentanone, and the carotenoid-derived 6-methyl-5-heptene-2-one, that were present in cod fillets throughout
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storage, but no obvious increase occurred until at the end of shelf-life and during continued storage. Ketones can influence the overall odor because of their low odor thresholds. The lipid-derived saturated aldehydes detected on day 12 at sensory rejection also contributed to the overall sweet aroma. The odor of ethyl butanoate, described as sickeningly sweet and nauseous, contributed to the sensory rejection of chilled cod fillets on day 12 and suggested the role of Pseudomonas fragi in the development of sweet, fruity off odors [37,38]. Pseudomonas species have also been found responsible for the formation of volatile sulfides, alcohols (3-methyl-1-butanol, 1-penten-3-ol), and ketones (2-butanone), contributing to the stale and putrid off odors in fish because of amino acid and lipid degradation [39].
8.4.1.2 Dried Fish, Ammonia-Like, and Stale Odors The development of dried fish, ammonia-like, and stale odors by amines during fish spoilage is well known, and measurements of volatile amines such as TMA or total volatile bases (TVB-N) have been used in the fish industry as indicators of quality for fish and fish products. Enzymically produced DMA (dimethylamine), which forms very early after harvest of fish, has been suggested as a freshness indicator along with its precursor TMAO (trimethylamine oxide) [27]. TMA is a potent odorant with a characteristic fishy, dried fish, ammonia-like odor. Figure 8.2 shows that TMA was detected in high levels on day 12. At this point there was an increase in the pH value, which may have influenced the overall odor perception leading to the sensory rejection of the fillets. Additionally, TMA has been noted for intensifying fishiness by a synergistic action with certain volatile unsaturated aldehydes derived from autoxidation of polyunsaturated fatty acids [40]. TMA is characteristic for the spoilage odors of fish, whereas DMA may influence the overall fresh flavor of fish in combination with oxidatively formed aldehydes from long-chain fatty acids in fish. The onset of stale odors can be explained by cis-4-heptenal and heptanal, which contributed to boiled potato-like odors (Table 8.1).
8.4.1.3
Putrid, Onion, and Cabbage-Like Odors
Low levels of sulfur compounds (Figure 8.2) indicated that they were not important in the spoilage of chilled cod fillets stored in styrofoam boxes. In whole fish stored in ice, volatile sulfur compounds such as hydrogen sulfide, methyl mercaptan, methyl sulfide, and dimethyl disulfide have been suggested as the main cause of putrid spoilage aromas [41]. Dimethyl trisulfide has also been associated with spoilage in fish and associated with the growth of Shewanella putrecfaciens [25,38,39]. Milo and Grosch [42] evaluated the headspace of boiled cod by gas chromatography olfactometry (GC–O) and found that dimethyl trisulfide was the most potent odorant contributing to off odors in cod formed when the raw material was inappropriately stored. The origin of the sulfur compounds is microbial degradation of cysteine and methionine to form hydrogen sulfide and methyl mercaptan, respectively [41]. Oxidative processes are involved in the formation of dimethyl sulfide from methyl mercaptan and further oxidation of dimethyl disulfide, and the incorporation of hydrogen sulfide yields dimethyl trisulfide [38].
8.4.1.4 Miscellaneous The concentration of the straight chain alkanes (nonane, decane, and undecane) appeared to be similar throughout storage in chilled cod fillets [22]. Additionally, numerous branched chain
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alkanes were detected, and overall the alkanes showed an increasing trend with storage time. However, they are not considered of interest as quality indicators, since they are not aroma active. A characteristic earthy odor in many species residing in ponds has been associated with piperidine and its reaction products, but the knowledge of the formation of these compounds is obscure. Piperidine levels have been reported to increase in spawning salmon and contribute to off odors [43]. Piperidine was tentatively identified in chilled cod fillets [22] and has also been suggested as a quality indicator in sea bream [29]. Several odor active terpene derivatives have been identified in fish. Limonene has low odor threshold and a fresh lemon odor was detected by GC–O analysis of cod, suggesting that it may have an impact on the overall odor of fish fillets [22]. Limonene has also been detected in sea bream during storage [29]. The origin of limonene in fish is most likely related to the diet derived from algae or plant source. Similarly, the feed may have influenced higher levels of aldehydes, ketones, aromatics, and terpenes found in wild sea bream compared with those of its cultured counterpart [29].
8.4.2 Oxidatively Derived Odors Initiation of lipid oxidation in fish is generally associated with the polyunsaturated fatty acids in phospholipids of muscle cell membranes [44], which are known to be more susceptible to oxidation than triacylglycerols in fat deposits [45]. Various pro and antioxidants influence the stability of the muscle and have been studied in relation to the oxidative stability of phospholipids [46]. Phospholipids are the main membrane-bound lipids, and because of their high unsaturation, they are in particular sensitive to oxidation, which is further enhanced by preprocessing and storage of fish. Oxidative processes occurring during storage of fish result in the accumulation of aldehydes, such as hexanal, cis-4-heptenal, 2,4-heptadienal, and 2,4,7-decadienal, that contribute to the development of rancid cold store flavors [47]. Our studies on the development of volatile compounds in chilled cod fillets packed in styrofoam boxes during storage at 0°C showed that oxidatively formed, lipid-derived saturated aldehydes, such as hexanal, heptanal, and decanal, were detected in the fillets throughout the storage time, in similar or slightly increasing levels. These oxidation products contributed to the overall characteristic sweet, fish-like odors of chilled cod fillets in combination with other carbonyls (3-hydroxy-2-butanone, 3-methyl-butanal, 2-butanone, 3-pentanone, and 6-methyl-5-heptene-2one). Aldehydes generally have low odor thresholds, and, therefore, their impact was greater than alcohols and ketones, although their overall levels were lower. 6-Methyl-5-heptene-2-one derived from carotenoids was described as spicy and flowery by GC–O and suggested to contribute along with other ketones and aldehydes to the characteristic sweet odor of cod fillets [22]. The influence of other aroma active compounds present in lower levels such as the unsaturated autoxidatively derived aldehydes (2,4-heptadienal and 2,4,7-decatrienal) should not be overlooked. These compounds have been associated with rancid and dried fish odors, but the sampling techniques used were not sensitive enough to allow quantification of these compounds.
8.4.2.1
Cooked Odor—Boiled Potato and Rancid Odors
Characteristic odors and key volatile compounds in boiled cod stored in closed plastic bags for 22 days compared with fresh boiled cod are shown in Figure 8.3 to demonstrate which odors are most dominating in the aroma profile [48]. Boiled potato- and potato-like odors contributed by
Volatile Aroma Compounds in Fish DMS Sulfur 5
Cucumber, sweet, melon Cucumber
2-Nonenal
Fishy
3-Pentanone 1-Penten-3-ol Flowery 2-Penten-1-ol
3
Flowery Fatty, green-like odors Grass Hexanal
2 1
2,4-Heptadienal Rancid
Heavy
Geranium-like 1-Octen-3-ol
107
Fishy odors
4
Fatty Fatty, green-like, rancid odors Flowery
◾
Mushroom, earthy
Mushroom
Potato-like
Earthy, pop-like
Boiled potato
cis-4-Heptenal Heptanal
Earthy-like odors
Figure 8.3 Odor profile (GC–O analysis) of boiled cod stored in plastic bags (-♦-) after 22 days of refrigerated storage (3°C) compared with freshly boiled cod (---▲---). (From Jónsdóttir, R. and Ólafsdóttir, G., Unpublished data, 2004.)
heptanal and cis-4-heptenal were the most potent odors. Overall earthy, sweet, sour, and fish oil notes were characteristic for fresh cooked salmon, and the most pronounced attribute was a boiled potato odor [49]. The fresh raw salmon odor was characterized as cucumber-like with weak sweet, sourish, and fish oil notes in the same study. The occurrence of cis-4-heptenal has been associated with the “cold storage flavor” of cod [47]; however, some confusion exists about the role of cis-4heptenal as the “cold-storage compound” [8]. In fact, this aldehyde does not exhibit a fishy-type aroma by itself, but it rather participates in the expression of the overall fishy odor. Its odor has been described both as cardboardy, paint-like [50], as well as boiled potato-like [51,52]. Other pronounced odors detected in boiled cod (Figure 8.3) were fatty, green-like, and rancid odors contributed by 2-nonenal and 2,4-heptadienal. Fatty, sweet, flowery, and green-like odors were associated with oxidatively derived 3-pentanone, 1-penten-3-ol and hexanal. Baltic herring has been reported to have a similar development of volatiles, although the level of the compounds may vary and explain the differences in the characteristic odor of these species. In fresh baked herring (200°C; 20 min) 3-methylbutanal, 2-methylbutanal, and hexanal were abundant in headspace, and after storage for 3 days the proportions of 4-heptenal, 2-heptanone, and octatriene increased significantly. Hexanal, heptanal, 1-penten-3-ol, and octadienes also increased many-fold during further storage, and after 8 days of storage at 6°C, microbial metabolites such as 3-methyl1-butanol and cresol were identified [53]. Ideally, quality indicators should demonstrate clear increasing or decreasing levels with storage time. However, this is not always the trend for dynamic microbial and oxidative changes and the formation of volatiles in fish during storage [22]. Taking into account the complexity of the spoilage processes, multivariate data analysis is useful to explore the overall trend of the main quality indicators. Principal component analysis (PCA) was performed (Figure 8.4) on data from our studies on volatiles in cod [22] during prolonged storage for 17 days and compared with corresponding
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1.0
PC2
B-D17
Bi-plot
1-Penten-3-ol
3-me-butanal Acetic acid 0.5
Undecanal Ethanol 3-me-1-butanol Ethylbutanoate B-D4 Heptanal Nonanal Acetaldehyde Ethylacetate 2-Butanone
B-D10 0
R-D4 R-D12 R-D7 R-D10
3-HO-2-Butanone 2-me-1-propanol TMA Decanal R-D14 6-me-5-h-2-one Hexanal
–0.5 –0.4 –0.2 Raw and boild c…, X-expl: 53%, 19%
0
0.2
0.4
0.6
R-D17
0.8
PC1 1.0
Figure 8.4 Principal component analysis of raw and boiled cod. Samples are labeled with R, raw and B, boiled and storage days, D (4, 7, 10, 12, 14, and 17 days).
samples after heating (see Table 8.1). The PCA demonstrates how volatile compounds can explain the variation in quality of samples according to storage time and handling (raw and boiled), in particular the role of volatile compounds derived from oxidation in heated/boiled samples. The characteristic pattern or trend in volatiles in raw and boiled fish is clearly different, as indicated by the arrows (Figure 8.4). It is in particular interesting to demonstrate that the influence of heating gives a very different volatile profile compared with that of the raw samples that are all clustered on the left of the PCA plot. Only the spoiled raw samples (R-D14 and R-D17) can be correlated with the freshly boiled (B-D4) sample. The effect of oxidation induced by cooking and formation of oxidation products such as heptenal and nonanal characterizes the (B-D4) sample. Other oxidatively formed compounds like 2-butanone and aldehydes were in higher levels in the B-D4 sample compared with the corresponding raw sample (R-D4). Sulfur compounds dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide were detected in higher levels in the boiled samples (data not shown). The oxidatively formed compounds, that is, hexanal, decanal, and 6-methyl-5-hepten-2-one, increased with time and were pronounced in the spoiled raw samples (R-D14 and R-D17). Autoxidatively produced unsaturated carbonyl compounds were the most abundant components in boiled and canned fish, especially in trout [15]. In boiled trout, methional with a characteristic boiled potato-like odor dominated the odor of the aldehyde fraction of the headspace volatiles. The malty flavor of 3-methyl butanal was suggested earlier to be mainly responsible for the malty off flavor defect of boiled cod [54]. Interestingly, 3-methyl-butanal was correlated to the boiled stored cod (B-D17) (Figure 8.4), in agreement with earlier studies [54]. On the basis of odor evaluation, 3-methyl-butanal in combination with acetaldehyde, methional, and oxidatively derived (Z)-1,5-octadien-3-one, (E,Z)-2,6-nonadienal, and (E,E)-2,4-decadienal from PUFA were determined as character impact odorants of boiled cod [54].
8.4.2.2
Washed Cod Muscle System
Rancid odor development during chilled storage of fish has commonly been associated with fatty species. However, oxidation of membrane-bound phospholipids in lean species can cause fishy,
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rancid, dried fish-like off odors as discussed before. Studies on the development of the odorous degradation compounds of phospholipid oxidation can lead to a better understanding of the kinetics and reaction pathways of oxidation in lean fish. Consequently, this may facilitate the selection of preventive measures to limit oxidation and guide new technological developments with the aim to ensure the delicate taste and nutritional value of lean fish products. To accurately evaluate the potential of antioxidants in foods, it is necessary to apply models that take into account the chemical, physical, and environmental conditions expected in food products. This is because the activity of antioxidants in food systems depends not only on the chemical reactivity of the antioxidant (e.g., free radical scavenging and chelation) but also on factors such as physical location, interaction with other food components, and environmental conditions (e.g., pH) [55,56]. The role of antioxidants (a-tocopherol, ascorbic acid, and glutathione peroxidase) and aqueous prooxidants in fish muscle, including blood components like inorganic metals iron (Fe) and copper (Cu), has been studied to understand better the mechanisms of oxidation in the muscle [57,58]. In lean fish such as cod, lipid oxidation of muscle phospholipids may be induced by several catalysts, including hemoglobin from blood [7,59]. Sohn et al. [60] studied lipid oxidation and rancid odor during the early stage of ice storage of ordinary and dark muscle of yellowtail and concluded that myoglobin was the main cause in the development of the unpleasant color and undesirable odor during ice storage of fish muscle. Washed cod muscle system has been widely used to study oxidation and the influence of prooxidative and antioxidative factors [59,61]. Odor development in lean fish studied by hemoglobininduced oxidation in washed cod muscle system showed that sweet, green, earthy, cucumber-like, floral, and rancid odors dominated the aroma profile [62]. The added hemoglobin was very effective as a prooxidant, and the overall odor was an intense dried fish, painty, rancid fish oil like. To monitor the development of rancidity, the concentration and composition of volatile oxidation products analyzed by GC were compared with TBARS measurements, sensory assessments, and color. The most potent odors detected in the model system were malty, sweet, and caramel-like odors contributed by 3-methylbutanal, 2,3-pentandione, and 1-penten 3-ol; grass odor contributed by hexanal; rancid, potato-like odor caused by cis-4-heptenal and heptanal; mushroom odor caused by 1-octen-3-ol; and spicy and flowery notes exhibited by 6-methyl-5-hepten-2-one. Furthermore, rancid, fatty, soapy, and lemon-like odors were explained by 2,4-heptadienal [62]. These odors were also detected in cod fillets during chilled storage (Table 8.1), but the compounds were detected in much lower levels [22]. Preconcentration techniques are necessary for the analysis of unsaturated aldehydes, which is not practical for rapid determination of oxidation. On the other hand, it is possible to detect the most volatile oxidation products like propanal and hexanal by rapid, static headspace sampling methods. These compounds can be used as indicator compounds for oxidation, as demonstrated by Boyd et al. [63]. They showed that direct analysis of propanal can provide a quick and economical method for the determination of oxidation of n-3 fatty acids and pentane and hexanal analysis can give an indication of the oxidation of linoleic acid. Similarly, we found in our studies on the washed cod muscle system that hexanal could be used as indicator for rancid odor development. The prooxidative effect of hemoglobin was evident by the formation of hexanal in high levels, and a similar trend was observed in the development of cis-4-heptenal (Figure 8.5) as well as 2,4heptadienal that contributed to rancid odor caused by oxidation, in agreement with TBARS and changes in color [62]. The effect of thermal treatment on hemoglobin-mediated oxidation in the phospholipid model system from cod muscle was studied by monitoring oxidative changes during chilled storage on ice by sensory analysis, TBARS (thiobarbituric reactive substances), and instrumental color changes.
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1000
Hexanal
25
800
20
600
ng/g
ng/g
cis-4-Heptenal
30
400
15 10
200
5 0
0 0
1 Blank-II
2 Hb-Char-II
3
0
4
1
3
Hb-Char-II
Blank-II
Hb-Cod-II
2
4
Hb-Cod-II
Figure 8.5 Gas chromatography analysis (FID) of characteristic volatile compounds contributing to rancid odor (hexanal and cis-4-heptenal) in hemoglobin (from Arctic char and cod) mediated oxidation in washed cod model stored at 0°C for 4 days (-♦-, Blank-II; -▲-, Hb-Char-II; -■-, HbCod-II). ( Adapted from Jónsdóttir, R. and Ólafsdóttir, G., J. Aquat. Food Prod., 16, 67, 2007.)
100 90 80 70 60 50 40 30 20 10 0
40 35
TBARS (μmol/kg)
Odor score (rancidity)
Thermal treatment of the cod model system significantly enhanced the oxidation of the model on day 1, as measured by rapid increase in rancid odor, described as rancid, painty, and dried fish odors, and in TBARS (Figure 8.6) as well as more rapid loss of red color (not shown) already on the first day of storage. The studies on the washed cod muscle system verify the importance of oxidation in off odor development in fish muscle and consequently the benefit of being able to control oxidation to prevent the formation of the aldehydes. Active research is ongoing on the application of various natural antioxidants based on polyphenols like flavonoids (i.e., catechins from tea) and cinnamic acid derivatives (i.e., caffeic acid) [65] as well as application of tocopherol, citric acid, and EDTA [66]. Studies on LOX inhibitors are of interest in preventing the initiation of oxidation in fish. Some promising results have been reported, where commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle [67].
30 25 20 15 10 5 0
0
1 Blank
2 Raw
3 Cooked
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2
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3 Cooked
Figure 8.6 Sensory analysis of rancid odor (odor score) and TBARS measurements in raw and cooked washed cod model stored at 0°C for 4 days, with added hemoglobin (raw and cooked, respectively) and raw without hemoglobin (blank). (From Jónsdóttir, R. et al., Matis Report 08, 73, 2008. With permission.)
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8.4.3 Processing Odors Flavor development in processed seafood is a result of complex proteolytic and lipolytic reactions induced by different processing parameters like enzymes and temperature. Maillard reaction, including Strecker degradation, thermal degradation, and lipid oxidation, plays important roles in the formation of complicated processing flavors. Thermally generated aroma-active compounds via the Maillard reaction such as pyrazines are characteristic for enzymatically hydrolyzed seafood products like crayfish processing by-products [68]. Volatile compounds like alkyl-pyrazines and sulfur-containing compounds have been found in cooked crustaceans, and furans have been found in spray-dried shrimp powder and shrimp hydrolysate [69]. Key volatile compounds identified in enzymatically produced seafood flavorants are formed via Maillard reaction and Strecker degradation of amino acids. These are compounds like methional, the Strecker aldehyde produced from methionine, which has a characteristic potato-like odor, and 2-acetyl-1-pyrroline, giving a popcorn-like odor that can be thermally generated. Lipid-derived components, like cis-4-heptenal, 1-octen-3-ol, 2,6-nonadienal, hexanal, 2,4-heptanal, and 2,4-decadienal, also contribute to the aroma of seafood flavorants [70]. Lipid-derived aldehydes play an important role in flavor formation and have been reported to contribute to the characteristic fish-like, sweet odors of processed seafood like those in smoked salmon [23,71,72].
8.4.3.1
Smoked Fish Odors
Degradation compounds from Maillard reactions and lipid oxidation are the main compounds contributing to the aroma of smoked salmon [72]. The typical smoked salmon aroma results from a number of chemicals found in the smoke, but it is mostly attributed to the phenols. Figure 8.7 illustrates the main odors that were present in smoked fish samples after 14 days of chilled storage, which is typical for products on the market [23]. Phenolic derivatives like guaiacol (2-methoxyphenol) and syringol (2,6-dimethoxyphenol) have been identified as the most characteristic smoke-related compounds in smoked fish-like herring (Clupea harengus) [73] and in smoked salmon (Salmo salar) [23,72]. Guillén et al. [74] analyzed headspace components of cod and swordfish, where groups of phenol pyrolysis were most noticeable in the smoke flavor volatiles. In addition to phenolic compounds, furan-like compounds have been reported to be responsible for the smoked odor in smoked salmon, whereas carbonyl compounds, such as heptanal and (E,Z)-2,6-nonadienal, were characteristic in unsmoked fish, giving the flesh its typical fishy odor [71,72]. The oxidatively derived compounds cis-4-heptenal and heptanal, giving rancid, potato-like odors, and 1-octen-3-ol, contributing to mushroom-like odor, gave the most intense odors of smoked salmon and contributed to the fish-like earthy odors and fatty and rancid odors (Figure 8.7) [23]. Other oxidatively derived compounds like 1-penten-3-ol, hexanal, nonanal, and decanal were among key volatiles, and although they contributed less to the odors, it is clear that their presence contributes to the characteristic fish odor of smoked salmon products. Microbially produced ketones, aldehydes, and alcohols were abundant in the headspace of cold smoked salmon products during storage, associated with spoilage off flavors, like 3-methyl butanal, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-penten-3-ol, and 1-propanol [28,75]. Some of these compounds were selected as key spoilage indicators for smoked salmon based on their high levels and contribution to sweet and fruity spoilage off odors in our study on smoked salmon (Figure 8.7) (e.g., ethanol, 2-butanone, 2-pentanone, 3-methyl-butanal, 3-hydroxy-2-butanone, and 3-methyl-1-butanol) [23]. Additionally, it was verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked, sweet/sour rancid, and off odor and flavor) than traditional chemical and microbial variables.
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Sweet and fruity-like odors
Smoked salmon odors Characteristic smoke odor Smoke-like
Sweet, fruity Flowery, sweet
2- and 3-Methyl phenol Guaiacol 4-Methyl-guaiacol
Wood, burnt, smoke
3-Methyl butanal Sweet, caramel
Smoke-house, sweet
Wood, smoke, sweet
Flowery, sweet
Burnt, smoke
Flowery, earthy, mushroom 2, 4-Heptadienal Sweet, fatty
Mushroom, geranium
Boiled potato-like Fatty, and rancid-like odors
Rancid cis-4-Heptenal Heptanal Earthy-like odors
1-Octen-3-ol
Figure 8.7 GC–O evaluation of volatile compounds detected in cold smoked salmon after 14 days of storage at 5°C. (Modified from Jónsdóttir, R. et al., Food Chem., 109, 184, 2008.)
8.4.3.2
Ripening Odor—Salted and Dried Fish Odor
Numerous volatile compounds have been detected in ripened products like dry cured ham, most of them generated from chemical or enzymatic oxidation of unsaturated fatty acids and further interactions with proteins, peptides, and free amino acids. [76–78]. Similar processes have been reported in ripened seafood products, where methional derived from methionine and 2,6-nonadienal from fatty acid oxidation were the main odorants in sugar salted, ripened roe products [79] Similarly, Triqui and Reineccius [80] found that 2,4-heptadienal and 3,5-octadien-2-one were associated with the development of the typical flavor obtained after anchovy ripening. Thus, they suggested that lipid autoxidation during ripening was primarily responsible for aroma development. However, manufacturers of ripened products have observed that some degree of proteolysis is necessary before flavor can develop. Methional and (Z)-1,5octadien-3-one were also identified as potent odorants in ripened anchovy [81], and aldehydes such as acetaldehyde, 2-methylpropanal and 3-methylbutanal were the key, highly volatile components of ripened anchovy, probably originating from amino acids. Salted cod are traditional products from the North-Atlantic fisheries and are highly regarded as ripened fish products in many countries, especially those in the Mediterranean. During ripening of salted cod, the desired flavor and texture develop as a consequence of protein and fat degradation. In our study, where the ripening of salted cod (Gadus morhua) produced by different salting methods was studied, the highest odor scores were given for boiled potato and rancid, potato-like odors together with cucumber-like odor [82]. The rancid, potato-like odor was identified as cis4-heptenal and the boiled potato-like odor, as heptanal, both oxidatively derived compounds.
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Methional, derived from methionine and eluting at a similar time as cis-4-heptenal and heptanal, could also be responsible for the boiled potato-like odor. The cucumber-like odor detected is possibly 2,6-nonadienal, according to retention index (RI) of standard and odor evaluation, although the compound could not be identified by GC–MS. Other key volatile compounds in salted cod are derived form lipid oxidation, for example, 1-penten-3-ol, hexanal, and 2-butanone. A certain degree of lipid oxidation is both necessary and desirable for sufficient ripening of the products but the process should be controlled to obtain a desirable degree of ripening based on consumer preferences [82,83].
8.5 Conclusions Although aldehydes, such as heptanal and (E,Z)-2,6-nonadienal, cause off odors in fish during storage, their presence at nominal levels gives the characteristic and desirable fishy odor in fresh and processed fish. Lipid oxidation during ripening appears to be primarily responsible for desirable aroma development in processed fish. The oxidatively derived compounds cis-4-heptenal and heptanal, exhibiting rancid, potato-like odors, and 1-octen-3-ol, contributing to mushroom-like odor, were the most intense character impact compounds of salted cod and smoked salmon. Studies on hemoglobin-induced oxidation in the washed cod model system and enhanced oxidation after heating verified the role of the oxidatively derived compounds contributing to off odors in chilled stored and boiled cod. Proper handling and application of natural antioxidants to control oxidative processes caused by lipoxygenase, hemoglobin, and myoglobin, and other prooxidants in combination with mild heating treatment are important factors to maintain the delicate flavor and odor of fish products. In addition, microbial growth can be limited by effective cooling techniques, temperature control, proper handling, and new packaging technologies. Therefore, careful control of handling and processing conditions should open up possibilities for fish to become a favored choice in new product development of convenience food and in functional food because of its health beneficial properties. However, careful evaluation of the quality of product is needed to ensure acceptable flavor. Volatile compounds as indicators of freshness quality and spoilage can be monitored to determine the quality of fish products. Development of smart sensor technologies like the electronic nose to detect microbial metabolites and oxidation products is of interest to verify the quality of products to facilitate process management, to increase trust between buyers and sellers in trade, and in retail for consumers as smart sensors imprinted on packaging. Detection of microbial metabolites originating mainly from soluble aqueous fractions of the muscle can be directly related to the quality of products. Knowledge of the spoilage pattern of volatile compounds is the basis for the development of rapid techniques like smart sensor technologies. A similar set of sensors with selectivity and sensitivity toward the main quality-indicating classes of compounds, such as ketones, amines, alcohols, aldehydes, acids, esters, and sulfur compounds, can be used for a variety of fish species that are stored and processed by different techniques.
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41. Herbert, R.A., Ellis, J.R., and Shewan, J.M. Isolation and identification of the volatile sulphides produced during chill-storage of North sea cod (Gadus morhua). J. Sci. Food Agric., 26, 1195, 1975. 42. Milo, C. and Grosch, W. Detection of defects in boiled cod and trout by gas chromatographyolfactometry of headspace samples. J. Agric. Food Chem., 43, 459, 1995. 43. Yamanaka, H. Offensive odour of fish and shellfish, in Odour of Marine Products, Koizumi, C., Ed., Koseisha-Koseikaku, Tokyo, Japan, 1989, p. 53. 44. Decker, E.A. and Xu, Z. Minimizing rancidity in muscle foods. Trends Food Sci. Technol., 9, 241, 1998. 45. Frankel, E. M. Lipid Oxidation. The Oily Press, Dundee, Scotland, 1998, 303. 46. Hultin, H.O. Oxidation of lipids in seafoods, in Seafoods: Chemistry, Processing Technology and Quality, Shahidi, F., and Botta, J.F., eds., Blackie Academic and Professional, Glasgow, U.K., 1994, 49. 47. McGill, A.S., Hardy, T., Burt, R.J., and Gunstone, F.D. Hept-cis-4-enal and its contribution to the off-flavour of cold-stored cod. J. Sci. Food Agric., 25, 1477, 1974. 48. Jónsdóttir, R. and Ólafsdóttir, G. Unpublished data, 2004. 49. Refsgaard, H.H.F., Brockhoff, P.B., and Jensen, B. Sensory and chemical changes in farmed Atlantic salmon (Salmo salar) during frozen storage. J. Agric. Food Chem., 46, 3473, 1998. 50. Hardy, R., McGill, A.S., and Gunstone, F.D. Lipid and autoxidative changes in cold stored cod (Gadus morhua). J. Sci. Food Agric., 28, 999, 1979. 51. Josephson, D.B. and Lindsay, R.C. Retro-aldol degradations of unsaturated aldehydes: Role in the formation of c4-heptenal from t2,c6-nonadienal in fish, oyster, and other flavours. J. Food Sci., 64, 1186, 1987. 52. Josephson, D.B. and Lindsay, R.C. c4-Heptenal: An influential volatile compound in boiled potato flavour. J. Food Sci., 52, 328, 1987. 53. Aro, T., Tahvonen, R., Koskinen, L., and Kallio H. Volatile compounds of Baltic herring analysed by dynamic headspace sampling-gas chromatography-mass spectrometry. Eur. Food Res. Technol., 216, 483, 2003. 54. Milo, C. and Grosch, W. Changes in the odourants of boiled salmon and cod as affected by the storage of the raw material. J. Agric. Food Chem., 44, 2366, 1996. 55. Decker, E.A., Warner, K., Richards, M.P. and Shahidi, F. Measuring antioxidant effectiveness in food. J. Agric. Food Chem., 53, 4303, 2005. 56. Jacobsen, C., Let, M.B., Nielsen, N.S., and Meyer, A.S. Antioxidant strategies for preventing oxidative flavour deterioration of foods enriched with n-3 polyunsaturated lipids: A comparative evaluation. Trends Food Sci. Technol., 19, 76, 2008. 57. Hultin, H.O. and Kelleher, S.D. Surimi processing from dark muscle fish, in Surimi and Surimi Seafood, Park, J.W., Ed., Marcel Dekker Inc., New York, 2000, p. 59. 58. Undeland, I., Hall, G., and Lingnert, H. Lipid oxidation in fillets of herring (Clupea harengus) during ice storage. J. Agric. Food Chem., 47, 524, 1999. 59. Richards, M.P. and Hultin, H.O. Rancidity development in a fish model system as affected by phospholipids. J. Food Lipids, 8, 215, 2001. 60. Sohn, J.H., Taki, Y., Ushio, T., Kohata, T., Shioya, I., and Ohshima, T. Lipid oxidations in ordinary and dark muscles of fish: Influences on rancid off-odor development and colour darkening of yellowtail flesh during ice storage. J. Food Sci., 70, 7, 490, 2005. 61. Undeland, I., Kristinsson, H.G., and Hultin, H.O. Hemoglobin-mediated oxidation of washed minced cod muscle phospholipids: Effect of pH and hemoglobin source. J. Agric. Food Chem., 52, 4444, 2004. 62. Jónsdóttir, R., Bragadóttir, M., and Ólafsdóttir, G. The role of volatile compounds in odor development during hemoglobin-mediated oxidation of cod muscle membrane lipids. J. Aquat. Food Prod., 14, 67, 2007. 63. Boyd, L.C., King, M.F., and Sheldon, B. A rapid method for determining the oxidation of n-3 fatty acids. J. Am. Oil Chem. Soc., 69, 325, 1992.
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64. Jónsdóttir, R., Bragadóttir, M., and Ólafsdóttir, G. Oxidation in fish muscle: The role of phosholipids, proteins, pro-oxidants, antioxidants and the effect of heating. Matis report 08, Reykjavík, Iceland, 2008, 73. 65. Medina, I., Gallardo, J.M., Gonzalez, M.J., Lois, S., and Hedges, N. Effect of molecular structure of phenolic families as hydroxycinnamic acids and catechins on their antioxidant effectiveness in minced fish muscle. J. Agric. Food Chem., 55, 3889, 2007. 66. Jittrepotch, N., Ushio, H., and Ohshima, T. Effects of EDTA and a combined use of nitrite and ascorbate on lipid oxidation in cooked Japanese sardine (Sardinops melanostictus) during refrigerated storage. Food Chem., 99, 70, 2006. 67. Banerjee, S. Inhibition of mackerel (Scomber scombrus) muscle lipoxygenase by green tea polyphenols. Food Research International, 39, 486, 2006. 68. Baek, H.H. and Cadwallader, K.R. Volatile compounds in flavour concentrates produced from crayfish-processing byproducts with and without protease treatment. J. Agric. Food Chem., 44, 3262, 1996. 69. Pan, B.S. and Kuo, J.M. Flavour of shellfish and kamaboko flavourants, in Seafoods: Chemistry, Processing, Technology and Quality, Shahidi, F., and Botta, J.R., Eds., Blackie Academic and Professional, Glasgow, U.K., 1994, 85. 70. Jónsdóttir, R., Ólafsdóttir, G., Hauksson, S., and Einarsson, J.E. Flavorants from seafood byproducts, in Handbook of Food Products Manufacturing: Health, Meat, Milk, Poultry, Seafood, and Vegetables, Vol 2, Hui, Y.H., Ed., John Wiley & Sons, Hoboken, NJ, 2007, 931. 71. Varlet, V., Knockaert, C., Prost, C., and Serot, T. Comparison of odour-active volatile compounds of fresh and smoked salmon. J. Sci. Food Agr., 54, 3391, 2006. 72. Varlet, V., Prost, C., and Serot, T. Volatile aldehydes in smoked fish: Analysis methods, occurrence and mechanisms of formation. Food Chem., 105, 1536, 2007. 73. Sérot, T., Baron, R., Knockaert, C., and Vallet, J.L. Effect of smoke processes on the content of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus). Food Chem., 85, 111, 2004. 74. Guillén, M.D., Errecalde, M.C., Salmerón, J., and Casas, C. Headspace volatile components of smoked swordfish (Xiphias gladius) and cod (Gadus morhua) detected by means of solid phase microextraction and gas chromatography–mass spectrometry. Food Chem., 94, 151, 2006. 75. Joffraud, J.J., Leroi, F., Roy, C., and Berdagué, J.L. Characterisation of volatile compounds produced by bacteria isolated from the spoilage flora of cold-smoked salmon. Int. J. Food Microbiol., 66, 175, 2001. 76. Toldrá, F. Proteolysis and lipolysis in flavour development of dry-cured meat products. Meat Science, 49, 101, 1998. 77. Toldrá, F. and Flores, M. The role of muscle proteases and lipases in flavour development during the processing of dry-cured ham. Crit. Rev. Food Sci., 38, 331, 1999. 78. Toldrá, F., Aristoy, M.C., and Flores, M. Contribution of muscle aminopeptidases to flavour development in dry-cured ham. Food Res. Int., 33, 181, 2000. 79. Jónsdóttir, R., Ólafsdóttir, G., Martinsdóttir, E., and Stefánsson, G. Flavour characterization of ripened cod roe by gas chromatography, sensory analysis and electronic nose. J. Agric. Food Chem., 52, 6250, 2004. 80. Triqui, R. and Reineccius, G.A. Changes in flavour profiles with ripening of anchovy (Engraulis encrasicholus). J. Agric. Food Chem., 43, 1883, 1995. 81. Triqui, R. and Guth, H. Determination of potent odourants in ripened anchovy (Engraulis encrasicholus L.) by aroma extract dilution analysis and by gas chromatography-olfactometry of headspace samples, in Flavour and Lipid Chemistry of Seafoods, Shahidi, F., and Cadwallader, K.R., Eds., ACS Symposium Series 674 American Chemical Society, Washington, DC, 1997, 31. 82. Jónsdóttir, R., Lauritzesen, K., and Thórarinsdóttir, K. Unpublished data, 2007. 83. Lauritzesen, K. and Olsen, R.L. Effects of antioxidants on copper induced lipid oxidation during salting of cod (Gadus morhua). J. Food Lipids, 1, 105, 2004.
PROCESSING CONTROL
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Chapter 9
Basic Composition: Rapid Methodologies Heidi Nilsen, Karsten Heia, and Margrethe Esaiassen Contents 9.1 Near-Infrared Spectroscopy............................................................................................ 122 9.1.1 Determination of Basic Composition.................................................................. 122 9.1.2 Theory, Measurement Principles, and Data Analysis ........................................... 122 9.1.3 Analysis of Basic Constituents ............................................................................ 124 9.2 Imaging Spectroscopy .................................................................................................... 128 9.2.1 Theory, Measurement Principles, and Analysis ................................................... 128 9.2.2 Analysis of Basic Constituents ............................................................................ 128 9.3 NMR Spectroscopy ........................................................................................................ 130 9.3.1 Determination of Basic Composition.................................................................. 130 9.3.2 Theory and Measurement Principles ................................................................... 130 9.3.3 Analysis of Basic Constituents .............................................................................131 9.4 X-Ray Imaging ................................................................................................................132 9.4.1 Theory and Measurement Principles ....................................................................132 9.4.2 Analysis ...............................................................................................................132 9.5 Summary ........................................................................................................................133 Acknowledgment .................................................................................................................... 134 References ............................................................................................................................... 134
Fish and seafood consumption has gained increased attention during the last years as a consequence of increased focus on nutritional quality as well as aspects related to healthy living. 121
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Compared with the production and distribution of meat from the agricultural sector, seafood is considered highly fragile and perishable with a short shelf life and delicate texture, and hence these issues must be considered during the processing and characterization of the material. Another aspect to be considered is the increased consumer awareness regarding the quality of their food; frequently consumers want readily accessible information about nutritional parameters and food quality. The documentation of basic nutritional composition of foods is a legal requirement in many countries, and so the need for measurement and documentation of such parameters is both a consumer requirement and also issued by law. In this perspective there is an obvious need for objective methods for evaluating and documenting the basic composition of fish and seafood. Regarding industrialized food production, requirements for such a method would preferably be that it is rapid and nondestructive. In this context seafood is particularly challenging as it comprises a vast number of different species with their own characteristics and qualities. In this chapter, we review some of the most relevant methods for assessing the basic composition of fish and seafood as presented in scientific literature. These methods are near-infrared (NIR) spectroscopy, magnetic resonance, imaging techniques, as well as x-rays. The basic principles of the techniques are described as well as a presentation of the use and applicability of quality measures of fish. The four methods presented fit with the requirements of speed and nonobtrusiveness; hence, these techniques may be applied in or at a production line.
9.1 9.1.1
Near-Infrared Spectroscopy Determination of Basic Composition
The development and usage of near-infrared spectroscopy (NIR) as an analytical tool has proven useful in areas varying from food quality, pharmaceutical applications, to analysis related to the environment and the petrochemical sector [1]. During the last 30 years the use of NIR spectroscopy has gained increased importance in the evaluation of a number of different food quality parameters [2–7]. The work in food analysis tends to have a focus within the agricultural sector [1]. However, throughout the years the method has also proven useful for the analysis of seafood and seafood products [8]. There are several reasons why NIR as a food analytical tool has caught attention and approval during the last decennia. The measurements are based on light interaction with material, facilitating a rapid response, which is a prerequisite for a methodology to be applied along a production line. Another benefit is the potential of simultaneous measurements of more parameters, and additionally the method may be applied with little or no obtrusion to the material sample. In the following section, we give a short introduction to the principles of NIR spectroscopy, followed by a presentation of the usage of NIR measurements for the rapid determination of basic constituents in fish and seafood products.
9.1.2 Theory, Measurement Principles, and Data Analysis The electromagnetic range applied in NIR spectroscopy spans from 700 to 2500 nm, comprising the frequencies just below those of visible light. A food sample exposed to emission in this wavelength range will absorb certain parts of the energy depending on the chemical composition of the sample. The absorption of light is due to the response of the molecular bonds O−H, C−H, C−O,
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and N−H [9] and corresponds mainly to overtones and combinations of fundamental vibrations. A thorough theoretical description of the NIR theory as well as the designation of numerous bands of absorptions may be found in Osborne and Fearn [2] and reviews on the subject [1,9,10]. Over the years there has been a steadily ongoing development of instrumentation for NIR spectroscopy, both with respect to the detectors and the capture of the spectral information [10,11]. In the context of rapid methodologies, we view this in terms of the measurement setup enabled by technology, developed toward the facilitation of nondestructive, nondisruptive, and noncontact methods. Different measurement modes for NIR spectroscopy are illustrated in Figure 9.1. A setup as shown in (a), where the light passes through the sample from one side to another, enables “transmission” measurements. The amount of light entering the detector unit depends on the scattering and absorption features of the sample as well as the sample thickness and lamp characteristics. If the light source and the detector are placed on the same side of the sample as shown in (b), the system is operated in “reflection” mode. For both (a) and (b), the transmission and reflection may be either direct or diff use, depending on the scattering properties of the medium under investigation. Finally, (c) illustrates how measurements are performed in “transflection” mode, placing the light source and the detector at the same side of the sample, but focusing the two devices so as to ensure that the light has traversed some region of the sample before detection. NIR spectroscopy is an indirect measurement technique. The broad spectral bands may be an indication of the material constituents, but an immediate look at an NIR spectrum is not sufficient to quantify the different substances. Hence, the spectral readings must be correlated to a relevant reference method such as, for example, traditional chemical determination of the constituents. NIR spectroscopy would not have had such an impact as an analytical tool had it not been for the development of mathematical tools for spectral analysis. A common methodology is chemometrics or
Shelter
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Figure 9.1 Different measurement setups for NIR spectroscopy. In (a) the transmission setup is shown; light from the source penetrates the sample and enters the detector. The setup in (b) displays the reflection setup where light reflected from the sample surface enters the detector. In (c) the light source and detector are located to register light that has traversed the sample before detection. In order to prevent direct reflection from the surface, a screen is placed between the directly emitted area and the area of inspection.
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multivariate data analysis. If there is good correlation between the spectral measurements and the method of reference, the reference method may be replaced by the spectral reading and the analytical model. Typically, a model based on several wavelengths is required to extract useful information from the spectroscopic data. Among the most used multivariate techniques are principal component analysis (PCA), partial least square (PLS) regression, and soft independent modeling of class analogies (SIMCA) [12].
9.1.3 Analysis of Basic Constituents As found in NIR analysis of foods in general, a substantial part of the work related to NIR analysis of fish and food from fish concerns the quantification of the chemical constituents, fat, protein, and water. Being the basic nutritional components of any food, an easy, reliable, and rapid method for the assessment and quantification of these constituents is considered a valuable tool in the quality evaluation of any foodstuff. In the following paragraphs, we give several examples of the use of NIR spectroscopy for the determination of basic food constituents in fish and seafood and how the method has been applied and developed over the last 20 years. The earliest reports of NIR spectroscopy to measure chemical components in fish appeared more than two decades ago. In 1987 Gjerde and Martens [13] demonstrated the applicability of NIR to predict water, fat, and protein content in rainbow trout. The same year Mathias et al. [14] reported the use of NIR spectroscopy to determine lipid and protein content in freshwater fish, namely, fingerling Arctic charr and rainbow trout. In both studies reflectance measurements were performed, and the sample preparation included mincing and freeze drying of the material to be evaluated. Both of these early reports concluded that the method was promising in terms of speed and efficiency when measuring a large number of samples. Darwish and others [15] used the technique in 1989 to measure fat, water, and protein in cod, mackerel, and tuna. For the measurement of fat and protein, the samples were minced and dissolved in a milk-like emulsion, whereas water determination was made on the water extracted from the fish mince. In spite of the rather cumbersome sampling procedure, the study concluded that the method could be a useful tool for rapid quality control. Consecutive research articles proved the feasibility of the tool in developing the method to apply with simpler procedures of sample preparation. Sollid and Solberg [16] measured the fat content in salmon by transmission spectroscopy on raw minced muscle. As early as in 1992 Lee and others [17] showed how NIR spectroscopy could be used noninvasively to estimate the lipid content of small-sized, intact rainbow trout. The measurements were performed by use of fiber optic bundles conveying the light to and from the sample site. Based on measurements through scales and skin, it was possible to estimate the lipid content of the intact muscle. This measurement setup clearly displayed how NIR spectroscopy could be used in a nondestructive way. Downey [18] applied a similar spectroscopic setup to measure fat and water content of intact farmed salmon; and, as in the work of Lee et al. [17], the measurement locations for obtaining the best calibration results were also addressed. In addition, by use of NIR in connection with fiber optics Solberg et al. [19] performed a study on live anesthetized farmed salmon, demonstrating the possibility to determine fat content in live fish. The prospect of measuring the chemical composition of intact fish could facilitate the use of the method in connection with selection in breeding programs [17] as well as for quality grading in terms of nutritional quality [19]. Farmed salmon is of high commercial value and a worldwide favorable product. This could account for the many studies relating to the rapid analysis of the basic chemical composition of
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salmon. Isaksson et al. [20] conducted a study in which they compared NIR measurements on intact salmon fillet, as well as on minced salmon muscle. In this work they applied a fiber optic measurement setup. Spectroscopic readings obtained on the minced samples correlated better with the reference measurements on fat, water, and protein than those made on intact muscles. The study concluded that NIR is well suited for nondestructive quality evaluation of salmon fillets. Wold et al. [21,22] also conducted studies documenting the efficiency of applying NIR spectroscopy in different measurement modes to assess fat and water content in salmon. An example illustrating the use of NIR spectroscopy for assessing fat content in farmed salmon is given in Figure 9.2. NIR spectroscopy has been used for evaluating the chemical composition of several other fish species as well. Nortvedt, Torrissen, and Tuene [24] made use of NIR transmission spectroscopy to assess protein, fat, and dry matter in halibut fillet. Transmission spectroscopy was also employed for the analysis of fat and dry matter in capelin [25], applying minced samples for the spectral readings. This work also emphasized the impact of the conditional state of the fish when making calibration models, whether pre-, in, or postspawning. In a research article published in 2004, Xiccato et al. [26] showed that NIR spectroscopy could be used to estimate lipid, water, and protein content of European sea bass, and additionally the spectroscopic measurements could be used for origin identification or authentication of the samples. In a recent work by Khodabux et al. [27], NIR spectroscopy was proven to be a useful tool for the evaluation of basic constituents of different types of tuna. The fat content of herring has also been assessed by the use of NIR spectroscopy. In both the works of Vogt et al. [28] and Nielsen et al. [29] one question of interest was the comparison of different methods for measuring fat content, Torry fatmeter, microwave, and NIR spectroscopy,
Measured Y Elements: Slope: Offset: Correlation: RMSEP: SEP: 21 Bias: 24
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Figure 9.2 The plot shows the predicted versus measured fat content in farmed salmon based on multivariate analysis of 78 spectra from salmon fillets and the respective chemical analyses of the fillets. (From Nilsen, H. and Sørensen, N.K., Unpublished data, 1998.) Spectral measurements were performed on intact fillets by transflection measurements by use of the fiber optic probe of the instrument NIRS6500 (Perstorp Analytical Inc., Silver Spring).
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and Distell fatmeter, NIR, and NMR, respectively. In both studies NIR spectroscopy resulted in favorable outcomes with respect to speed and accuracy. Vogt et al. [28] however, also commented on the cost aspect of the different methods as part of the feasibility of the methods. The use and results described above were all on raw fish samples, either intact fish/muscle or minced muscle. NIR spectroscopy has proven applicable also for the analysis of frozen products as well as processed and refined products. Shimamoto et al. [30] used NIR spectroscopy in connection with an interactance probe as a means of determining the fat content in frozen horse mackerel nonintrusively. A few years later the same group used NIR to assess the fat content in frozen skipjack [31]. Smoked and cured fish have also been subject to investigation by the use of NIR spectroscopy. Huang et al. [32] performed a study to show that moisture and salt content in cold smoked salmon could be evaluated using NIR measurements. Similar findings were made on hot smoked portions of salmon fillets by Lin et al. [33]. The salting, smoking, and exposure to elevated temperatures, about 63°C for the hot smoking process, alter the physical and chemical properties as well as the textural properties of the fish muscle. NIR spectroscopy, however, still proved viable for assessing the chemical constituents of the samples. Moisture and sodium chloride in cured Atlantic salmon were measured nondestructively by NIR diff use reflectance spectroscopy [34], although the assessment of salt did not prove as effective as that of water content. Of the most recent studies in the field is work by Wold et al. [35] applying the NIR technique to determine water content in salted dried cod—clipfish. They addressed the sampling/measurement location and the method of performing measurements in a representative way. They did, however, combine the NIR technique with imaging—further described later in this chapter—which facilitates a novel way of measuring and analyzing fish quality. In addition to the analysis on raw fish and processed fish material, NIR spectroscopy has also been applied for the analysis of basic chemical constituents in other types of fish products. In 2001 Huang et al. [36] presented a study where NIR spectroscopy was used for the investigation of salt content in cured salmon roe. It was argued that the sensitivity of the method could have been better; however, the nonintrusive method would still be an interesting alternative for rapid testing of high-value food products. The spectroscopic method has been used to assess moisture, fat, and protein content in another roe-based product, namely, the Greek dish taramosalata. A work by Adamopoulos and Goula [37] showed that the chemical composition could be assessed with a high degree of accuracy in addition to the obvious benefit of the ease and simplicity of the measurement method. For surimi products, refined fish-based products made by washing mechanically deboned fish to remove constituents such as blood, lipids, enzymes, and certain proteins, NIR spectroscopy was applied to determine water and protein content [38]. A further use of NIR measurements for the evaluation of basic food constituents was suggested by Svensson et al. [39]. In this work it was demonstrated how NIR spectroscopy could be used to assess the protein content in brine from salted herring and thus indirectly be a measure of the maturity and ripening of the salted herring. In addition to the many studies assessing the basic chemical constituents in fish and seafood, the spectroscopic method has confirmed its applicability for the evaluation of several other quality issues in fish. Examples of these are nondestructive texture analysis of farmed salmon [40], differentiation between fresh and frozen-thawed fish [7], storage time of frozen fish [41], evaluation of freshness or storage time of fresh fish [41,42], and the detection of bruises in the fish muscle [33]. The versatility of the method is one reason for its relevance and growing popularity during the recent years. The broadbanded spectra contain information about several parameters, and
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the possibility of simultaneously monitoring a number of different issues, with one reading, is considered intriguing. As illustrated by the above, the ease of use of the methodology has increased through instruments facilitating little or no sample preparation as well as measurement setups for rapid and nonintrusive registration. Instrument development has come from the grand-size laboratory desktop versions to portable or handheld instruments as illustrated in Figure 9.3. These developments have enabled the use of at-line or online methodology. The technique has, however, not yet become an everyday instrumental tool for food-quality control nor, say, fish-quality inspection. There may be several reasons for this. The high price of the instrumentation, on one side, has been a reason for the method not gaining a broader range of applicability. High-cost instrumentation designed for versatile use and flexibility has probably better met the requirements of laboratory use than those of industrial application. Another issue is the need for modeling the correlation between the spectroscopic reading and the quality parameter in question. Th is is a challenging task in view of the variety and the heterogeneity of the material and so may have contributed to the reluctance in investing in and developing this technology to a commercial tool for assessment of fish quality. The development in recent years in instrumentation, combining imaging techniques with the spectral information, may promote the future applicability and usefulness of the information in
Figure 9.3 Prototype version of handheld spectroscopic instrument for quality assessment of fish. This instrument was used for the determination of freshness of cod as well as the assessment of frozen storage time of hake.
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the near-infrared spectra. A novel example of this is the development of the QMonitor (QVision AS, Oslo, Norge), an analytical tool for industrial quality control of clipfish and salmon fillets. The realization of a commercial processing analytical tool for the simultaneous analysis of several parameters makes the technology interesting for a broad range of fish and seafood processing industries.
9.2 Imaging Spectroscopy 9.2.1
Theory, Measurement Principles, and Analysis
Imaging spectroscopy, also known as multispectral imaging or hyperspectral imaging, is a new technique that has been developed during the last decade [43,44]. It has become a widely used technique within fields spanning microscopy to satellite remote sensing. In addition to what traditional spectroscopy can facilitate, this technique also provides spatial information. Th is means that for each spatial location it is possible to access the full spectral information. To simplify the concept, this can be illustrated as simultaneously recording information about shape and color. This implies that this technique is a powerful tool for segmentation and classification and that it may also map the chemical composition into the spatial domain [45]. Imaging spectroscopy can be implemented for transmission, reflection, as well as transflection measurements. Depending on the applied sensor technology, the spectra may be recorded in the visible and near-infrared region. Typically, an imaging spectrograph operates in the following way; it uses a two-dimensional sensor, and each frame captured provides full spectral information for one line across the object to be imaged. Between each captured frame, the spectrograph and the object must move relative to each other. In this way an image of the object is built line by line. Typically, the relative motion is accomplished by mounting the imaging spectrograph above a conveyer belt where each captured frame images a line perpendicular to the direction of motion. As described in Section 9.1 on NIR spectroscopy, this method is an indirect measurement technique. The analytical techniques described in that section are also applied to imaging spectroscopy data. As these techniques only use the spectral information, improved results can be obtained by combining these techniques with more traditional image processing techniques. For instance, the hyperspectral data can be preprocessed based on spatial features before applying analytical spectral techniques, or the result from these techniques can be postprocessed to utilize the spatial information [46].
9.2.2
Analysis of Basic Constituents
During the last decade several applications within food-quality inspection have been developed based on imaging spectroscopy. Most of them are on foods such as fruits, vegetables, and meat. There are still relatively few reports on imaging spectroscopy applied for the analysis of fish and seafood. However, the feasibility of the method for the analysis of basic composition of foods, in general, demonstrates the potential of the method in the seafood sector as well. In order to illustrate the potential parameters to be assessed by imaging spectroscopy, some examples related to the agricultural sector are referred. For fruits and vegetables more articles report on determination of chemical constituents such as moisture content, total soluble solids, and acidity (expressed as pH) [47–49]. Several solutions have also been developed for detection of defects and contaminations on fruits. It has been shown that NIR hyperspectral imaging techniques are
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useful for automatic online detection of surface defects and contaminations on apples [50–52]. A thorough review of imaging spectroscopy applications within fruits and vegetables is presented by Nicolai et al. [53]. Inspection systems based on hyperspectral imaging have been tested for poultry carcass inspection focusing on classification of carcasses into normal, septicemic, and cadaver [54,55]. Further on, imaging spectroscopy solutions for detection of contaminants such as fecal and ingesta on poultry carcasses have been studied [56–58]. A recent work on quality assessment of pork has been reported by Qiao et al. [59,60] where several quality parameters were evaluated by imaging spectroscopy. The parameters included were drip loss, pH, color, and different texture features. The first article addressing analysis of fish or seafood by imaging spectroscopy was published in 2000 by Sigernes et al. [61]. Peeling of shrimps and detection of nematodes were mentioned as possible applications for the future. Since 2000, the main activities within imaging spectroscopy and fish analysis have been focused on online solutions for assessing chemical composition and detection of quality defects in fish products. Regarding the determination of basic chemical composition of fish and seafood, there is one recent publication on assessing water content in salted dried cod by Wold et al. [35]. In this publication the importance of including spatial information is illustrated. When drying fish, the moisture content of the fish varies from the thinner parts to the thicker parts of the fish. Hence, measuring the water content in one spot is not necessarily representative for the whole fish. QVision (Oslo, Norway) has also developed an industrial solution based on multispectral imaging for measuring the fat content in salmon fi llets (see Figure 9.4).
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Figure 9.4 Fat distribution in salmon fillet measured by the multispectral imaging system QMonitor fabricated by QVision (Oslo, Norway). The color bar to the right indicates the correspondence between color and fat content in percentage. The mean fat content for this fillet is 18.3%, whereas the local fat content varies from approximately 6% up to 43%.
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In addition to the measurement and documentation of basic composition, imaging spectroscopy of fish has been applied to address other quality issues. For detection of flaws or defects in fish, a lot of effort has been invested in the detection of nematodes, blood spots, black lining, and skin remnants in whitefish fillets [62–64]. Still the number of imaging spectroscopy applications with fish and seafood is low, but looking at reported applications within other areas the potential for new applications is high. For NIR spectroscopy several applications within fish and seafood are reported, and the methods that are feasible by spot measurements may also be implemented using imaging spectroscopy. Even more important is that for some applications imaging spectroscopy can provide better results, since it is possible to use spectra from dedicated relevant areas on the sample. For instance, if blood oxidation should be quantified spectra from blood-infested area of a fillet can easily be extracted for analysis based on imaging spectroscopy data. Furthermore, experience with NIR spectroscopy shows that more than one attribute can be estimated based on one recording, but this requires that the same spot be used. With imaging spectroscopy this is not a problem since spectra are available for all spatial locations. Imaging spectroscopy is well suited for application in the fish processing industry as an online technique. Using the interaction between light and the sample object, measurements may be performed at high speed as well as in noncontact mode. With respect to commercial implementation of imaging spectroscopy, this is a relatively new field, and currently there are a limited number of equipment suppliers. A low-resolution (spectral and spatial) instrument is available for industrial assessment of chemical composition such as fat and water content (QMonitor, QVision, Oslo, Norway) in fish. In addition to this a high-resolution prototype imaging spectrograph has been developed for detection of defects as well as determination of chemical constituents in fish fillets as reported by Heia et al. [63].
9.3 NMR Spectroscopy 9.3.1
Determination of Basic Composition
Nuclear magnetic resonance (NMR) has evolved from being an expensive and academic analytical technique into being a technique applicable for the food industry in both size and price of the equipment as well as speed of analyses. The main technique used is NMR spectroscopy, but during the last few years magnetic resonance imaging (MRI) has also been explored for its usefulness in food analyses.
9.3.2 Theory and Measurement Principles NMR provides a large amount of information regarding composition and structure of components in food. NMR techniques use electromagnetic radiation and magnetic fields to obtain chemical information, and they are based on the magnetic properties of atomic nuclei. All nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. The most commonly measured nuclei are 1H and 13C. Additionally, 31P-NMR and 23Na-NMR have also been used for food analyses. When an external magnetic field is applied, NMR active nuclei absorb at a frequency characteristic of the isotope. The energy absorptions of the atomic nuclei are also affected by the nuclei of neighboring atoms within the same molecule as well as nuclei in surrounding molecules. Hence, NMR spectroscopy may provide detailed
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information regarding the molecular structure of a food sample. Numerous applications of NMR in food analyses have been reported in the literature, and some examples of analyses of seafood are given here. Today, different NMR equipments are available, and they may provide different information regarding the food properties. For analyses of seafood products, low-resolution NMR (LR-NMR) and high-resolution NMR (HR-NMR) spectroscopy as well as MRI and NMR-mobile universal surface explores (NMR-MOUSE) have been used. HR-NMR has been used in many studies and has the advantage over LR-NMR that it is possible to obtain detailed information regarding the molecular structure.
9.3.3 Analysis of Basic Constituents For several years 1H- and 13C-NMR have been applied to measure the lipid or water content of many different foods including fish, and there are numerous reports available. Low-field (LF) NMR spectroscopy requires little or no sample preparation, and it has mainly been used for analyses of water in food samples, but the technique has been applied in the recent years for determination of both fat and water content in different food products and also seafood. For example, LF-1H-NMR has been used for studying water distribution in smoked salmon [65], cod [66], water distribution, and mobility in herring [67] and oil and water content of salmon and cod [68]. Studies of large objects like whole fish are impossible using most traditional LF-NMR instruments. A new type of LF-NMR instrument, the Bruker Professional MOUSE ® (Bruker Optik GmbH, Rheinstetten, Germany) has been developed to handle such samples. Aursand et al. [69] demonstrated that this equipment could be applied to determine fat in homogenates from salmon, whereas Veliyulin et al. [70] demonstrated that NMR-MOUSE could also be used for in vivo determination of fat content in Atlantic salmon. Additionally, in a study focusing on both 23Na-NMR and low-field 1H-NMR spectroscopy, it was shown that 23Na-NMR has proven useful for quantitative salt determinations in salted cod, whereas LF- 1H-NMR seems to correlate to fillet pH and water-holding capacity [71]. High-resolution NMR can be used to provide information on lipid classes, degree of saturated/ unsaturated fatty acids, fatty acid composition, and studies of lipid degradation processes in lipid mixtures such as fish oils. As recent examples, Tyl et al. [72] used HR-NMR to measure the content of n-3 polyunsaturated fatty acids in four types of unoxidized fish oils, whereas Siddiqui et al. [73] used HR-1H- and HR-13C-NMR for multicomponent analyses of encapsulated marine oil supplements. Falch et al. [74] reported the use of HR-NMR to determine oxidation products in marine lipids. Due to the provision of very detailed information regarding the molecular structure of a food sample, high-resolution NMR has been applied in many food authenticity studies. Extensive reviews on different techniques, including NMR, used for seafood authenticity have been provided by Martinez et al. [75] and Arvanitoyannis et al. [76]. Among more recent work, Standal et al. [77] used NMR to discriminate cod liver oil according to whether the origin was wild/ farmed as well as geographic origin. Rezzi et al. [78] demonstrated the use of NMR lipid profiling for classification of gilthead sea bream according to geographic origin, whereas Thomas et al. [79] and Masoum et al. [80] used this technique to determine the origin of Atlantic salmon. Additionally, 1H NMR spectroscopy has been explored to identify the fate of some bioactive compounds during processing of seafood. Martinez et al. [81] showed that it was possible to identify taurine, betaine, anserine, creatine, trimethylamine oxide, and dimethylamine in extracts
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from processed cod. Gribbestad et al. [82] showed that it was possible to identify single chemical compounds such as hypoxanthine, amino acids, anserine, lactate, and some fatty acids in extracts and muscle from salmon using high-resolution 1H NMR spectroscopy. As illustrated here, NMR is a versatile tool for the identification and quantification of numerous compounds in fish related to nutritional quality. An objection to the method, however, has been that conventional NMR is an expensive technique. However, the high spectral resolution is not always required, and lately many low-field, low-resolution NMR spectrometers have been developed and commercialized. Such equipment is cheaper, smaller, and less sensitive to fluctuations in the environment and thus more applicable in industry as well as in many research fields.
9.4
X-Ray Imaging
9.4.1 Theory and Measurement Principles X-ray imaging is a technique based on the emission of x-rays through a sample and recording the amount of attenuation. For online applications this can be implemented as a line-by-line imaging or a frame-by-frame imaging. The decrease in x-ray intensity inside a sample will be due to absorption by different materials. A more dense material will absorb more x-ray energy, and the attenuation will also be influenced by the sample thickness. It is not possible to accurately characterize the observed sample by applying only one x-ray energy level. There are two interactions, the photoelectric effect and the Compton scattering that causes the x-ray attenuation, and their relative contributions are energy dependent [83]. By using two x-ray energy levels, more specific information about the sample can be revealed. This technique is referred to as dual-energy x-ray absorptiometry (DXA, previously DEXA) and may be implemented using a two-layer detector, one layer for each energy level. Within the field of medicine, computed tomographic (CT) scanning is widely used. This is also an x-ray imaging system, but it provides a three-dimensional image of the sample. This is achieved by rotating the x-ray/detector unit around the sample. Making profiles from different angles and then combining them by software, a two-dimensional cross section of the sample can be made. Then the third dimension is accomplished by the sample movement. Th is is a powerful imaging technique that can be used both as a single-energy and a dual-energy module.
9.4.2 Analysis X-ray imaging provides spatial information in two dimensions (2D) or three dimensions (3D) (CT). Typical applications within fish and fish products are related to the detection of bones and bone fragments as well as chemical composition and localization. A study has been conducted on the applicability of CT scanning as a nondestructive and rapid way of measuring muscle dry matter content and liquid leakage in cod fillets [84]. The results obtained showed that CT scanning could be used as a rapid method for the assessment of these attributes and would add valuable information to be used in genetic studies and breeding programs. Further on the CT scans gave significant information about dry matter distribution from head to tail of the cod. In another study Kolstad et al. [85] tested CT scanning as a tool for estimating the relative size of fat deposits and lean tissue and fat content in Atlantic halibut. Based on the results obtained the
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Figure 9.5 Detection of pin bones in fish fillets by x-ray imaging using the SensorX instrumentation (Marel, Iceland). To the left is the original x-ray image of one cod fillet and to the right is the processed image where only the bones identified in the fillet are shown.
authors recommended CT scanning as an online technique for carcass evaluation. A similar work has been carried out by Hancz et al. [86] showing good results predicting fat content of common carp based on CT scanning. With respect to bone detection in fish fillets there are commercial solutions available today (Marel Hf, Iceland). Marel developed an X-ray-based bone detection unit (SensorX) that was commercially available on the market in 2003 [87]. This instrument can detect bones and bone fragments down to a diameter of 0.3 mm when operating at industrial speed (see Figure 9.5 for an example).
9.5 Summary The methods and applications presented in the above clearly illustrate that there are more tools and techniques that could serve as an easy and useful way of rapid quality determination of fish and seafood; instrumental means capable of objective and rapid determination of basic composition are also available. Throughout development all presented techniques have met the requirements
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of simplicity in sample preparation, and progress in data processing and analytical tools has facilitated usability and ease of interpretation of measurement results. In addition, NIR, NMR, imaging, and x-rays are operated at a speed that makes it possible to perform measurements at or in a processing line, hence allowing for measurements to be performed on large-scale quantities. This chapter, however, also makes it clear that although proven useful and promising in laboratory-scale trials, these techniques have—with a few commercial exceptions—still not been shown to be commercially valid for quality determination in the fish and seafood processing industry. Part of the explanation for this could be the cost level of the equipment in question. The price of measurement equipment for NIR, NMR, and x-rays is considerable and, therefore, not easily applicable for small-scale industries as is often the case in the fish processing industry. Another issue is the substantial variety and heterogeneity of the material to be analyzed. Due to the spread and diversity in fish species and sizes as well as the seasonal difference in bodily composition, the finding of a universal measurement tool to meet with this variety is a challenging task. However, the technological development exemplified by SensorX (Marel hf, Reykjavik, Iceland) and QMonitor (QVision AS, Oslo, Norway) confirms that these techniques may be applied in commercial and industrial high-speed fish processing applications. Introducing and applying these methods to industrial applications and enabling production of well-documented, quality seafood products will contribute to retaining the good reputation of fish and seafood in the years to come.
Acknowledgment The authors would like to thank Dr Jens Petter Wold, Nofima Food, Norway, for providing the example picture used in Figure 9.4.
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32. Huang, Y. et al., Nondestructive prediction of moisture and sodium chloride in cold smoked Atlantic salmon (Salmo salar), Journal of Food Science, 67(7), 2543–2547, 2002. 33. Lin, M. et al., Bruise detection in pacific pink Salmon (Oncorhynchus gorbuscha) by visible and shortwavelength Near-Infrared (SW-NIR) Spectroscopy (600–1100 nm), Agricultural and Food Chemistry, 51, 6404–6408, 2003. 34. Huang, Y. et al., Nondestructive determination of moisture and sodium chloride in cured Atlantic salmon (Salmo salar) (Teijin) Using short-wavelength Near-infrared Spectroscopy (SW-NIR), Journal of Food Science, 68(2), 482–486, 2003. 35. Wold, J.P. et al., Non-contact transflectance near infrared imaging for representative on-line sampling of dried salted coalfish (bacalao), Journal of Near Infrared Spectroscopy 14(1), 59–66, 2006. 36. Huang, Y. et al., Detection of sodium chloride in cured salmon roe by SW-NIR spectroscopy, Journal of Agricultural Food Chemistry, 49, 4161–4167, 2001. 37. Adamopoulos, K.G. and Goula, A.M. Application of near-infrared reflectance spectroscopy in the determination of major components in taramosalata, Journal of Food Engineering, 63, 199–207, 2004. 38. Uddin, M. et al., Nondestructive determination of water and protein in surimi by near-infrared spectroscopy, Food Chemistry, 96, 491–495, 2006. 39. Svensson, V.T., Nielsen, H.H., and Bro, R. Determination of the protein content in brine from salted herring using near-infrared spectroscopy, Lebensmittel- Wissenschaft und- Technologie, 37, 803–809, 2004. 40. Isaksson, T. et al., Non-destructive texture analysis of farmed Atlantic salmon using visual/ near-infrared reflectance spectroscopy, Journal of the Science of Food and Agriculture, 82, 53–60, 2001. 41. Heia, K. et al., Visible spectroscopy—Evaluation of storage time of ice stored cod and frozen hake, in Quality of Fish from Catch to Consumer, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G. Eds., Wageningen Academic Publishers, Wageningen, the Netherlands, 2003, pp. 201–209. 42. Nilsen, H. et al., Visible/Near-Infrared spectroscopy—A new tool for the evaluation of fish freshness, Journal of Food Science, 67(5), 1821–1826, 2002. 43. Herrala, E. and Okkonen, J., Imaging spectrograph and camera solutions for industrial applications, International Journal of Pattern Recognition and Artificial Intelligence, 10, 43–54, 1996. 44. Hyvarinen, T.S., Herrala, E., and Dall’Ava, A., Direct sight imaging spectrograph: A unique add-in component brings spectral imaging to industrial applications, in Digital Solid State Cameras: Designs and Applications, Williams, G.M. Jr. ed., Proc. SPIE 3302, 165–175, 1998. 45. Colarusso, P. et al., Infrared spectroscopic imaging: From planetary to cellular systems, Applied Spectroscopy, 52(3), 106A–120A, 1998. 46. Kohler, A. et al., Multivariate image analysis of a set of FTIR microspectroscopy images of aged bovine muscle tissue combining image and design information, Analytical and Bioanalytical Chemistry, 389, 1143–1153, 2007. 47. Lu, R.F., Multispectral imaging for predicting firmness and soluble solids content of apple fruit, Postharvest Biology and Technology, 31(2), 147–157, 2004. 48. Peng, Y. and Lu, R., Modeling multispectral scattering profiles for prediction of apple fruit firmness, Transactions of the ASAE, 48(1), 235–242, 2005. 49. ElMasry, G. et al., Hyperspectral imaging for nondestructive determination of some quality attributes for strawberry, Journal of Food Engineering, 81(1), 98–107, 2007. 50. Lu, R., Detection of bruises on apples using near-infrared hyperspectral imaging, Transactions of the ASAE, 46(2), 523–530, 2003. 51. Mehl, P.M. et al., Development of hyperspectral imaging technique for the detection of apple surface defects and contaminations, Journal of Food Engineering, 61(1), 67–68, 2004. 52. Nicolai, B.M. et al., Non-destructive measurement of bitter pit in apple fruit using NIR hyperspectral imaging, Postharvest Biology and Technology, 40(1), 1–6, 2006.
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53. Nicolai, B.M. et al., Nondestructive measurement of fruit and vegetable quality by means of NIR spectroscopy: A review, Postharvest Biology and Technology 46, 99–118, 2007. 54. Chao, K. et al., On-line inspection of poultry carcasses by a dual-camera system, Journal of Food Engineering, 51(3), 185–192, 2002. 55. Park, B., Chen, Y.R., and Huffman, R.W., Integration of visible/NIR spectroscopy and multispectral imaging for poultry carcass inspection, Journal of Food Engineering, 30(1–2), 197–207, 1996. 56. Lawrence, K.C. et al., A hyperspectral imaging system for identification of faecal and ingesta contamination on poultry carcasses, Journal of Near Infrared Spectroscopy, 11(4), 269–281, 2003. 57. Windham, W.R. et al., Algorithm development with visible/near-infrared spectra for detection of poultry feces and ingesta, Transactions of the ASAE, 46(6), 1733–1738, 2003. 58. Park, B. et al., Hyperspectral imaging for detecting fecal and ingesta contaminants on poultry carcasses, Transactions of the ASAE, 45(6), 2017–2026, 2002. 59. Qiao, J. et al., Prediction of drip-loss, pH, and color for pork using a hyperspectral imaging technique, Meat Science, 76(1), 1–8, 2007. 60. Qiao, J. et al., Pork quality and marbling level assessment using a hyperspectral imaging system, Journal of Food Engineering, 83(1), 10–16, 2007. 61. Sigernes, F. et al., Multipurpose spectral imager, Applied Optics, 39(18), 3143–3153, 2000. 62. Wold, J.P., Westad, F., and Heia, K., Detection of parasites in cod fillets by using SIMCA classification in multispectral images in the visible and NIR region, Applied Spectroscopy, 55(8), 1025–1034, 2001. 63. Heia, K. et al., Detection of nematodes in cod (Gadus morhua) fillets by imaging spectroscopy, Journal of Food Science 72, E11–E15, 2007. 64. Stormo, S.K. et al., Effects of single wavelength selection for anisakid roundworm larvae detection through multispectral imaging, Journal of Food Protection, 70(8), 1890–1895, 2007. 65. Loje, H. et al., Water distribution in smoked salmon, Journal of the Science of Food and Agriculture, 87(2), 212–217, 2007. 66. Andersen, C.M. and Rinnan, A., Distribution of water in fresh cod, LWT-Food Science and technology, 36(8), 807–812, 2002. 67. Jensen, K.N. et al., Water distribution and mobility in herring muscle in relation to lipid content, season, fishing ground and biological parameters, Journal of the Science of Food and Agriculture, 85(8), 1259–1267, 2005. 68. Jepsen, S.M., Pedersen, H.T., and Engelsen, S.B., Application of chemometrics to low-field H-1 NMR relaxation data of intact fish flesh, Journal of the Science of Food and Agriculture, 79(13), 1793–1802, 1999. 69. Aursand, I.G., Veliyulin, E., and Erikson, U., Low Field NMR Studies of Atlantic Salmon (Salmo salar), in Modern Magnetic Resonance, Webb GA, Ed., Springer, Dordrecht, the Netherlands, 2006. 70. Veliyulin, E. et al., In vivo determination of fat content in Atlantic salmon (Salmo salar) with a mobile NMR spectrometer, Journal of the Science of Food and Agriculture, 85(5), 1299–1304, 2005. 71. Eriksson, U. et al., Salting and desalting of fresh and frozen-thawed cod (Gadus morhua) fillets: A comparative study using Na-23 NMR, Na-23 MRI, low-field H-1 NMR, and physicochemical analytical methods, Journal of Food Science, 69(3), 107–114, 2004. 72. Tyl, C.E., Brecker, L., and Wagner, K.H., H-1 NMR spectroscopy as tool to follow changes in the fatty acids of fish oils, European Journal of Lipid Science and Technology, 110(2), 141–148, 2008. 73. Siddiqui, N. et al., Multicomponent analysis of encapsulated marine oil supplements using highresolution H-1 and C-13 NMR techniques, Journal of Lipid Research, 44(12), 2406–2427, 2003. 74. Falch, E. et al., Correlation between H-1 NMR and traditional methods for determining lipid oxidation of ethyl docosahexaenoate, Journal of the American Oil Chemists Society, 81(12), 1105–1110, 2004. 75. Martinez, I. et al., Destructive and non-destructive analytical techniques for authentication and composition analyses of foodstuffs, Trends in Food Science and Technology, 13, 489–498, 2003.
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76. Arvanitoyannis, I.S., Tsitsika, E.V., and Panagiotaki, P., Implementation of quality control methods (physicochemical, microbiological and sensory) in conjunction with multivariate analyses towards fish authenticity, International Journal of Food Science and Technology, 40, 237–263, 2005. 77. Standal, I.B. et al., Discrimination of cod liver oil according to Wild/Farmed and geographical origins by GC and C-13 NMR, Journal of the American Oil Chemists Society, 85(2), 105–112, 2008. 78. Rezzi, S. et al., Classification of gilthead sea bream (Sparus aurata) from H-1 NMR lipid profiling combined with principal component and linear discriminant analysis, Journal of Agricultural and Food Chemistry, 55(24), 9963–9968, 2007. 79. Thomas, F. et al., Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin, Journal of Agricultural and Food Chemistry, 56, 989–997, 2008. 80. Masoum, S. et al., Application of support vector machines to H-1 NMR data of fish oils: Methodology for the confirmation of wild and farmed salmon and their origins, Analytical and Bioanalytical Chemistry, 387(4), 1499–1510, 2007. 81. Martinez, I. et al., Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses, Journal of Agricultural and Food Chemistry, 53(17), 6889–6895, 2005. 82. Gribbestad, I.S., Aursand, M., and Martinez, I., High resolution 1H magnetic spectroscopy of whole fish, fillets and extracts from farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses, Aquaculture, 250, 445–447, 2005. 83. Rebuffel, V. and Dinten, J.M., Dual-energy X-ray imaging: Benefits and limits, Insight, 49(10), 589–594, 2007. 84. Kolstad, K., Morkore, T., and Thomassen, M.S., Quantification of dry matter % and liquid leakage in Atlantic cod (Gadus morhua) using computerised X-ray tomography (CT), Aquaculture, 275(1–4), 209–216, 2008. 85. Kolstad, K. et al., Quantification of fat deposits and fat distribution in Atlantic halibut (Hippoglossus hippoglossus L.) using computerised X-ray tomography (CT), Aquaculture, 229(1–4), 255–264, 2004. 86. Hancz, C. et al., Measurement of total body composition changes of common carp by computer tomography, Aquaculture Research, 34(12), 991–997, 2003. 87. Andersen, K., X-ray techniques for quality assessment, in Quality of Fish from Catch to Consumer, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G. Eds., Wageningen Academic Publishers, Wageningen, the Netherlands, 2003, 283–286.
Chapter 10
Microstructure Isabel Hernando, Empar Llorca, Ana Puig, and María-Angeles Lluch Contents 10.1 Main Microscopy Techniques for Studying Seafood .....................................................139 10.2 Fish Muscle Microstructure...........................................................................................140 10.2.1 Herring ............................................................................................................145 10.2.2 Hake ................................................................................................................146 10.3 Processed Fish Microstructure .......................................................................................148 10.3.1 Smoked Salmon............................................................................................... 148 10.3.2 Salted Cod....................................................................................................... 149 10.3.3 Surimi ..............................................................................................................150 10.4 Squid Microstructure .................................................................................................... 151 References ................................................................................................................................ 151
10.1 Main Microscopy Techniques for Studying Seafood The microstructure of foods forms a link between the molecular and macroscopic levels and constitutes a key factor for studying the properties of foods and for improving and optimizing food processes. The organization of the chemical components of foods (proteins, carbohydrates, fats, etc.) is responsible for their microstructure, so any chemical or enzymatic change that takes place in the chemical components has an effect on the microstructural organization of the food matrices and their functionality. Several strategies can be used to study food microstructure. Pérez-Munuera et al. (2008) gave an overview of the most important techniques for studying muscle food structure. This chapter 139
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provides a detailed description of the protocols often followed to obtain information about seafood microstructure. The light microscope (LM) is a very versatile tool that works in different applications such as bright field, phase contrast or differential interference contrast (Nomarski), polarizing microscopy, or fluorescence microscopy. The most useful application for studying seafood structure is bright field microscopy. For this, the sample has to be prepared in semithin sections of about 0.1–2 mm (Figure 10.1). The sections are obtained using a microtome after embedding the food in paraffin or resin or using a cryotome after freezing the sample with CO2 or liquid N2. Once the semithin sections are obtained, they are mounted in glass slides and stained with different dyes (toluidine blue, sudan, red oil, iodine, light green, etc.) before examination in the LM. Electron microscopy (EM) allows food structures to be studied at higher magnifications than those used in LM. Two types of microscopes use electron beams as their source of illumination: transmission electron microscopes (TEM) and scanning electron microscopes (SEM). In both methods, the samples need to be prepared first. The steps in preparing samples for TEM observation (Figure 10.2) are primary fi xation with aldehydes such as glutaraldehyde, secondary fi xation with osmium tetroxide, dehydration in a series of ethanol dilutions of increasing concentration, infiltration and embedding in resin, cutting ultrathin sections (5–100 nm) in an ultramicrotome, and staining the ultrathin sections with heavy metal solutions such as lead citrate or uranyl acetate. The SEM method observes the surface of the sample, so there is no need to section it. There are two ways of preparing samples for SEM: chemical fi xation and physical fi xation (Figure 10.3). In the former, the sample preparation steps are chemical fi xation (with aldehydes and osmium tetroxide, as for TEM), dehydration in a series of ethanol dilutions of increasing concentration, critical point drying, and coating with a conducting metal for SEM imaging or with carbon for x-ray. When physical fixation is used, the sample is frozen in liquid nitrogen and then freeze-dried before being coated and observed. In recent years considerable progress has been made in the field of SEM through vitrification techniques. In Cryo-SEM, the sample is frozen in slush nitrogen (Figure 10.4) and quickly transferred under vacuum to a cold stage fit on a microscope where the frozen sample is fractured, etched, coated, and observed; in this way, the sample can be observed with all its constituent water. Besides the secondary electrons, other emanations or signals such as x-rays, backscattered electrons, and so on, may be generated as a result of the electron beam striking the specimen (Pérez-Munuera et al., 2008); these different signals can be captured by the appropriate detector in each case. In this way, ions or molecules can be identified and quantified in situ using specific detectors coupled to the electron microscope, so microanalysis can be carried out by means of x-ray. Finally, image analysis relies heavily on computer technology to obtain quantitative results from microscopy observation.
10.2
Fish Muscle Microstructure
Fish muscle consists of myotomes. They are arranged in concentric circles forming subdivisions of striated muscle (Figure 10.5). At each subdivision there are macroscopic collagenous dividing lines (myocommata). The muscle cells are short and 0.02–1.0 mm in diameter. They are each surrounded by the sarcolemma membrane and by a thin layer of connective tissue (endomysium). Many of the endomysia are connected to the perymisium, which is contiguous to the myocommata (Ofstad et al., 2006). The fibers are essentially the same as those of terrestrial animals in terms of the arrangement of the thick and thin filaments, showing alternate arrangements of
Microstructure ◾
Food
Specimen portions
Embedding in paraffin or resin
Freezing (CO2, liquid N2)
Cold knife
Preparation for slicing
Slice
CO2
Semithin sections (0,11–2 μm)
Knife Cold stub Microtome
Cryotome
Mounting in glass slides
Staining specimen
1% Toluidine blue 1% Lugol, 1% red oil, ...
LM observation
Figure 10.1 Preparation of samples for LM observation.
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Food
Specimen portions
Fixation 2,5% Glutaraldehyde
2% Os O4
Dehydration Ethanol (30%, 50%, 70%, 90%, 100%)
Infiltration and embedding in resine Epoxi resin, Araldite Spurr’s, LR white
Cured resin
Glass or diamond knife
Ultrathin sectioning (5–100 nm)
Ultramicrotome Specimen block
Trimmed block
Tweezers Section collection Ultrathin section staining
Specimen block face Grid Knife
4% Lead citrate 2% Uranyl acetate
TEM observation
Figure 10.2 Preparation of samples for TEM observation.
Microstructure ◾ 143
Food
Specimen portions
Fixation Physical fixation
Quick freezing in liquid N2
Chemical fixation
2,5% Glutaraldehyde
2% Os O4
Ethanol (30, 50, 70, 90, 100%) Sublimated H2O
P
T Dehydration
(To vaccum) Freeze dryer
CO2
Critical point dryer
(To transformer) (To pumps) Sputter metal coater (or evaporation coater)
Coating (Au, Pd, for SEM imaging) (C, for X-ray)
SEM observation
Figure 10.3
Preparation of samples for SEM observation.
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Food
Specimen portions
Quick freezing in slush N2 (T < –210°C)
Freezing
Transfer to Cryo-SEM
Specimen transfer
Knife Specimen fracturing (into Cryo-SEM) Specimen fracturing (–180°C, vaccuum)
Etched H2O
Specimen fracturing (–90°C, vaccuum)
Au deposition
Etching (into Cryo-SEM)
Coating (Au, C, ...) (into Cryo-SEM)
(–130°C, vaccuum)
Cryo-SEM observation
Figure 10.4
Preparation of samples for Cryo-SEM observation.
Microstructure ◾ 145
a
b
Figure 10.5 Schematic representation of fish muscle with myotomes. a: myotome, b: myocommata.
A and I bands (Pérez-Munuera et al., 2008), but the total collagen content is lower, since the water in which the fish live lends support for the body (Lampila, 1990). Examples of different fresh fish tissues observed by several techniques are described here.
10.2.1 Herring Figure 10.6A shows a cross section of herring tissue fi xed with glutaraldehyde and observed by SEM. The typical fish muscle fibers can be seen, surrounded by the sarcolemma and by the endomysial connective tissue, which is mainly composed of collagen. The separation that can be observed between the muscle cells is usually attributed to the effect produced by chemical fixation and dehydration during preparation for SEM. The myofibrils are shown in longitudinal section in this sample (Figure 10.6B), where the Z disks can be distinguished. Fixing in osmium tetroxide shows the distribution of fat in the herring tissue. At low magnification, the fat can be observed covering the fibers in a longitudinal section of herring muscle (Figure 10.6C). Figure 10.6D shows the microstructure of herring tissue at a higher magnification, where the fat is viewed as globules on the surface of the fiber. The fiber is composed of myofibrils in which Z disks are distinguished in the areas where the sarcolemma is broken. In a cross section of the sample fi xed in osmium tetroxide (Figure 10.6E), fat globules of different sizes are observed occupying the interfibrillar spaces and myofibrils are distinguished inside the cells. When the muscle fibers are observed using the Cryo-SEM technique, the aggregation of solutes produced during the etching of the sample generates the typical eutectic artifact observed in Figure 10.6F. A micrograph cross section of the fibers shows them surrounded by the sarcolemma, with the endomysial connective tissue keeping the muscle fibers firmly attached to one another. Figure 10.7A shows a herring sample stained with toluidine blue and observed by LM; the perymisial connective tissue that surrounds the muscle bundles can be seen. Figure 10.7B, obtained by the same technique but observed at a higher magnification, reveals the myofibrils inside each cell; the myofibrils at the cell edges have a less rounded section than the central myofibrils and are arranged like a palisade. When ultrathin sections of herring muscle tissue are studied by TEM, it is possible to observe ultrastructural details. The longitudinal section in Figure 10.7C shows the inside of a muscle fiber with the myofibrils perfectly bundled. The layouts of the Z disks that mark the length of the sarcomere are visible. The myofibrils are
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Z
300 μm
(A)
6 μm
(B)
I 40 μm
(C)
a
f
f
f (E)
10 μm
(D)
c 60 μm
(F)
30 μm
Figure 10.6 Herring tissue. (A–E) SEM, (F) Cryo-SEM. Z, Z disk; f, fat globule; a, eutectic artifact; c, connective tissue.
connected to each other at the Z disk level by the costameres, which are the components of the cytoskeletal network that links the myofibrils to one another and to the sarcolemma. The structural elements that constitute the sarcomere, the A and I bands, can be seen, along with the M and Z lines. The same structure has also been observed in different meat products, for example, pork meat (raw ham) (Larrea et al., 2007). The TEM technique allows images to be obtained at higher magnification and with better resolution than other microscopy techniques (Figure 10.7D).
10.2.2
Hake
The observation of hake muscle by SEM after fi xing with glutaraldehyde allows distinguishing that the fibers of hake muscle tissue are very similar to those of herrings. The main difference is their size: hake fibers are thicker than herring ones. Hake fibers surrounded by connective tissue can be observed in Figure 10.8. The cytoskeletal ultrastructure of hake was studied by Pagano et al. (2005) after depleting the thick and thin filaments with a potassium iodide treatment. TEM and SEM studies demonstrated an extensive network of filaments connecting Z structures that were regularly spaced and connected by sets of longitudinal, continuous, and roughly parallel filaments (Figure 10.9).
Microstructure ◾
147
p m m
30 μm
10 μm
(A)
(B) m
c M
m A Z (C)
(D)
Figure 10.7 Herring tissue. (A and B) LM, (C and D) TEM. P, perymisial connective tissue; m, myofibrils in a “palisade” ringing the edge; Z, Z disks; c, costameres; A, A band; I, I band; M, M line.
100 μm
Figure 10.8 Hake tissue observed by SEM.
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LZ
Z
IZ DZ
8.000 X
Figure 10.9 Cytoskeletal structure of hake observed by TEM. Z, Z-disk; LZ, longitudinal filament connecting Z-Z. (Reprinted from Pagano, M.R. et al., Com. Biochem. Physiol. B, 141, 13, 2005. With permission.)
10.3 Processed Fish Microstructure 10.3.1
Smoked Salmon
A cross section of a smoked salmon sample obtained using the Cryo-SEM technique is seen in Figure 10.10A. The micrograph shows geometrically shaped fibers surrounded by a connective tissue. Figure 10.10B shows a detail of an intercellular space created by the conjunction of three fibers or cells. Sigurgisladottir et al. (2000) used LM to observe the changes that occurred in the salmon during the smoking process and quantified them by image analysis. The data of the average cross-sectional area of muscle fibers showed that the smoking process produces shrinkage of the fibers; the higher the smoking temperature, the greater the shrinkage. The fiber shrinkage and the space between the fibers both increased to a greater extent in the muscle from the salmon that were frozen before smoking than in muscle smoked from fresh salmon. Gudmundsson and
(A)
Figure 10.10
100 μm
(B)
Smoked salmon. (A and B) Cryo-SEM.
10 μm
Microstructure ◾ 149
Hafsteinsson (2001) studied the effect of pulsed electric fields (PEF) and a combination of PEF and high pressure on smoked salmon microstructure; these treatments decreased the cell size compared with fresh salmon, and gaps were formed in the tissue structure. A combination of PEF and high pressure had a more detrimental effect on smoked salmon microstructure than PEF treatment alone.
10.3.2 Salted Cod The presence of salt deposits in the cod tissue can be observed by SEM (Figure 10.11A) in samples that have been obtained using physical fi xing (freeze-drying) instead of chemical fi xing. Figure 10.11A shows a longitudinal section of salted cod, where two fibers can be observed completely covered by salt deposits. Figure 10.11B shows a cross section of salted cod tissue
(A)
Figure 10.11
100 μm
(B)
300 μm
Salted cod. (A) SEM, (B) Cryo-SEM.
(A)
(B)
(C)
Figure 10.12 Seafood stick (surimi) observed by SEM. (A) longitudinal section, (B) cross section, and (C) protein network.
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observed by Cryo-SEM, where the presence of salt makes the etching of the sample for observation difficult and masks the underlying structures.
10.3.3 Surimi One of the most common surimi products on the market is artificial crab muscle. Such a product is often sold as “crab sticks” or “seafood sticks.” Lean fish meat is minced to a paste; after adding different additives, the paste is shaped and an “artificial fish muscle” is obtained. Figure 10.12A, obtained by SEM, shows a longitudinal section of a crab stick where the “artificial fibers” can be observed. The cross section (Figure 10.12B) shows the typical concentric layers of this type of surimi product. The formation of a new network with the myofibrillar protein (Figure 10.12C) is responsible for the water-holding capacity and functional properOuter lining Outer tunic Muscle tunic Inner tunic Visceral lining Radial fibers
Circumferential fibers
(A) Radial fibers
Circumferential fibers (B)
Figure 10.13 Schematic representation of (A) squid mantle and (B) arrangement of muscle cells. (From Lluch, M.A. et al., The Chemical and Functional Properties of Food Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 2001. With permission.)
Microstructure ◾ 151 –200 μm
(A) 10 μ
–10 μm
(B) 3.75 μ
m
l
s (C)
(D)
Figure 10.14 Squid. (A and B) SEM, (C) LM, and (D) TEM. s, central sarcoplasm; m, myofibril; l, sarcolemma. (From Llorca, E. et al., Eur. Food Res. Technol., 225, 807, 2007. With permission.)
ties of surimi. Th is gel network structure gives surimi its characteristic elasticity and texture (Sikorski, 1990).
10.4
Squid Microstructure
The squid mantle is composed of muscle tissue sandwiched between two tunics of connective tissue (Figure 10.13). The inner and outer tunics are covered by a visceral lining and outer lining, respectively. Muscle fibers are grouped in bands that are arranged orthogonally. Circumferential muscle bands (100–200 mm) comprise fibers running about the entire circumference of the mantle cone. Radial bands (10–15 mm thick) comprise fibers that connect two tunics of connective tissue. Regardless of their orientation, all the muscle fibers are thin, approximately 3.5 mm in diameter (Lluch et al., 2001). The fibers arranged in circumferential and radial bands were observed by SEM in samples fi xed with glutaraldehyde (Figure 10.14A and B) (Llorca et al., 2001). This fiber distribution can also be observed by LM in samples stained with toluidine blue (Figure 10.14C). LM makes it possible to distinguish a peripheral area in blue and a central core in white inside each cell. When TEM is used to study the ultrastructure of fresh squid (Figure 10.14D), a central sarcoplasm is shown to be surrounded by myofibrils; the intermyofibrillar spaces between these can be observed. Each fiber is surrounded by a sarcolemma (Llorca et al., 2007).
References Gudmundsson, M. and Hafsteinsson, H., Effect of electric field pulses on microstructure of muscle foods and roes, Trends Food Sci. Tech., 12, 122–128, 2001. Lampila, L.E., Comparative microstructure of red meat, poultry and fish muscle, J. Muscle Foods, 1, 247–267, 1990.
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Larrea, V., Pérez-Munuera, I., Hernando, I., Quiles, A., Llorca, E., and Lluch, M.A., Microstructural changes in Teruel dry-cured ham during processing, Meat Sci., 76, 574–582, 2007. Llorca, E., Hernando, I., Pérez-Munuera, I., Fiszman, S.M., and Lluch, M.A., Effect of frying on the microstructure of frozen battered squid rings, Eur. Food Res. Technol., 213(6), 448–455, 2001. Llorca, E., Hernando, I., Pérez-Munuera, I., Quiles, A., Larrea, V., and Lluch, M.A., Protein breakdown during the preparation of frozen batter-coated squid rings, Eur. Food Res. Technol., 225(5–6), 807–813, 2007. Lluch, M.A., Pérez-Munuera, I., and Hernando, I., Proteins in food structures, in The Chemical and Functional Properties of Food Proteins, Sikorski, Z.E. (Ed.), Technomic Publishing Co., Inc., Lancaster, PA, 2001, chap. 2. Ofstad, R., Olsen, R.L., Taylor, R., and Hanneson, K.O., Breakdown of intramuscular connective tissue in cod (Gadus morhua L.) and spotted wolfish (Anarhichas minor O.) related to gaping, Lebens. Wiss. Tech., 39, 1143–1154, 2006. Pérez-Munuera, I., Larrea, V., Quiles, A., and Lluch, M.A., Microstructure, in Handbook of Muscle Foods Analysis, Nollet, L. and Toldrá, F. (Eds.), CRC Press, Boca Raton, FL, 2008. Pagano, M.R., Paredi, M.E., and Crupkin, M., Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and post spawned female hake (Merluccius hubbsi), Com. Biochem. Physiol. Part B 141, 13–21, 2005. Sigurgisladottir, S., Ingvarsdottir, H., Torrisen, O.J., Cardinal, M., and Hafsteinsson, H., Effects of freezing/ thawing on the microstructure and the texture of smoked Atlantic salmon (Salmo salar), Food Res. Int., 33, 857–865, 2000. Sikorsi, Z.E., Seafood: Resources, Nutritional Composition and Preservation. CRC Press, Boca Raton, FL, 1990, chap. 1.
Chapter 11
Chemical Sensors Corrado Di Natale Contents 11.1 Introduction ..................................................................................................................153 11.2 Sensor Parameters..........................................................................................................154 11.3 Chemical Sensor Technologies ......................................................................................157 11.3.1 Sensors Based on Conductance Changes ..........................................................157 11.3.1.1 Metal-Oxide Semiconductors ..........................................................157 11.3.1.2 Conducting Polymers and Molecular Aggregates .............................158 11.3.2 Amperometric Gas Sensors ..............................................................................158 11.3.3 Mass Transducers ............................................................................................158 11.3.4 Field-Effect Transistors ....................................................................................159 11.3.5 Color Indicators ...............................................................................................159 11.4 Electronic Noses ............................................................................................................160 11.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement ...................................................................................................160 11.6 Conclusions ...................................................................................................................164 References ................................................................................................................................165
11.1
Introduction
Among the thousands of molecules composing food complex mixtures, some are of great importance to define overall properties such as freshness or quality [1]. The relationship between chemistry and food properties is particularly interesting in the case of fish and seafood in general, for which the human perception of airborne chemicals, called odor, is one of the most used method to assess freshness by both consumers and industries [2]. For these reasons the knowledge of the chemical 153
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profile of food is considered of great value, and the development of rapid and reliable chemical analyzers has been pursued since decades. Chemical analysis of foodstuff is a large part of the analytical chemistry discipline, and a number of methods and protocols for different food are available. Analytical chemistry is naturally based on “separation” approaches: namely, it develops methods to decompose complex mixtures (foods contains thousands of different molecules) in order to target either a single molecular species or a molecular family. These methods require in some cases complex sample treatments and instrumentation such as gas chromatography or spectrophotometers. On the other hand, it is known that Nature provides living beings chemical senses that, in order to be reliable, do not require any sample treatment. Differently from analytical instrumentations, natural senses are not analytical, in the sense that the interaction of human senses with complex mixtures provides a global perception rather than a list of compounds. Global perceptions may be enough in many cases to detect freshness or edibility, and ultimately they are of paramount importance to determine the acceptance of foodstuff [3]. A sort of combination of natural and analytical approaches has been pursued since decades, and it resulted in a class of chemical analyzers that have the advantage of interacting directly with samples and of providing signals bearing the notion of the chemical composition of a sample being a liquid or gas. These analyzers are chemical sensors. In the rest of this chapter an overview of the technologies related to these devices is provided together with examples of their use for fish freshness and quality determination.
11.2
Sensor Parameters
A sensor is an electronic device whose parameters depend on some external quantity of whatever nature [4]. As an example, according to this definition there are resistors whose resistance is a function of external temperature (thermistors) or diodes whose current–voltage relationship is strongly altered once they are illuminated by light (photodiodes). In the same way there are devices that from the electronic point of view are resistors, capacitors, or even field effect transistors, whose electrical parameters may depend on the chemical composition of the environment in which they are in contact. Electronic properties of materials may hardly be directly influenced by the ambiental chemistry; in order to achieve chemical sensors, a complex structure is necessary. Figure 11.1 shows what can be considered as the general structure of a chemical sensor. The device is composed of two parts. The first is a chemically interactive material, namely a solid-state layer of molecules that can interact with the molecules in the environment. These interactions can be of different nature, and the more utilized are adsorption and reaction phenomena. The interaction with a target molecule (hereafter called analyte) and a solid-state layer is a chemical event that, as a consequence, can modify the physical properties of the sensing layer. Properties such as conductivity, work function, mass, or optical absorbance are among those that can be transduced into an electric signal by suitable transducers. These transducers are the second component of a chemical sensor, and they are sometimes called “basic devices.” The matching between sensitive material and transducer is not univocal: a single sensitive material can be coupled with many different transducers and vice versa. In practice, there are many possibilities of assembling a chemical sensor. The optimal matching between a sensitive layer and transducer is fundamental to achieving a well-performing sensor.
Chemical Sensors
Environment
Chemically interactive material
◾
155
Quantity to be measured (concentration)
ΔT
Δm
Δσ
Δn
ΔΦ
Intermediate quantity
Basic device Δi
Δv
Δf
ΔΦ
Electrical or optical signal
Figure 11.1 Schematic representation of a generic chemical sensor. Targeted molecules interact with a chemically interactive material. As a consequence of the interaction, one or more physical properties of the interactive material change. These quantities can be the temperature (DT), mass (Dm), electric conductance (Ds), refraction index (Dn), or work function (DF). For each, and many others, of these quantities, there are a number of devices that, once properly connected in an electric circuit, provide an electrical signal that is a function of the quantity of interactions occurring at the interface between the sensor and the environment.
Before illustrating the technological basis of chemical sensors, it is important to introduce the fundamental parameters that allow a correct interpretation of the performance of any sensor. These parameters are sensitivity, resolution, and selectivity. The fundamental action of a chemical sensor is the conversion of the information about the concentration of a chemical species into an electric signal. The relationship between the signal and the chemical concentration can be represented by an analytical function that embodies the sensor operation. V = f (C ) where V is a generic signal C is the analyte concentration The knowledge of the response function is necessary to estimate from the sensor signal the amount of concentration. This estimation is straightforward if the response function is linear, and in more general cases, the estimation may require the solution of a nonlinear equation. Besides response function, other important quantities are necessary to be known to appreciate sensor performances [5]. One of these quantities is sensitivity. The sensitivity expresses the capability of a sensor to modify its signal as a consequence of a change in concentration. Analytically, it is the first derivative of the response function S=
dV df (C ) = dC dC
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Only in case of a linear response function, the sensitivity is a constant quantity. In all the other cases, it is a function of the concentration. Let us consider the generic case of a chemical sensor based on a sensitive material characterized by a limited number of adsorption sites. The amount of adsorbed molecules as a function of the concentration is ruled by the Langmuir isotherm [6]. The response curve is almost linear at low concentration and tends to saturation corresponding to the complete occupation of available adsorption sites. A sensor containing such a sensing material and a basic transducer simply providing a signal proportional to the number of adsorbed molecules is represented by the response curve shown in Figure 11.2a. In Figure 11.2b the corresponding sensitivity is shown. The sensitivity is larger at low concentrations, and it tends gradually to zero as the sensor response reaches saturation. In order to fully appreciate the importance of sensitivity, it is necessary to evaluate the influence of measurement errors. The knowledge of the signal V is affected by an error and this error is propagated in an error on the estimation of the concentration. Simple mathematical considerations lead to the conclusion that given an error ΔVerr affecting the signal V, the error ΔC on the estimated concentration is given by the following relationship: ΔC =
ΔVerr S
The error in concentration is then inversely proportional to the sensitivity. It is worth mentioning that in case of electrical signals, the error ΔV is limited by the electronic noise that determines the ultimate uncertainty of any electric signal. The previously mentioned quantities are totally general, and their importance holds for any kind of sensor. For chemical sensors, an additional parameter of great importance is selectivity. Selectivity defines the capability of a sensor to be sensitive only to one quantity rejecting all the others. In the case of physical sensors, the number of quantities is limited to a dozen, and the selectivity can be achieved in many practical applications. For chemical sensors, it is important to consider that the number of chemical compounds is in millions and that the structural differences among them may be extremely subtle. With these conditions, the selectivity of a chemical sensor can be obtained only in very limited conditions. Lack of selectivity means that the sensor responds with comparable intensity to different species, and with such a sensor, it is not possible to deduce
Saturation
Sensitivity
Signal
Nonlinear region
Linear region Concentration
Concentration
Figure 11.2 Typical response curve (left) and sensitivity (right) of a generic chemical sensor based on adsorption of target molecules in a sensing layer characterized by a limited amount of adsorption sites.
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any reliable information about the chemical composition of the measured sample. Selectivity will be reconsidered in Section 11.4.
11.3
Chemical Sensor Technologies
In this section the basic principles of the most popular categories of chemical sensors are illustrated. Sensors are here classified according to the physical intermediate quantity.
11.3.1 Sensors Based on Conductance Changes 11.3.1.1 Metal-Oxide Semiconductors Changes in conductance become appreciable in materials characterized by a limited number of mobile charge carriers. In practical, semiconductors are subjected to large changes of conductance also in the presence of a modest variation in the number of conductance electrons or holes. The most popular materials undergoing a conductance change on interaction with gases are metaloxide semiconductors. These are oxides of transition metals, the most known and studied of which is SnO2 [7]: a wide band gap n-type semiconductor. The main sensitivity mechanism is related to the role played by oxygen. At sufficiently high temperature (above 200°C), dissociative adsorption sites of molecular oxygen are active on the oxide surface. A charge transfer occurs between the material and the adsorbed oxygen atom with the consequence that the conductance band in proximity of the surface becomes depleted, and a surface potential barrier is formed. The amount of depletion and the barrier height are proportional to the number of adsorbed molecules. Since the material is a semiconductor, the number of conductance electrons is limited, and then the amount of oxygen molecules that can be adsorbed at the surface is also limited. The consequence of the exposure to oxygen is a reduction of the surface conductance. The exposure to any molecule interacting on the sensor surface with adsorbed oxygen atoms may result in a release of electrons back to the conductance band, a reduction of the surface conductance band depletion, and a lowering of the potential barrier. Paradigmatic, in this regard, is the case of carbon monoxide, which reacts with the bounded oxygen to form carbon dioxide, releasing an electron back to the conductance band. This is only one of the many interactions taking place on the surface of metal oxides, and the sensitivity of these devices is extended to many different kinds of volatile compounds [8]. The sensitivity can be further modified adding ultrathin amounts of noble catalytic metal atoms on the surface. It is important to remark that this kind of sensors needs to be operated at high temperature, and as a consequence, an electrically actuated heater is integrated in the device. Metal-oxide semiconductor sensors can be prepared in many different ways; in any case, the general advice is to produce a nanocrystalline material in such a way that the modulation of the surface conductance band population becomes dominant in the whole sensor, providing the maximum sensitivity. Recently, metal oxide growth in regular shapes such as nanosized belts [9] has shown peculiar properties. The characteristics of these structures, although interesting, have not yet resulted in practical improvements of performances. Metal oxide semiconductor chemoresistors have been used several times in fish freshness applications. For instance, the sensitivity to trimethylamine and dimethylamine of aluminum-doped ZnO films was demonstrated [10] as well as the sensitivity to trimethylamine of SnO2 and CuO [11,12].
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11.3.1.2 Conducting Polymers and Molecular Aggregates The conductance properties of organic materials based either on polymers or on molecular aggregates have been studied since several years, with broader scopes related to the possibility of developing a novel sort of electronics based on carbon chemistry [13]. Chemical sensors based on conducting polymers may be considered as a lateral result of these studies. Indeed, aggregates of polypyrrole or polythiophene have a semiconducting character, and their conductance can change after exposure to volatile compounds. With respect to metal oxides these sensors have two important advantages: they are operated at room temperature and, most important, their chemical sensitivity can be changed at synthesis level modifying the chemical structure of the monomer [14]. Thanks to this versatility, conducting polymers sensors can be prepared for different applications, and food freshness is among them [15]. One of the drawbacks of these sensors is the instability mainly due to the degradation of doping radicals that are added to increase the conductance.
11.3.2 Amperometric Gas Sensors Electrolytic cells based on either solid-state or liquid-ionic conductors are used to detect several kinds of gases. The main mechanism is the catalytic reaction occurring on the surface of a noble metal electrode. Although designed for polluting gases, these sensors demonstrated a good sensitivity for compounds relevant for fish freshness. For instance, sensors designed for CO are found to be sensitive toward alcohols, aldehydes, and esters; a sensor for ammonia can detect amines; and a sensor for SO2 can detect volatile sulfides. Due these cross-selectivities, these sensors were properly used to detect fish freshness [16].
11.3.3 Mass Transducers The adsorption of molecules into a sorbent layer produces a change of mass; the measurement of these mass shifts can allow the evaluation of the amount of adsorbed molecules. The measurement of small mass changes is made possible by piezoelectric resonators. A piezoelectric resonator is a piece of piezoelectric crystal properly cut along a well-specified crystalline axis. Due to the piezoelectric effect, the mechanical resonance of the crystal is coupled with an electric resonance. Since crystal resonance is extremely efficient, the electric resonance is characterized by a very large quality factor (Q). This property is largely exploited in electronics to build stable oscillators as clock references. The same effect is exploited for chemical sensing adopting particularly shaped crystals such as in quartz microbalances (QMB). These are thin slabs of AT cut quartz oscillating at a frequency between 5 and 50 MHz approximately [17]. The frequency of the mechanical oscillation decreases almost linearly with the mass gravitating onto the quartz surface. If the quartz is connected to an oscillator circuit, the electric frequency decreases linearly with the mass. A typical QMB has a limit of detection around 1 ng, an amount that is sufficient in many practical applications. QMB coated by sensitive layers was used for many applications. As an example, the possibility of using these sensors to measure fish freshness was demonstrated with metalloporphyrin coating [18]. Piezoelectric effect can also be exploited in other configurations such as those based on surface acoustic waves. More sophisticated mass transducers were proposed by using resonant cantilevers similar to those adopted in atomic force microscopy [19]. In spite of the claimed properties, these sensors were never demonstrated in practical applications.
Chemical Sensors
11.3.4
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Field-Effect Transistors
Most of the properties of field-effect transistors (FET) depend on the difference between the work function of electrons in the metal gate and in the semiconductor. This difference can be modulated by a layer of electric dipoles that can reach the metal–oxide interface. The principle was adequately exploited with a palladium gate FET exposed to hydrogen gas [20]. H2 molecules dissociate into atomic hydrogen at the palladium surface, and hydrogen atoms can diffuse through the palladium film until they reach the oxide surface, where they form an ordered dipoles layer. As a result, although under constant bias, the current flowing in the FET changes revealing the chemical interaction. This basic structure was successively modified changing the gate metal and thickness to extend the range of measured gases. In this way sensitivity to ammonia, an important gas for fish freshness and quality, was also obtained [21]. FET structures were also modified to accommodate, as a sensing part, organic molecular layers, such as metalloporphyrins [22], whose sensitivity toward amine was also recently measured [23].
11.3.5 Color Indicators Although known for several years [24], the colorimetric detection of fish freshness recently received a novel interest. In particular, the importance of amines as spoilage markers leads to consider their reducing role and then the possibility to detect them with functional layers sensitive to pH changes. The feasibility of this approach has been demonstrated using as sensitive layer a film of a sodium salt (bromocresol green) [25]. This salt exhibits a rather large change in color, also appreciable by eye. Nonetheless, the use of pH indicators is limited by the fact that mainly amines are considered (limiting the detection not to freshness but rather to spoilage), and, furthermore, the visual determination limits the performance and may greatly vary between individuals. Chemical sensing based on optical sensitive layers is a captivating strategy due to the strong influence of target chemicals on the absorption and fluorescence spectra of chosen indicators [26]. Nonetheless, the chemical practice of this approach is badly balanced by the transducer counterpart. Indeed, standard optical instrumentations are usually expensive. On the other hand, in the last decade we have seen rapid growth in performance in fields such as consumer electronics, giving rise to a number of low-cost advanced optical equipments such as digital scanners, cameras, and screens, whose characteristics largely fit the requirements necessary to capture change in optical properties of sensitive layers in many practical applications. The first demonstration in this direction was given by Suslick and colleagues when they showed that a digital scanner has enough sensitivity to detect the color changes in chemical dyes due to the adsorption of volatile compounds [27]. The method demonstrated also the possibility to identify a number of different amines [28]. Furthermore, Lundström and Filippini proved that it is possible to assemble a sort of spectrophotometer using the computer screen monitor as a programmable source and a web camera as detector [29]. This last technique, known as computer screen photo assisted technique (CSPT), is based on the fact that a computer screen can be easily programmed to display millions of colors, combining wavelengths in the optical range. Compared with the use of digital scanners, to probe the sample with a variable combination of wavelengths instead of using the white light of scanners gives the possibility of performing an optical fingerprint measurement, allowing a simultaneous evaluation of absorbance and fluorescence of samples. Due to the large diffusion of portable computers, PDAs, and cellular phones all endowed with color screen,
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camera, and an even more extended computation capabilities, the application of the CSPT concept may be foreseen as greatly extending the analytical capacity worldwide. CSPT has demonstrated its utility in particular to classify airborne chemicals reading absorbance and fluorescence changes in chemical dyes such as metalloporphyrins [30]. Standard optochemical sensors are based either on absorbance or on fluorescence, whereas CSPT arrangement gives the possibility of evaluating at the same time both the effects.
11.4 Electronic Noses As discussed above, the lack of selectivity of many chemical sensors was considered as one of the main problems limiting their diff usion for practical applications. Nonetheless, observation of Nature offered a useful suggestion about the use of such devices. Investigations about olfaction receptors show that Nature strategies for odor recognition are completely different from those of analytical chemistry. The physiology of olfaction has made considerable advances, models of receptor mechanisms explaining the sensitivity to volatile compounds are now available, and the genes expressed by olfactive receptors are known [31]. Recent studies are also beginning to unveil the signal pathways leading from the generation of olfactory neuron signal to the conscious identification of odors [32]. Receptors were found to be rather unselective; each receptor senses several kinds of molecules, and each molecule is sensed by many receptors [33]. After this discovery, it was proposed that arrays of nonselective chemical sensors may show properties similar to those of natural olfaction [34]. After this conjecture, the possibility of developing artificial olfaction systems became possible, and such systems were soon dubbed as “electronic noses.” This denomination is currently given to any array of unselective chemical sensor coupled with some multicomponent classifier. Since the 1980s, almost all sensor technologies were used to build such systems. Odor classification properties of artificial systems were tested on several different fields proving that electronic noses could be in principle used to replace human olfaction in practical applications such as food quality and medical diagnosis [35]. The features of electronic noses are fundamentally dependent on the sensing properties of the artificial receptors. The possibility having some versatile tool to tailor the sensitivity and selectivity of sensors is of primary importance to make arrays capable of capturing either large or narrow ranges of chemicals, allowing for electronic nose application oriented optimizations. To this point of view, organic synthetic receptors offer an unlimited number of possibilities to assemble molecules endowed with differentiated sensing features.
11.5 The Application of Electronic Noses for Fish Freshness and Quality Measurement The composition of fish headspace is a source of information about its freshness. Previous investigations evidenced that the headspace composition is a result of the balance between the “fresh fish” odor and the microbial spoilage produced compounds [36]. The most important chemicals involved in the fresh fish odor are long-chain alcohols and carbonyls, bromophenols, and N-cyclic compounds. Their concentration and the presence of other compounds are rather typical of each species. On the other side, microbial spoilage produces short-chain alcohols and carbonyls, amines, sulfur compounds, and aromatic, N-cyclic, and acid compounds. The concentrations of these chemicals are directly correlated to the degree of spoilage. Among these compounds amines
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are considered as the typical markers for fish freshness detection. Standard analytical methods for volatile amines and also sensors for some specific amines have been used to inspect fish freshness. Nevertheless, amines become instrumentally appreciable only when spoilage processes take place. Minor contributions to the fish headspace come from contamination of the environment (e.g., petroleum in sea), from fish processing, and finally from products of lipid oxidation [37]. The number of compounds whose concentrations are only partially correlated makes this application particularly appealing for sensor arrays of partially selective chemical sensors. Such sensor arrangement consists in the application of a number of sensors characterized by a broad sensitivity toward species that are relevant for a certain application. When properly analyzed by pattern recognition methods, the data produced by a sensor array can classify samples according to some of their global features; in case of fishes, typically according to the freshness or more precisely according to the balance between fresh and spoilage produced compounds. In recent years attempts to use electronic nose technology to track the spoilage processes occurring in fishes have been reported in numerous articles. Most of these are feasibility studies, showing the ability of the electronic nose to track the different spoilage levels occurring at different storage times. Instruments based on different sensor technologies have been used, such as metal-oxide chemoresistor sensors [38–40], MOSFET sensors [41], amperometric sensors [42], conducting polymer sensors [43], quartz microbalance sensors [44,45], and optical indicators [46]. In addition, hybrid electronic noses were used combining different sensor technologies such as QMB and amperometric sensors [47]. In order to understand the potential of electronic noses to detect fish freshness, let us discuss a simulation of a case study. In Figure 11.3 the time evolution of the major families of volatile compounds found in the headspace of fishes is shown. Data are extrapolated from an investigation by Strachan and Nicholson [48]. Let us consider the use of an array of sensors absolutely selective for each individual family of compounds mentioned in Figure 11.3. Each sensor then provides a signal proportional to the concentration of each family. Sensor data can be conveniently represented by a principal component analysis (PCA) scores plot. PCA is a data analysis method allowing the representation of a multidimensional dataset in a reduced dimensionality space, for example, a plane. The representation plane is determined as that where the data variance is maximized and then the statistical properties of the dataset are, as much as possible, preserved [49]. Results shown in Figure 11.4 demonstrate a continuous progress after the 8th day, but the behavior at the beginning is absolutely nonlinear, with a super impression of 6th and 1st days. Apparently, with an array of selective sensors it is not possible to distinguish between fresh and flat fishes. The sensitivity of chemical sensors is not immediately related to the molecular family but rather to the interaction mechanism. In this regard, it is more realistic to consider an array of sensors specific for a single interaction mechanism. An imperfect application of this method was demonstrated with engineered polymer-coated QMB [50]. In gas chromatography, the interaction between polymers and volatile compounds is often described by the linear sorption energy relationship (LSER) model [51]. In LSER, five different kinds of interactions are considered: dispersion, polarity, dipolarity, hydrogen bond basic, and acid. Since LSER was fruitfully used to model polymer-based chemical sensors [52], let us consider an array specific for each LSER interaction and one compound for family. Analyzing the data with PCA the plot of Figure 11.5 is obtained. As a result, the progress of spoilage is less linear with respect to Figure 11.4, and fresh, flat, and sweet conditions are hardly identified. This result is rather surprising because fish spoilage is in general expected to be a linear and somewhat straightforward process; nonetheless, the chemical complexity of the problem, the accumulation of some compound, and the decrease in others result in a nonlinear problem. The same nonlinearity is observed with electronic noses; Figure 11.6
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Figure 11.3 Time evolution of the major families of volatile compounds in fish headspace. The typical sensorial description is also reported.
shows the scores plot of a partial least-squares discriminant analysis model related to an array of metalloporphyrin-coated QMBs. The experiment was related to COD fishes, and original data were previously published [53]. Results are qualitatively similar to those shown in Figure 11.5, with a folding back of the spoilage process in the representation plane. This feature that can be interpreted as a failure of the electronic nose is likely due to an intrinsic nonlinearity of the studied problem. Nonetheless, humans provide a more reliable identification of fish freshness. It is important to consider that sensorial methods of freshness appraisal involve the use of sight (to evaluate the skin appearance and the color and the global aspect of eyes), tactile (to test the flesh firmness and elasticity), and olfaction (to smell the gill odor) [54], and the use of only one sense (e.g., olfaction) provides several errors of evaluation. As a consequence, in order to measure the quality of fish instrumentally, an integration of instruments is necessary, each able to capture different aspects of fishes. The fusion of multi-instrumental information can then be treated as the descriptors provided by a trained panel providing a sort of artificial quality index [55]. The possibility of developing a multisensor device to measure and/or estimate fish freshness with a combination of instrumental techniques (electronic noses, spectroscopic methods, texture meters, image analyzers, color meters, and devices measuring electrical properties) has been illustrated in different applications related to cods [56,57], sardines [58], and groupies [59].
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Scores plot 2 30 1.5 2 4 6
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Figure 11.4 PCA scores plot of a simulated experiment where sensors selective for the compound family in Figure 11.3 are used. Data show the impossibility of distinguishing the spoilage process in the first 6 days and an abrupt change between 6th and 8th days. Scores plot 1.5 10 12
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Figure 11.5 PCA scores plot of data related to a virtual array of sensors, each specific for a single interaction mechanism among those modeled by LSER.
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Figure 11.6 PCA score plot of metalloporphyrin-coated QMB; data are related to cod fish fillets, and labels indicate the storage days in ice.
11.6
Conclusions
The conversion of chemical information into electric signals that can be measured, stored, analyzed, transmitted and integrated with other data can be performed by several different technologies. These technologies are sometimes equivalent in terms of performances, and for some specific applications, one technology may outperform the others. It is important in any application to design the optimal sensor array to determine quality and quantity of the relevant chemical species and to select sensors optimizing sensitivity and resolution. In this regard, the application of arrays of sensors can greatly improve the performance in terms of prediction of quality and freshness. Chemical sensors are an almost mature technology for many practical applications. In the case of fish and seafood freshness and quality determination, all the actors of the food chain (producers, processors, and consumers) are potential users of chemical sensor technology. Each step of the food chain has peculiar needs that a proper chemical sensor approach can in principle contribute to satisfy. As an example, at producer level the increment in quality and yield, at processors level the screening of quality of incoming products to optimize the processing and to sort processed food, and finally at consumer level, the control of quality and safety both on the market and at home. All these applications require instruments able to work on-site. Food-related sites are usually highly contaminated from the point of view of odor. At the current state of the art, sensors are
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not able to distinguish between background and relevant odor, so that the performance of the sampling of an application is difficult. From this perspective, portable systems without any conditioning of sample are of limited use for fish inspection. Let us imagine, for instance, measuring the odor of a fish in a typical storage room among dozens of stacks of fish crates. On the other hand, there are applications, interesting at industrial level, where existing chemical sensors can be specialized, in terms of sampling and data presentations, in order to fulfill user requirement. For this a strong cooperation between sensor developers and end users is necessary in order to optimize practical solutions. At this level a correct and careful analysis of user needs and expectations and an education effort toward the users are important to disseminate the intrinsic novelty carried by sensor systems such as those widely belonging to the class of artificial olfaction. It is also important that developers and users are aware of the intrinsic limit of information that is carried by the volatile part of a food. For instance, it is important to consider that sensory analysis is almost never confined to only olfactory perception. Actually, synesthetic action among the senses is required to form a full judgment over a certain food sample. As an example, in fish analysis, quality index, linearly correlated with the days in ice, is calculated considering at the same time visual, tactile, and olfactory perceptions. This suggests that to fully reproduce the perceptions of humans with artificial sensors, the electronic nose has to be compared and integrated with instruments providing information about visual aspects, texture, and firmness. This opens a further novel investigation direction involving again researchers from different areas, confirming that interdisciplinarity is the most strong added value for food analysis.
References 1. Coultate, T.P. Food: The Chemistry of its Components, RSC Press, Cambridge, U.K., 2002. 2. Olafsdottir, G. et al. Methods to evaluate fish freshness in research and industry, Trends Food Sci. Tech., 87, 258, 1997. 3. Bremner, H.A. Toward practical definitions of quality for food science, Crit. Rev. Food Sci., 40, 83, 2000. 4. Fraden, J. Handbook of Modern Sensors, AIP Press, New York, 2004. 5. D’Amico, A. and Di Natale, C. A contribution on some basic definitions of sensors properties, IEEE Sens. J., 1, 183, 2001. 6. Alberty, R., Physical Chemistry, John Wiley & Sons, New York, 1982. 7. Madou, M. and Morrison, S. Chemical Sensing with Solid State Devices, Academic Press, San Diego, CA, 1989. 8. Barsan, N., Koziej, D., and Weimar, U. Metal oxide based gas sensor research: How to? Sens. Actuators B, 121, 18, 2007. 9. Comini, E. Metal oxide nano crystals for gas sensing, Anal. Chim. Acta, 568, 28, 2006. 10. Roy, S. and Basu, S. ZnO thin film sensors for detecting dimethyl- and trimethyl-amine vapors, J. Mater. Sci. Mater. Electron., 15, 321, 2004. 11. Egashira, M., Shimizu, Y., and Takao, Y. Trimethylamine sensor based on semiconductive metaloxides for detection of fish freshness, Sens. Actuators B, 1, 108, 1990. 12. Hammond, J. et al. A semiconducting metal-oxide array for monitoring fish freshness, Sens. Actuators B, 84, 113, 2004. 13. Heeger, A.J. Semiconducting and metallic polymers (Nobel lecture), Angew. Chem. Int. Ed., 40, 2591, 2001. 14. Persaud, K. Polymers for chemical sensing, Mater. Today, 8, 38, 2005.
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15. Du, W.X. et al. Microbiological, sensory, and electronic nose evaluations of yellowfin tuna under various storage conditions, J. Food. Prot., 64, 2027, 2001. 16. Olafsdottir, G., Martinsdóttir, E., and Jónsson, E.H. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus). J. Agric. Food Chem., 45, 2654, 1997. 17. Ballantine, D.S. et al. Acoustic Wave Sensors, Academic Press, San Diego, CA, 1997. 18. Brunink, J. et al. The application of metalloporphyrins as coating material for quartz microbalance based chemical sensors, Anal. Chim. Acta, 325, 53, 1996. 19. Battiston, F.M. et al. A chemical sensor based on a microfabricated cantilever array with simultaneous resonance-frequency and bending readout, Sens. Actuators B, 77, 122, 2002. 20. Lundstrom, I. et al. A hydrogen sensitive MOS field effect transistor, Appl. Phys. Lett., 26, 55, 1975. 21. Winquist, F. et al. Modified palladium metal-oxide semiconductor structure with increased ammonia gas sensitivity, Appl. Phys. Lett., 43, 839, 1983. 22. Andersson, M. et al. Development of a ChemFET sensor with molecular films of porphyrins as sensitive layers, Sens. Actuators B, 77, 567, 2001. 23. Takulapalli, B. et al. Electrical detection of amine ligation to a metalloporphyrin via a hybrid SOIMOSFET, J. Am. Chem. Soc., 130, 2226, 2008. 24. Tozawa, H., Enokihara, K., and Amano, K. Proposed modification of dyer’s method for trimethylamine determination in cod fish, Technical Conference on Fish Inspection and Quality Control, Halifax (Canada), 15–25 July, 1969. 25. Paquit, A. et al. Development of a smart packaging for the monitoring of fish spoilage, Talanta, 102, 466, 2007. 26. Gauglitz, G. Optical sensing looks to new field. Trends Anal. Chem., 25, 748, 2006. 27. Rakow, N. and Suslick, K. A colorimetric sensor array for odour visualization, Nature, 406, 710, 108, 705, 2000. 28. Rakow et al. Molecular recognition and discrimination of amines with a colorimetric array, Angew. Chem. Int. Ed., 44, 4458, 2005. 29. Filippini, D., Svensson, S., and Lundström, I. Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays, Chem. Commun., 240, 2003. 30. Filippini, D. et al. Chemical sensing with familiar devices, Angew. Chem. Int. Ed., 45, 3800, 2006. 31. Buck, L. and Axel, R. A novel multigene family may encode odorant receptors: A molecular basis for odor recognition, Cell, 65, 175, 1991. 32. Friedrich, R.W. and Stopfer, M. Recent dynamics in olfactory population coding, Curr. Opin. Neurobiol., 11, 468, 2001. 33. Sicard, G. and Holley, A. Receptor cell responses to odorants: Similiarities and differences among odorants, Brain Res., 292, 283, 1984. 34. Persaud, K. and Dodds, G. Analysis of discrimination mechanisms in the mammalian olfactory system using a model nose, Nature, 299, 352, 1982. 35. Röck, F., Barsan, N., and Weimar, U. Electronic nose: Current status and future trends, Chem. Rev., 108, 705, 2008. 36. Josephson, D., Lindsay, R., and Olafsdottir, G. Measurement of volatile aroma constituents as a means for following sensory deterioration of fresh fish and fishery products; in Seafood Quality Determination Symposium, D. Kramer, L. Liston (eds.), 10–14, November 1986, Elsevier, Amsterdam, the Netherlands, 1986. 37. Ólafsdóttir, G. and Fleurence, J. Evaluation of fish freshness using volatile compounds: Classification of volatile compounds in fish, in Methods to Determine the Freshness of Fish in Research and Industry, Proceedings of the Final meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. Nantes, November 12–14, 1997. International Institute of Refrigeration, 55–69. 38. Ólafsson, R. et al. Monitoring of fish freshness using tin oxide sensors, in Sensors and Sensory Systems for an Electronic Nose, Gardner, J.W., Bartlett, P.N., (eds.), Kluwer, Dordrecht, the Netherlands, 1992, p. 257.
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39. Schweizer-Berberich, P.M., Vaihinger, S., and Göpel, W. Characterisation of food freshness with sensor arrays, Sens. Actuators, 18, 282, 1994. 40. Ólafsdóttir, G. et al. Prediction of microbial and sensory quality of cold smoked Atlantic salmon (Salmo salar) by electronic nose, J. Food Sci., 70, 563, 2005. 41. Haugen, J.E. and Undeland, I. Lipid oxidation in herring fillets (Clupea harengus) during ice storage measured by a commercial hybrid gas-sensor array system, J. Agric. Food Chem., 51, 752, 2003. 42. Ólafsdóttir, G., Martinsdóttir, E., and Jónsson, E.H. Rapid gas sensor measurements to predict the freshness of capelin (Mallotus villosus), J. Agric. Food Chem., 45, 2654, 1997. 43. Du, W.X. et al. Potential application of the electronic nose for quality assessment of salmon fillets under various storage conditions, J. Food Sci., 67, 307, 2002. 44. Di Natale, C. et al. Recognition of fish storage time by a metalloporphyrins-coated QMB sensor array, Meas. Sci. Technol., 7, 1103, 1996. 45. Zhao, C.Z. et al. Assay of fish freshness using trimethylamine vapor probe based on a sensitive membrane on piezoelectric quartz crystal, Sens. Actuators B, 81, 218, 2002. 46. Alimelli, A. et al. Fish freshness detection by a computer screen photoassisted based gas sensor array. Anal. Chim. Acta, 582, 320, 2006. 47. Olafsdottir, G., Di Natale, C., and Macagnano, A. Measurements of quality of fish by electronic noses, in Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G., (eds.), Wageningen Academic Publishers, Wageningen, the Netherlands. 2003, 225. 48. Strachan, N. and Nicholson, F. Gill air analysis as an indicator of cod freshness and spoilage, Int. J. Food Sci. Tech., 27, 261, 1992. 49. Johnson, R. and Wichern, D. Applied Multivariate Statistical Analysis, Prentice Hall Inc., Englewood Cliffs, NJ, 1982. 50. Hierlemann, A. et al. Polymer based sensor array and mulicomponent analysis for the detection of hazardous organic vapours in the environment, Sens. Actuators B, 26, 126, 1995. 51. Grate, J. and Abrahams, H. Solubility interactions and the design of chemically selective sorbent coatings for chemical sensors and arrays. Sens. Actuators B, 3, 85, 1991. 52. Houser, E. et al. Rational materials design of sorben coatings for explosives: Applications with chemical sensors, Talanta, 54, 469, 2001. 53. Di Natale, C. et al. Comparison and integration of different electronic noses for freshness evaluation of cod-fish fillets, Sens. Actuators B, 77, 572, 2001. 54. Luten, J.B. and Martinsdottir, E. QIM an European tool for fish freshness evaluation in the fishery chain, in Methods to Determine the Freshness of Fish in Research and Industry, Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3 CT94 2283. Nantes, November 12–14, 1997. International Institute of Refrigeration, 287. 55. Di Natale, C. Data fusion in Mustec: Towards the definition of an artificial quality index, in Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability, Luten, J.B., Oehlenschlager, J., and Olafsdottir, G., (eds.), Wageningen Academic Publishers, Wageningen, the Netherlands, 2003, 273. 56. Olafsdottir, G. et al. Multisensor for fish quality determination, Trends Food Sci. Technol., 15, 86, 2004. 57. Kent, M. et al. A new multivariate approach to the problem of fish quality estimation, Food Chem., 87, 531, 2004. 58. Macagnano, A. et al. A model to predict fish quality from instrumental features, Sens. Actuators B, 111, 293, 2005. 59. Di Natale, C. et al. Ubiquitous chemical sensing and optical imaging for ubiquitous environments, IEEE International Conference on Robotics and Automation, Rome, April 10–14, 2007.
Chapter 12
Physical Sensors and Techniques Ruth De los Reyes Cánovas, Pedro José Fito Suñer, Ana Andrés Grau, and Pedro Fito-Maupoey Contents 12.1 Sensors for Quality Assessment .....................................................................................170 12.2 The Importance of Quality Control—Advances in the Online Control Techniques ....................................................................................................................170 12.3 New Technologies for Online Control ..........................................................................171 12.3.1 Ultrasounds—Acoustic Spectroscopy ..............................................................172 12.3.2 Visible Spectroscopy .........................................................................................173 12.3.3 IR Spectroscopy ...............................................................................................173 12.3.4 RF Spectroscopy—Impedance Spectroscopy ...................................................174 12.3.5 Microwave Spectroscopy—Dielectric Spectroscopy .........................................174 12.3.6 Advantages and Benefits of Microwave Methods .............................................175 12.4 Overview of Microwave Theory .................................................................................... 176 12.5 Applications of Microwave Technology in the Assessment or the Control of Processes ...................................................................................................................179 12.5.1 Determination of Moisture Content ................................................................180 12.5.2 Freshness and Salting/Desalting Process Quality Control of Fish and Seafood, by Microwaves: Methods and Equipments ........................................182 12.6 Conclusions ...................................................................................................................184 References ................................................................................................................................184
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12.1 Sensors for Quality Assessment A food quality sensor is a device that can respond to some physical or chemical property or properties of food and transform the response(s) into a signal, often an electric signal. This signal provides direct information about the quality factor(s) to be measured or may have a known relation to the quality factors. Usually, sensors are classified according to their mode of use: online, at-line, or off-line. Online sensors operate directly in the process, and they give a real time signal, which relates to the quality factors. Thus, an online sensor has the advantage of giving an immediate quality measurement and provides possibilities for regulating the process by adjustments. At-line sensors are devices to be used for instance in split-flow measurements, requiring reagent additions or equilibrations/reaction times. They often have short-response times (minutes or seconds) and also allow process corrections. Off-line sensors are laboratory devices, responding within hours or days. Traditionally the on/at-line quality control was restricted to external properties (weight, size, color, etc.) that can be measured by a simple balance or by a sophisticated video camera, and the internal properties were determined off-line by destructive and time-consuming technologies. This chapter tries to show the increasing growth of new and efficient online and at-line control methods that can provide important information about the internal quality of foods, focusing on the seafood sector advances.
12.2
The Importance of Quality Control—Advances in the Online Control Techniques
Quality control is essential in the food industry, and efficient quality assurance is becoming increasingly important. Since consumers expect good shelf life and high-safety products with an adequate ratio of quality–price, the food industry is progressively investing more and more capital in quality control, research, and development, as well as in machinery for the separation of products by their varying degrees of quality (i.e., the calibration lines for fruit processing). However, quality in food products is very difficult to define. Consumers perceive the quality of a product on the basis of a feeling of satisfaction that some sensory properties produce in them, such as color, taste, ease of consumption, or flavor. Th is perception is used to choose the product one wishes to buy. In this way, different physical and chemical parameters related to the quality of foodstuffs have been selected [1]. The acquisition of these parameters that characterize the abstract concept of “quality perceived by the consumer” leads to the development of the necessary technology for application in the classification of products. Existing techniques in food quality assessment, either instrumental or sensory evaluation, can provide reliable information about food quality; however, these techniques are destructive, timeconsuming, and unsuitable for online application. Because of that, traditionally, the development of in-line calibrators was restricted to external properties (weight, size, etc.). This was due to the absence of nondestructive technologies that would allow the product classification by its properties (internal properties). Therefore, quality control in manufacturing lines was limited to destructive off-line analyses that determine the acceptance or disposal of much of the production of the day, as a result of not being able to perform online nondestructive measures that would correct the manufacturing process in real time.
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New analytical techniques have been (and they are still being) developed to study the quality of complex food materials and to monitor the properties of foods during processing; these techniques can provide new quality control systems of the internal (and external) properties of foods that act in real time and in a nondestructive way. This kind of system not only permit an assessment of quality in terms of their properties but also, with the appropriate hardware and software, allow input from the manufacturing line with information obtained from the measurement of quality parameters selected (feedback); therefore it is possible to apply to the product under development the necessary corrective measures while it is still in the manufacturing line. Thus, it is able to obtain a final product that will always be within the margins of quality predetermined. These systems will reach three milestones, the new sensors’ concept of being easy-to-use, an excellence in accuracy, and low cost in the sensor’s compounds. In addition to the requirements of consumers, food inspectors require good manufacturing practices, safety, labeling, and compliance with the regulations. In this way many new food safety concepts and key quality parameters have arisen during the last decade: Hazard analysis critical control points (HACCP), total quality management (TQM), ISO 9000 Certifications, traceability, and authentication all require improved control methods. In addition, they all call for intime and online sensors for control, new data systems, warning systems, tight feedback loops for automation of the production, and so forth. Further, food producers are increasingly asking for efficient control methods, in particular through online or at-line quality sensors, first to satisfy the consumer and regulatory requirements and second to improve the production feasibility, quality sorting, automation, and reduction of production cost and production time (increased throughputs). The great challenge is indeed to focus on the real time and online sensors and data systems surveying processes and products, controlling the automated process and the raw material stream, sensing the final product quality, and typing the product labels, nutritional and health information, and much more. Concretely, the safety and quality of fishery products has been of particular concern in recent years. With the increasing globalization of fishery product sales, processors, consumers, and regulatory officials have been seeking improved methods for determining freshness and quality [2]. It is necessary to stress that fish quality is a complex concept involving a whole range of factors, which for the consumer include, for example, safety, nutritional quality, availability, convenience and integrity, freshness, eating quality, the obvious physical attributes of the species, size, and product type [3,4]. A study performed by Consumers Union found that more than one-quarter of the fish samples tested were on the brink of spoilage [5]. Information about handling, processing, and storage techniques, including time/temperature histories that can affect the freshness and quality of the products, is very important for the partners in the chain. One of the most unique characteristics of fish as food is that it is a highly perishable commodity. Consequently, time passed after catch and the temperature “history” of fish are very often the key factor determining the final quality characteristics of a fish product [6].
12.3 New Technologies for Online Control The quality of almost all the industrial processes depends on the modification of a few parameters, which are commonly structural, physical, or chemical properties, such as water content for drying processes. In general, these properties need slow and destructive methods to be controlled, but online methods are required for industrial quality control.
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Given the premise that online control requires a nondestructive method, which, moreover, must act in real time and without producing permanent effects on the food; it is almost imperative to resort to elastic (sonic) waves such as ultrasounds or to nonionizing electromagnetic radiation, such as radio frequency (RF), microwave, thermal and near-infrared (NIR), and visible. The interaction between wave radiation and matter as a function of wavelength or frequency is called spectroscopy. The spectroscopic techniques use the information found in the spectrum that is emitted for the food to predict certain of its qualities. It is also necessary to work at very low power in order to not cause permanent effects such as heating. When these waves pass through foods (or are refracted by them), some of their propagation parameters are modified. Thanks to advancing technology, the modification of these parameters can be measured in real time, fulfilling the initial premise. Normally the modification of any quality parameter is macroscopically correlated to the change in any wave parameter that can be controlled; below are cited some examples of the use of these new technologies in the quality control of foodstuffs. Ultrasonic velocity in fish tissues, chicken, and raw meat mixtures can be related to its composition using semiempirical equations [7]. Visible (and near UV) transmittance method has been investigated to inspect the internal quality (freshness) of intact chicken egg [8]; NIR measurements are widely used in the food industry to determine the sugar content in fruits [9]; impedance measurements (RF) can determine salt and water content in salmon filets [10]; and dielectric measurements at microwave frequencies can be used to analyze water activity [11] and water content [12,13] in foodstuffs; The salt and water content are related to dielectric properties of cod at microwave frequencies [14–16]. It is impossible to address all these techniques with precision, the reason is, in this chapter, we concentrate on electromagnetic methods at microwave frequencies. Nevertheless, the other techniques that enable online control have been briefly commented on below, exposing their main disadvantages and highlighting the advances in the field of seafood.
12.3.1 Ultrasounds—Acoustic Spectroscopy Ultrasonic is a rapidly growing field of research, which is finding increasing use in the food industry for the analysis of food products. Ultrasound is a form of energy generated by sound (really pressure) waves of frequencies that are too high to be detected by human ear, i.e., above 16 kHz [17]. Ultrasound attenuation spectroscopy (acoustic spectroscopy) is a method for characterizing properties of fluids and dispersed particles. Ultrasound imaging is a versatile, well-established, and widely used diagnostic tool. This technique encompasses a wide range of imaging modes and techniques that use the interaction of sound waves with living tissues to produce an image of the tissues or, in the case of Doppler-based modes, determine the velocity of a moving tissue. Ultrasound, when propagated through a biological structure, induces compressions and depressions of the medium particles, and a high amount of energy can be imparted. Depending on the frequency used and the sound wave amplitude applied, a number of physical, chemical, and biochemical effects can be observed, which enable a variety of applications [18,19]. Highfrequency, low-energy diagnostic ultrasounds are used as a nondestructive analytical technique for quality assurance and process control with particular reference to physicochemical properties such as composition, structure, and physical state of foods [20]. For fish samples, Suvanich et al. [21] published a report on how the ultrasonic velocity measurements show potential for analyzing fish composition. The main disadvantage of ultrasound is that the energy propagates poorly through a gaseous medium. It is virtually impossible for
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ultrasound to pass through air; thus, ultrasound transducers must have airless contact with the sample during examinations [22]. This complicates the noncontact measurements.
12.3.2 Visible Spectroscopy In recent years, the usefulness of visible spectroscopy/near infrared spectroscopy (VS/NIRS) has been researched for many quality aspects [23–25]. This technique measures the reflectance of light from the product in the visible and NIR wavelength range; the visible spectrum is a function of the entire structure of the compound rather than specific bonds. Other information should be used in conjunction with visible spectra in determining the specific properties of interest. For example, the freshness of cod was estimated by Heia et al. [26] using the visible wavelengths only; the main disadvantage of this method is that only the surface of the sample is examined.
12.3.3 IR Spectroscopy In the recent years, NIR technology has been widely developed as an analytical tool. NIR spectroscopy is based on the absorption of electromagnetic radiation at wavelengths in the range 780–2500 nm. NIR spectra of foods comprise broad bands arising from overlapping absorptions corresponding mainly to overtones and combinations of vibration modes involving C–H, O–H, and N–H chemical bonds [27]. This makes it very feasible for measurements to be made in organic and biological systems. Focusing on fish products, Uddin et al. [28] applied NIR spectroscopy to assess the end point temperature (EPT) of heated fish and shellfish meats; a multispectral imaging NIR transflectance system was developed for online determination of moisture content in dried salted codfish [29]. A rapid, NIR spectroscopic method has been developed by Zhang and Lee [30] to directly determine free fatty acids (FFA) in fish oil and for the assessment of mackerel quality. All these techniques have been gradually implemented as monitoring systems in food processing [31], but their use is limited by their low penetration in the product (it depends on the wave length, but it is measured in terms of tenths of a millimeter [32] and is dependent on less-precise reference methods [27]. The most popular IR spectroscopy is the NIR one, but it is not the only one. Mid-infrared (MIR) and Raman spectroscopy have high structural selectivity and contain more of the type of information needed in structural elucidation studies. MIR spectroscopy concerns the region of the spectrum lying between 4,000 and 400 cm−1 (2,500–25,000 nm). When radiation with energy corresponding to the MIR range interacts with a molecule, the energy at defined frequencies can be partially absorbed. The region of the electromagnetic spectrum under consideration in Raman spectroscopy is similar to that in MIR, but it involves a scattering process. Raman spectroscopy is based on the shift of an excited incident beam of radiation that results from inelastic interactions between the photons and the sample molecules. In the fish sector, Karoui et al. [33] applied MIR spectroscopy combined with chemometric tools to determine whether fish has been frozen–thawed. Marquardt and Wold [34] concluded that Raman spectroscopy might be a useful tool for rapid and nondestructive analysis of fish quality. Most industrial processes require the measurement of temperature. The far IR, which is also called thermal infrared (TIR) refers to electromagnetic waves with a wavelength of between 3.5 and 20 micrometers, and it is able to provide thermal information. Thermal infrared imagers translate the energy transmitted in the infrared wavelength into data that can be processed
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into a visible light spectrum video display. Thermography (infrared; thermal scans) uses specially designed infrared video or still cameras to make images (called thermograms) that show surface heat variations. This technology has a number of applications, for example, recent studies conducted by Fito et al. [35] lay the groundwork for the use of TIR image for the control of the optimum drying time in a citrus line. Focusing on fish industry, Jacobsen and Pedersen [36] developed a method based on infrared measurement of temperature changes in cold-water prawns during the glazing process studied in a small-scale controlled experiment. The method is thus remote and physically based on the heat transfer between prawns and glazing water.
12.3.4
RF Spectroscopy—Impedance Spectroscopy
Radio frequency is an electromagnetic radiation within the range of 3 Hz to 300 GHz. This range corresponds to the frequency of alternating current electrical signals used to produce and detect radio waves. Different techniques have been developed for quality control based on the response of foods to waves in the RF region. The technique called “bioelectrical impedance analysis” (BIA) is highly effective for measuring human body composition such as fat content, lean muscle, or total water [37] and nutritional status [37,38] and there is abundant supporting literature from medical studies demonstrating the effectiveness of the approach. This technique works at 50 kHz and is also an accurate predictor of the composition of fish [39,40] as the amount of water or proportion of fat tissue to lean tissue is correlated to BIA measurements through regression equations built on multiple measurements of control groups [41]. Impedance spectroscopy measures the dielectric properties (see Section 12.4) of a “food material” as a function of frequency; this term usually applies to the range of RF frequencies, sometimes extended to low microwaves. Impedance spectroscopy has been widely used to estimate the physiological state of various biological tissues [42,43]. In studies of a biological tissue, it is of great importance to establish an appropriate equivalent circuit model to relate the measured data to the physical and physiological properties. A number of spectroscopic methods in RF have been used quite recently to measure the quality-determining properties of frozen fish [44,45]. Haddock muscle showed significant changes in its dielectric properties during rigor mortis at frequencies between 1 Hz and 100 kHz [46]. In quality control of fish, the principal method of data analysis of impedance results has been to calculate indices with the measurements conducted at one or two frequencies [44,47]. With living tissues and in the postmortem period, impedance data have been analyzed by regression at each measured frequency and at several selected frequencies, by Cole-Cole analysis, and so on [48], but multivariate techniques of data analysis are still not widely used. The main disadvantages of RF for online monitoring are related to the physical size of its hardware, which is very voluminous and difficult to manage; moreover, interactions with metals and other materials can be problematic, and ionic conduction effects (i.e., due to dissolved salts) are highly significant (masking other effects).
12.3.5 Microwave Spectroscopy—Dielectric Spectroscopy The actual state of art of microwave technology permits measuring in real time and in a nondestructive way most of the parameters that are related to quality control. For instance, in the late
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sixties, microwave sensors emerged as a plausible solution for real-time, nondestructive sensing of moisture content in a variety of materials [49–51]. Moreover, in recent years, the price of microwave components has dropped drastically because of a surge in demand from the wireless telecommunications sector. This, with new developments in solid-state and planar circuit technologies, provides an opportunity to develop reasonably priced microwave/RF sensors. Therefore, the application of microwave technologies to food quality control is a growing interest for the industry. Until recently, the interest of the food industry in microwave applications had been fi xed mainly in dielectric heating. These applications appeared in the years following the end of the Second World War, but the development of microwaves stopped due to technological reasons and the high cost of investment. At the beginning of the 1980s, the possibilities of microwave applications and their considerable advantages were recognized, and microwave ovens become more popular. This increase in the use of domestic microwave ovens gave rise to a reduction in the cost of the relatively high-power magnetron. However, the cost of these elements increases exponentially when the power is on an industrial scale [35]. Presently, domestic microwave ovens are universally accepted by consumers, and other microwave heating applications are widely used in industry; baking, drying, blanching, thawing, tempering, and packaging are the most important. Therefore, considerable experience has now been accumulated in this field and can be used in the design of sensor systems based on microwaves. These sensors are viable and affordable for online control in food industrial processes. Dielectric spectroscopy measures the dielectric properties (see Section 12.4) of a “material” as a function of frequency; this term usually applies to the range of microwave frequencies, sometimes extended to high RF. Dielectric spectroscopy is considered to be a very useful tool in food quality determinations, because, as will be explained in Sections 12.4 and 12.5, dielectric properties of biological tissues are closely correlated with water content and the aggregation state of it. Furthermore, the dielectric properties depend not only on water binding in foods but also on its composition. The interplay between molecular composition, presence of ions, electrical charges on proteins, and pH variations leads to a complex dielectric spectrum regulated by several phenomena. Dielectric properties are also related to structure, and the structural organization and composition of a muscle makes it a highly anisotropic dielectric material. This dielectric anisotropy was modeled by Felbacq et al. [52] to provide insight into microwave–muscle interactions. It tends to decrease during ageing or process-related cellular degradation. The main theoretical aspects of microwaves are treated in Section 12.4. In Section 12.5 some interesting applications of microwave technology in quality control are cited.
12.3.6 Advantages and Benefits of Microwave Methods A very important benefit of microwave sensing is that the bulk property (i.e., moisture or density) is determined, in contrast to surface determination provided, for example, with infrared (IR) or NIR techniques. This is particularly important in monitoring operations, for example, drying, where moisture gradients exist in the material; variations in moisture can exist within a few microns of the surface, but their effects are substantially reduced or insignificant at microwave frequencies. Another decided advantage is logistical flexibility in installation. With a wide variety of sensors from which to choose, placement can be on conveyors or in hoppers, shakers, pipes,
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chutes, and so on. Installation is generally minimally intrusive. Moreover, results can be obtained almost in real time, because the measurement time ranges from a few milliseconds to one second. A further advantage is that microwave radiation is noncontaminating and environmentally safe at power levels typically used for online sensing. Human exposure is usually less than that from common consumer electronic devices such as cordless and cellular telephones. Finally, microwave sensors are insensitive to environmental conditions such as dust, color, or ambient light, vapors, and machine vibrations, in contrast to IR and NIR techniques.
12.4
Overview of Microwave Theory
Microwaves are a common designation for electromagnetic waves at frequencies between 300 MHz and 300 GHz. These waves travel through the free space with a given energy (E) and propagation parameters, which are mainly magnitude (A) and phase (q). When they find a different “dielectric material” (in this case, food), one part of the radiation is refracted and another one passes through it (see Figure 12.1). The amount of radiation refracted or transmitted by food as well as its new propagation parameters are governed by the dielectric properties of the material. Therefore, the measurement of these properties allows both the characterization of food and the control of the process (see Figure 12.1). In the communications argot, “materials” are usually divided into the categories of conductors, insulators, and dielectrics. “Dielectric materials” cover the whole spectrum of anything between conductors and insulators. Therefore, dielectrics can consist of polar molecules or nonpolar molecules, or very often both. According to this classification, foods are “dielectric materials” (or really an addition of dielectric materials) susceptible to be defined by their dielectric properties. Complex permittivity (e r) (Equation 12.1) is the dielectric property that describes food behavior under an electromagnetic field [53].
E1, A1, θ1
, θ3 E 3, A 3
E1, A1, θ1
, θ5 E 5, A 5
Material permittivity εr1 = ε΄r1 –j.ε˝r1 Natural or industrial process
Modified material permittivity εr2 = ε΄r2 –j.ε˝r2
E2, A2, θ2
Product characterization
E4, A4, θ4 Processes control (or monitoring)
Figure 12.1 Scheme of the possibilities of the measurement of dielectric properties in quality control applications.
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The real part of complex permittivity is called the dielectric constant (e′), and the imaginary ′′ ). The subscript r indicates that values are related to part is called the effective loss factor ( ε eff vacuum, and the variable is therefore dimensionless: ′′ εr = ε ′ − j ε eff
(12.1)
Under a microwave field, the charges of certain food components (water, salts, etc.) try to displace from their equilibrium positions to orientate themselves following the field, storing microwave energy that is released when the applied field stops. This behavior is called polarization; e′ denotes the material’s ability to store this electromagnetic energy (or the ability to be polarized). Only a ′′ is perfect dielectric can store and release wave energy without absorbing it. The parameter ε eff related to absorption and dissipation of the electric energy from the field. Such energy absorptions are caused by different factors that depend on structure, composition, and measurement ′′ can be expressed by Equation 12.2 [53]: frequency, thus ε eff ε ′eff = ε ′′d + ε ′′MW + ε ′′e + ε ′′a + σ/ε o ω
(12.2)
In this equation the last term is called ionic losses. The symbols s, e o, and w refer to material conductivity, vacuum permittivity, and angular frequency, respectively. Subscripts d, MW, e, and a indicate dipolar, Maxwell–Wagner, and electronic and atomic losses, respectively. The different contributing mechanisms to the loss factor of a moist material are schematically represented in Figure 12.2.
ε˝
+ i
+
+ –
+ –
–
–
+ –
+ – + + –
dw
MW a e
da
1.8E10 3E8 3E11 Radio frequency Microwaves IR AC L–M–K wave VHF dm
cm
mm
μm
log f (Hz) 3E14 V
UV
nm
Figure 12.2 Schematic representation of the different effects that contribute to effective loss factor (e″e ff ) along the electromagnetic spectrum (logarithmic scale). i, ionic losses; MW, Maxwell–Wagner effect; dw, dipolar losses of water; da, dipolar losses of isopropyl alcohol; a, atomic losses; e, electronic losses.
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Under a microwave field, molecules with an asymmetric charge distribution (permanent dipoles such as water) rotate trying to align themselves with the electric field, storing part of the wave energy [54]. The dipolar contribution to total losses is one of the most important at microwave frequencies due to the fact that water is an abundant and common component in foods. Otherwise, as frequency is increased (the highest microwave frequencies and above them), the electromagnetic field can affect smaller particles, inducing dipoles even in neutral molecules (atomic polarization) and neutral atoms (electronic polarization). Atomic and electronic losses have behavior similar to that of permanent dipolar losses. At RF and the lowest microwave frequencies, charged atoms and molecules (ions) are affected by the field. Such ions move trying to follow the changes in the electric field. In case ions do not find any impediment (aqueous solutions, conducting materials), ionic conductivity gives rise to an increment in effective losses. At these frequencies, the ionic losses are the main contributors to the loss factor (supposing ions to be present in the material). Foods are complex systems and usually present conducting regions surrounded by nonconducting regions, for example, foods with a cellular structure have cytoplasm (conducting region) surrounded by the membrane (nonconducting region). In these cases, ions are trapped by the interfaces (nonconducting regions) and, as the ion movement is limited, the charges are accumulated, increasing the overall capacitance of the food [55] and the dielectric constant (Maxwell– Wagner Polarization). This phenomenon is produced at low frequencies at which the charges have enough time to accumulate at the borders of the conducting regions. The Maxwell–Wagner losses curve vs. frequency has the same shape as the dipolar losses curve (see Figure 12.2). At higher frequencies, the charges do not have enough time to accumulate and the polarization of the conducting region does not occur. At frequencies above the Maxwell– Wagner relaxation frequency, both ionic losses and the Maxwell–Wagner effect are difficult to distinguish due to the fact that both effects exhibit the same slope (1/f ). Foods are multicomponent and multiphase systems; therefore, more than one mechanism contributes to the combined effects. Figure 12.3 shows different shape variations in effective loss factor curves vs. frequency for the case of combined dipolar and ionic losses. Type_0 represents a typical pure dipolar loss factor curve (without ionic contribution), s increases between type_0 and type_4 curves (the corresponding ionic contribution is marked in discontinuous trace), ε d″ max is the highest value of dipolar losses, and relaxation frequency is the inverse of relaxation time [53,16]. In general, foods are dielectric materials with high losses and, under a microwave field, they can absorb part of the wave energy. The power that can be dissipated in a given material volume ′′ by Equation 12.3, in which E is the electric field strength [53]: (Pv) is related to ε eff Pv = 2π f ε0 ε eff ·E 2 (W/cm3 )
(12.3)
The high-power dissipation in foods has given rise to numerous high-power heating applications that have been developed since the fi fties. The interest in improving heating applications has provided a great deal of knowledge on dielectric properties and wave parameter measurements. Th is detailed knowledge has been very useful in further research into new lowpower online sensors, which relate these properties or parameters to process variables of food industry.
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ε˝ 4
3 σ/ωε0 +
+ –
+ –
2
+ –
+
– +
– 1 εd˝ 0 log ( f )
Figure 12.3 Influence of salt content in systems with different proportions of dipoles (water) and ions (salts) in the shape of effective loss factor curve. Salt content increases in curves from 0 (water) and 4 (saturation). (Adapted from De los Reyes, R. et al., Medida de propiedades dieléctricas en alimentos y su aplicación en el control de calidad de productos y procesos, ProQuest (Ed.), 2007.)
12.5 Applications of Microwave Technology in the Assessment or the Control of Processes The applications of electromagnetic radiation in the microwave band are varied and cover broad fields, from the radar [56] and radiometry [57], to medical applications, such as the diagnosis of breast cancer [58] and other image applications. In addition, industrial applications have been developed, such as rubber vulcanization [59], soils, wood, and animal products disinfection [60–62], or food processing [63,64]. They are so many that some frequency bands have been reserved especially for industrial, scientific, and medical applications (ISM). These frequencies are detailed in Table 12.1. Microwave applications that are better known within the food industry are related to energy absorption and, therefore, are made at high power and usually at 2.45 GHz, which is the frequency often reserved in Europe for industrial applications. These applications are mainly used for heating, pasteurization, sterilization, dehydration, thawing, and scalding [65–67]. Recently, the application of microwaves in combination with warm air in drying of foods has been also studied, either during the whole drying process or in part of it [68,69]. Within this field, applications to the drying of fruits and vegetables are notable for their interest to the food industry [70,71]. However, as noted above, the development of the technology that brings this large number of applications has allowed the onslaught of new applications such as the assessment or the control of
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Wave Longitude (cm)
433.92 ± 8
69.14
915 ± 13
32.75
2,450 ± 50
12.24
5,800 ± 75
5.17
24,125 ± 125
1.36
processes by microwaves in a nondestructive way (MNDT or MNDE) which is receiving a growing interest in the food industry. In these applications, very low power is used to avoid permanent effects in foods. As a result of that, the methods for determining dielectric properties have experienced a spectacular expansion within the field of the analysis of materials by microwaves, which until relatively recently, was exclusively associated with the design of electronic equipment. As has been explained before, the measurement of the dielectric properties can provide important information during industrial processes due to the relationships between food properties and electromagnetic parameters. This is because low-power microwaves change their parameters (amplitude, phase) according to the food properties, and this change can be measured in real time. This is the basic principle on which food-quality microwave sensors are based. Complex permittivity can be correlated with structural, physical, and chemical properties such as humidity, soluble solids content, porosity, characteristics of solid matrix, and density [16]. The changes in these properties are usually related with the treatments applied to foods throughout the industrial process; for instance, water losses in drying processes [72] or salt losses in desalting processes [14,15]. In addition, the structural changes produced in macromolecules, such as protein denaturalization, can occur during processing, leading to a modification of the dielectric properties [73]. For all these reasons, the measurement of dielectric properties can be used as a tool for online food process control. This section provides an overview of the most important microwave applications as techniques in food control.
12.5.1
Determination of Moisture Content
Water represents the main component of foods influenced by microwave energy and, therefore, nowadays most methods of determining moisture content are based on electrical properties. The determination of moisture based on electromagnetic parameters has been used in agriculture for at least 90 years and has been in common use for 50 years [12,74,75]. Diverse studies have been carried out relating the dielectric constant and loss factor with moisture in foods [76,74]. Further researches in this field have occurred during recent years. Trabelsi and Nelson [77] studied a method of moisture sensing in grains and seeds by measuring their dielectric properties. The reliability of the method was tested for soybean, corn, wheat, sorghum, and barley. The frequency used was 7 GHz with the free space technique. In the same year, the authors used the same technique at 2–18 GHz to determine the dielectric properties of cereal grains and oilseeds in order to predict the moisture content by microwave measurements [78]. This article presents a unified
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grain moisture algorithm, based on measurements of the real part of the complex permittivity of grain at 149 MHz using the transmission line method. Trabelsi and Nelson [79] reported the moisture in unshelled and shelled peanuts using the free space method at a frequency of 8 GHz. In 2005, Joshi [80] reported a technique for online, time domain, nondestructive microwave aquametry (US Patent numbers 6,204,670 and 6,407,555); this technique was used for determining moisture levels in substances such as seeds, soil, tissue paper, and milk powder. Plaza-González et al. [81] have published a report about a microwave sensor intended for online measurements of paper moisture. Since most efforts have been directed to the moisture determination of different materials, commercial meters for online moisture measurements have already been developed. These moisture meters are based on automatic online calculations of the reflected wave and dielectric permittivity, yielding physicochemical properties, such as moisture, chemical composition, and density, without affecting the product. For instance, Keam Holdem® Industry (Auckland, New Zealand) provides online moisture testing and analyzing systems. This manufacturer provides devices for measuring moisture in processed cheese, moisture and salt in butter, moisture and density in dried lumber and whole kernel grain, and fat-to-lean ratio in pork middles. A microwave moisture meter has also been developed for continuous control of moisture in grains, sugar, and dry milk in technological processes [82]. A consortium of companies from different countries, Microradar®, produces a commercial microwave moisture meter for measuring moisture in fluids, solids, and bulk materials based on this method. The enterprise KDC Technology Corporation (www.kdctech.com) provides microwave sensors for monitoring industrial processes and quality control. KDC sensors work in a wide range of applications such as monitoring moisture and density of manufactured wood and wood-based products, construction, and agricultural and processed food products. Patented contact (MDA1000) and noncontact (MMA-2000) sensors are used for online, continuous process monitoring of solids, particulates, and liquids or for in situ nondestructive testing/inspection. Another interesting application for online moisture measurement is a sensor for green tea developed by Okamura and Tsukamoto [72], which can measure moisture as high as 160%–300% on dry basis by use of microwaves at 3 GHz with a microstripline (Figure 12.4). A Guided Microwave Spectrometer (Thermo Electron Corporation, Waltham, MA) has been developed for online measurements of multiphase products. This guide is used to measure Microwave source Receiver Microstripline
Tea leaves
Electric field
Figure 12.4 Schema of a microstripline used for tea leaves moisture measurement. (Adapted from Okamura, S. and Tsukamoto, S., New sensor for high moist leaves in green tea production, in Proceedings of ISEMA 2005, Kupfer, K. (Ed.), MFPA an der Bauhaus-Universität Weimar, Weimar, Germany, 2005, 340–346.)
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moisture in raw materials such as corn, rice, soybeans, and in processed materials such as tomato paste and ground meat. It can also measure content of soluble solids, pH, viscosity, and acidity in orange juice, soft drinks, mayonnaise, and tomato products; fat in ground meats, peanut butter, and milk and other dairy products; salt in mashed potatoes and most vegetable products and, lastly, alcohol in beverages.
12.5.2
Freshness and Salting/Desalting Process Quality Control of Fish and Seafood, by Microwaves: Methods and Equipments
The dielectric properties of fish products have been measured by different authors [83–86]; nevertheless, the electromagnetic determination of quality parameters in muscle tissues is still a complex challenge due to its complex matrix, heterogeneous composition, and anisotropic disposition. It is important to point out that the limitation of most dielectric probes is the volume of the sample that interacts with the field. The volume has to be representative of the whole piece of fish, due to the fact that the electromagnetic parameters in this kind of tissue vary in a heterogeneous way. It has been reported that it is possible to predict the fat composition in fish using electromagnetic measurements [87]; this is because it is clearly related to the water content of the product, so that if one is known the other can be determined; this is the knowledge base of the “Torrymeter” mentioned later. Moreover, this author [88,89] has studied the determination of added water in fish using microwave dielectric spectra measurements. Measurements of dielectric properties have been tested and used during almost 40 years for quality grading and remaining shelf life determination of various fish. These investigations have been mainly focused on freshness and self-life evaluation and detecting fishes previously thawed. However, a number of research studies have been carried out to control or monitor the processing of fish products. In this field, De los Reyes et al. [14,15] verified the viability of an online measurement system using low-power microwaves to determine the desalting point of salted cod. Dielectric spectroscopy was performed on cod samples at different desalting stages and on its desalting solutions in order to find the appropriate measurement frequency. Figure 12.5 shows the dielectric spectra (e′ and e″) from cod loin samples (2 cm/side parallelepipeds) at desalting times (t) yielding from 15 min to 48 h. Optimum frequencies were selected from the spectrum, and dielectric properties data were related to other physicochemical properties of cod samples measured at the same desalting stages, such as moisture and salt content. Good correlations were found between salt content in cod samples and their loss factor values at 200 and 300 MHz. These results indicated the viability of developing an online control system for a cod desalting process. Polarimetric measurements, that is, with a linearly polarized electric field, make it possible to evaluate anisotropy. This method has been applied to assess fish freshness [90]. This is because, after death, muscle is not able to use energy by the respiratory system. Postmortem changes lead to a temporary rigidity of muscles, decreasing the water-holding capacity [91]. The level of glycogen stored in the animal at the time of slaughter affects the texture of the future marketed meat. For all these reasons, during rigor mortis the dielectric properties are expected to change. The “Intellectron Fishtester” [92], the “Torrymeter” (Distell.com), and the “RT-Freshtester” (RT rafagnatækni), represent instruments with increasing degrees of sophistication invented for fish-quality evaluation. Readings from all these instruments are based in the reflected dielectric properties of fish, because they decrease with storage time, almost following a straight line. Based on these rapid and nondestructive measurements, the “RT-Freshtester” allows automatic grading of 60–70 fish per min. Nevertheless, electrical properties of fish are not directly responsible for
Physical Sensors and Techniques
ε΄, ε˝ 800
0.2 GHz 0.3 GHz
0.9 GHz 1.8 GHz 2.45 GHz
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183
10 GHz
700 600 500
ε˝ t
400 300 200 100
ε΄
t
0 1E + 08
1E + 09
1E + 10
Frequency
Figure 12.5 Dielectric spectra from cod samples at desalting times (t) yielding from 15 min to 48 h. The arrows beside t indicate the growth of the desalting time. Frequency axis is in the logarithmic scale, and broken lines mark the selected frequencies (0.2, 0.3, 0.9, 1.8, 2.45, and 10 GHz). (Adapted from De los Reyes, R. et al., Dielectric spectroscopy studies of “salted cod-water” systems during the desalting process, in Proceedings of the IMPI’s 40th Annual Symposium, 2006.)
sensory spoilage and it is, therefore, to be expected that numerous factors influence the relationship between such measurements and seafood spoilage. In fact, these instruments need calibration depending on the season and fish handling procedures, and they are unsuitable for grading frozen–thawed fish, partially frozen, that is, superchilled fish, fish chilled in refrigerated seawater, or for fish fillets. This and the high cost of the instruments limit their practical use in the seafood sector for freshness evaluation. However, electrical measurements can also be used to test if fish was previously frozen [2]. Kent et al. [93] studied the effect of storage time and temperature on the dielectric properties of thawed–frozen cod (Gadus morhua) in order to estimate the quality of this product. The same year, Kent et al. [94] developed a combination of dielectric spectroscopy and multivariate analysis to determine the quality of chilled Baltic cod (Gadus morhua). These researches yielded a prototype developed by SEQUID [95,96] for measuring and analyzing the quality of different seafood. The SEQUID project concentrated on the measurement of the dielectric properties of fish tissue as a function of time both in frozen and chilled storage. This project has shown that it is possible, using a combination of time domain reflectometry and multivariate analysis, to predict certain quality-related variables, both sensory and biochemical, with an accuracy comparable to existing methods. Kent et al. [97] have also reported a way to determine the quality of frozen hake (Merluccius capensis) by analyzing its changes in microwave dielectric properties. The above mentioned “Torrymeter” has been successfully improved as a sensor for measuring fish freshness as a result of these investigations. In further investigations, the SEQUID project has shown that it is possible to predict certain quality-related variables (with comparable accuracy to existing methods) using a combination of time-domain reflectometry at microwave and RF frequencies and multivariate analysis [98].
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Conclusions
It is possible to implant reliable online sensors in fish industry both for determining the freshness as well as for monitoring processes (salting/desalting, thawing, etc.). The future of control in fish processing is the analysis of the physical and chemical properties using the dielectric signal at different frequencies, using multisensors. Multivariable knowledge of the process yields a modeling of the product.
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40. Duncan, M., Craig, S.R., Lunger, A.N., Kuhn, D.D., Salze, G., and McLean, E. Bio-impedance assessment of body composition in cobia Rachycentron canadum (L. 1766). Aquaculture, 271, 432– 438 (2007). 41. Barbosa-Silva, M. and Barros, A. Bioelectric impedance and individual characteristics as prognostic factors for post-operative complications. Clin. Nutr., 24, 830–838 (2005). 42. Cole, K.S. Electric phase angle of cell membranes. J. Gen. Physiol., 15, 641–649 (1932). (Full Text via CrossRef.) 43. Damez, J.-L., Clerjon, S., Abouelkaram, S., and Lepetit, J. Dielectric behavior of beef meat in the 1 kHz to 1500 kHz range. Simulation with the Fricke/Cole–Cole Model. Meat Sci., doi: 10.1016/j. meatsci.2007.04.028 (2007). 44. Yu, T.H., Liu, J., and Zhou, Y.X. Using electrical impedance detection to evaluate the viability of biomaterials subject to freezing or thermal injury. Anal. Bioanal. Chem., 378, 1793–1800 (2004). 45. Vidačeka, S., Medića, H., Botka-Petrakb, K., Nežakc, J., and Petraka, T. Bioelectrical impedance analysis of frozen sea bass (Dicentrarchus labrax). J. Food Eng., 88, 263–271 (2008). 46. Martisen, O.G., Grimnes, S., and Mirtaheri, P. Noninvasive measurements of post-mortem changes in dielectric properties of haddock muscle–A pilot study. J. Food Eng., 43, 189–192 (2000). 47. Hennings, C. The “Interelectron Fish Tester V”–A new electronic method and device for the rapid measurement of the degree of freshness of “wet” fish. In: The Technology of Fish Utilization, R. Kreutzer, ed., Fishing News Ltd., London, U.K., pp. 154–157 (1964). 48. Thomas, B.J. Ward, L.C., and Cornish, B.H. Bioimpedance spectrometry in the determination of body water compartments: Accuracy and clinical significance. Appl. Radiat. Isotopes, 49, 447–455 (1998). 49. Taylor, H.B. Microwave moisture measurements. Ind. Electron., 3, 66–70 (1965). 50. Kraszewski, A. Microwave Aquametry, IEEE Press, Piscataway, NJ (1996). 51. Busker, L.H. Microwave moisture measurement, I & CS, 41, 89–92 (1968). 52. Felbacq, D., Clerjon, S., Damez, J.L., and Zolla, F. Modeling microwave electromagnetic field absorption in muscle tissues. Eur. Phys. J.–Appl. Phys., 19(1), 25–27 (2002). 53. Metaxas, A.C. and Meredith, R.J. Industrial Microwave Heating, IEE Power Engineering series 4, Peter Peregrinus Ltd., London, U.K. (1993). 54. Datta, A.K. and Anantheswaran, R.C. Handbook of Microwave Technology for Food Applications, eds., Datta, A.K. and Anantheswaran, R.C., Series of Food Science and Technology, Marcel Dekker, New York (2001). 55. Hewlett-Packard. Basic of measuring the dielectric properties of materials. Application note 1217–1. Hewlett-Packard Company, Palo Alto, CA (1992). 56. De los Reyes, E., Imágenes radar para el estudio de superficies agrícolas, 113, Dcbre. 1981, pp. 111–116 (1981). 57. Sempere, L. Radiometría interferométrica de microondas para la monitorización del contenido en humedad del suelo. Tesis doctoral de la Universidad Politécnica de Valencia. Director Elías De los Reyes (1999). 58. Fear, E.C., Hagness, S.C., Meaney, P.M., Okoniewski, M., and Stuchly, M.A. Enhancing Breast tumor detection with Near-Field Imaging. IEEE Microwave Magazine, 3(1), 48–56 (2002). 59. Catalá-Civera, J.M., Sánchez-Hernández, D., and y de los Reyes, E. Rubber vulcanisation for the footwear industry using microwave energy in a pressure-aided cavity. International Conference on Microwave Chemistry, Prague, Czech Republic (1998). 60. Plaza, P.J., Zona, A.T., Sanchís, R., Balbastre, J.V., Martínez, A., Muñoz, E.M., Gordillo, J., and de los Reyes, E. Microwave disinfestation of bulk timber. J. Microwave Power E.E., 41(3), 21–36 (2007). 61. Zona, A.T., Balbastre, J.V., Nuno, L., de los Reyes, E., Calderon, O., Perez, E., and Vivancos, M.V. Procedure to exterminate woodworm in wood timbers by microwave-power application. In Proceedings of Global Congress on Microwave Energy Applications GCMEA 2008 MAJIC 1st (2008). 62. WO/2005/009122. Microwave method of controlling mites In A Food Product Of Animal Origin (2005).
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63. Catalá-Civera, J.M. and de los Reyes, E. Enzyme inactivation analysis for industrial blanching applications: Comparison of microwave, conventional and combination heat treatments on mushroom polyphenoloxidase activity. ed., Acs., J. Agric. Food Chem., 47, 4506–4511 (1999) (ISSN 0021-8561). 64. Andrés, A., Bilbao, C., and Fito, P. Drying kinetics of apple cylinders under combined hot air-microwave dehydration. J. Food Eng., 63, 71–78 (2004). 65. Schiffmann, R.F. Microwave processes for the food industry. In: Handbook of Microwave Technology for Food Applications, Datta, A.K., and Anantheswaran, R.C., Cap. 9, 299–337. Marcel Dekker, Inc., New York (2001). 66. Anon, G. Tempers frozen fish blocks inside a cold storage warehouse, Quick frozen foods, 43(11), 64 (1981). 67. Ohlsson, T. Industrial uses of dielectric properties of foods. In: Physical Properties of Foods. 2. COST 90bis final seminar proceedings. eds., Jowitt, R., Escher, F., Kent, M., McKenna, B., and Roques, M., Elsevier Applied Science. London, U.K., pp. 199–211 (1987). 68. Catalá-Civera, J.M. Combined Microwave and air drying of apple (var. Granny Smith). In Proceedings of European Research towards Safer and Better Food, 74, 383–387 (1998). 69. Martín, M.E., Fito, P., Martínez-Navarrete, N., and Chiralt, A. Combined air-microwave drying of fruit as affected by vacuum impregnation treatments. In Proceedings of the 6th Conference of Food Engineering (CoFE’99), 465–470 (1999). 70. Bilbao, C, Albors, A, Gras, M.L., Andrés, A., and Fito, P. Shrinkage during apple tissue air-drying: macro and microstructural changes. Proceedings of the 12th International Drying Symposium IDS2000, Paper No. 330 (2000). 71. Sharma, G.P. and Prasad, S. Drying of garlic (Allium sativum) cloves by microwave-hot air combination. J. Food Eng., 50(2), 99–105 (2001). 72. Okamura, S., Tsukamoto, S. New sensor for high moist leaves in green tea production. In Proceedings of ISEMA 2005, ed., Kupfer, K., pp. 340–346. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 73. Bircan, C. and Barringer, S.A. Determination of protein denaturation of muscle foods using dielectric properties, J. Food Sci., 67(1), 202–205 (2002). 74. Nelson, S.O. Dielectric properties of agricultural products–Measurements and applications. Digest of Literature on Dielectrics, ed. A. de Reggie. IEEE Trans. Electr. Insul., 26(5), 845–869 (1991). 75. Nelson, S.O. Dielectric properties measurement techniques and applications. Trans. ASAE, 42(2), 523–529 (1999). 76. Nelson, S.O. Radio frequency and microwave dielectric properties of shelled corn. J. Microwave Power, 13, 213–218 (1978). 77. Trabelsi, S. and Nelson, S.O. Universal Microwave Moisture Sensor. In Proceedings of ISEMA 2005, ed., Kupfer, K., pp. 232–235. MFPA an der Bauhaus-Universität Weimar. May 29–June 1, Weimar, Germany (2005). 78. Trabelsi, S. and Nelson, S.O. Microwave dielectric properties of cereal grain and oilseed. In Proceedings of the American Society of Agricultural Engineers, St. Joseph, MI, Paper No. 056165 (2005). 79. Trabelsi, S. and Nelson, S.O. Microwave dielectric methods for rapid, nondestructive moisture sensing in unshelled and shelled peanuts. In Proceedings of the American Society of Agricultural Engineers, St. Joseph, MI, Paper No. 056162 (2005). 80. Joshi, K. High resolution, non-destructive and in-process time domain aquametry for FMCG and other products using microstrip sensors. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 384–390. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 81. Plaza-González, P.J., Canós, A.J., Catalá-Civera, J.M., and Peñaranda-Foix, F. Microwave non-contact sensor for on-line moisture measurement of laminate paper. International Conference on Sensor Technologies and Applications, pp. 52–55 (2007). 82. Lisovsky, V.V. Automatic Control of Moisture in Agricultural Products by Methods of Microwave Aquametry. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 375–383. MFPA an der BauhausUniversität Weimar. May 29–June 1, Weimar, Germany (2005).
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83. Kent, M. Microwave dielectric properties of fish meal. J. Microwave Power, 7, 109–116 (1972). 84. Kent, M. Complex permittivity of fish meal: A general discussion of temperature, density, and moisture dependence. J. Microwave Power, 12, 341–345 (1977). 85. Wu, H., Kolbe, E., Flugstad, B., Park, J.W., and Yongsawatdigul, J. Electrical properties of fish mince during multifrequency ohmic heating. J. Food Sci., 63, 1028–1032 (1988). 86. Zheng, M., Huang, Y.W., Nelson, S.O., Bartley, P., and Gates, K.W. Dielectric properties and thermal conductivity of marinated shrimp and channel catfish, J. Food Sci., 63, 668–672 (1998). 87. Kent, M. Hand-held instrument for fat/water determination in whole fish, Food Control, 1, 47–53 (1990). 88. Kent, M., MacKenzie, K., Berger, Knöchel, R., and Daschner, F. Determination of prior treatment of fish and fish products using microwave dielectric spectra. Eur. Food Res. Technol., 210, 427–433 (2000). 89. Kent, M., Knöchel, R., Daschner, F., and Berger, U. Composition of foods including added water using microwave dielectric spectra, Food Control, 12, 467–482 (2001). 90. Clerjon, S., and Damez, J.L. Microwave sensing for food structure evaluation. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 357–364. MFPA an der Bauhaus-Universität Weimar. May 29–June 1, Weimar, Germany (2005). 91. Hullberg, A. Quality of Processed Pork. Influence of RN genotype and processing conditions, P.H.G, Swedish University of Agricultural Sciences, Uppsala, Sweden (2004). 92. Oehlenschläger, J. The intellectron fishtester VI an almostforgotten powerful tool for freshness/spoilage determination of fish on inspection level. 5th World Fish Inspection & Quality Control Congress, The Hague, the Netherlands, 20.10.–22.10 (2003) 93. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Knöchel, R., Daschner, F., and Schimmer, O. Estimation of the quality of frozen cod using a new instrumental method. Eur. Food Res. Technol., 219, 540–544 (2004). 94. Kent, M., Oehlenschlager, J., Mierke-Klemeyer, S., Manthey-Karl, M., Knöchel, R., Daschner, F., and Schimmer, O. A new multivariate approach to the problem of fish quality estimation. Food Chemistry, 87, 531–535 (2004). 95. Knöchel, R., Barr, U.K., Tejada, M., Nunes, M.L., Oehlenschläger, J., and Bennink, D. Newsletter of the SEQUID (Seafood Quality Identification) project. European Commission Framework Programme V Quality of Life and Management of Living Resources RTD Project QLK 1-200101643 (2004). 96. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Albrechts, C., Oehlenschläger, J., Mierke-Klemeyer, S. et al. Intangible but not Intractable: The prediction of food ‘quality’ variables using dielectric spectroscopy. In Proceedings of ISEMA 2005, ed. Kupfer, K., pp. 347–356. MFPA an der Bauhaus-Universität Weimar, Weimar, Germany (2005). 97. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Tejada, M., Huidobro, A., Nunes, L., Batista, I., Martins, A. Determination of the quality of frozen hake using its microwave dielectric properties. Int. J. Food Sci. Technol., 40, 55–65 (2005). 98. Kent, M., Knöchel, R., Daschner, F., Schimmer, O., Oehlenschläger, J., Mierke-Klemeyer, S., Kroeger, M. et al. Intangible but not intractable: The prediction of fish ‘quality’ variables using dielectric spectroscopy. IOP Publ. Meas. Sci. Technol., 18, 1029–1037 (2007).
Chapter 13
Methods for Freshness Quality and Deterioration Yesim Ozogul Contents 13.1 Introduction ..................................................................................................................190 13.2 Sensory Methods ...........................................................................................................190 13.2.1 The European Union Freshness Grading (EU or EC Scheme) ..........................191 13.2.2 The Quality Index Method ..............................................................................191 13.2.3 The Torry Scheme ............................................................................................192 13.2.4 The Quantitative Descriptive Analysis .............................................................192 13.3 Physical Methods ..........................................................................................................194 13.3.1 Texture Analysis ...............................................................................................194 13.3.2 The Torrymeter ................................................................................................194 13.3.3 The Intellectron Fischtester VI .........................................................................195 13.3.4 The RT-Freshtester ...........................................................................................195 13.3.5 The Cosmos .....................................................................................................195 13.3.6 Electronic Nose ................................................................................................196 13.3.7 Near-Infrared Reflectance Spectroscopy...........................................................196 13.4 Chemical and Biochemical Methods .............................................................................197 13.4.1 ATP and Its Breakdown Products ....................................................................197 13.4.2 Biogenic Amines ..............................................................................................199 13.4.3 pH....................................................................................................................199 13.4.4 Total Volatile Basic Nitrogen........................................................................... 200 13.4.5 Trimethylamine .............................................................................................. 200 13.4.6 Dimethylamine ................................................................................................201 189
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13.4.7 Formaldehyde ..................................................................................................201 13.4.8 Lipid Oxidation Indicators ...............................................................................201 13.4.9 Lipid Hydrolysis .............................................................................................. 203 13.5 Microbiological Methods ............................................................................................. 203 References ............................................................................................................................... 204
13.1
Introduction
Seafood is generally considered to be a high-protein food, low in fat and saturated fat when compared with other protein-rich animal foods. It is well known that fish oil is the major and the best source of polyunsaturated fatty acids (PUFA), called omega-3 fatty acids, especially eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Scientific evidence suggests that omega-3 fatty acids are essential for normal growth and development throughout the life cycle and inhibit the formation of atherosclerotic plaques, prevent arrhythmias, and contribute to the prevention or amelioration of autoimmune disorders, Crohn’s disease, breast, colon and prostate cancers, rheumatoid arthritis, and particularly cardiovascular diseases [1–6]. The Nutrition Committee of the American Heart Association recommends consumption of any type of fish two or three times a week. Therefore, it is important to prevent their loss due to oxidation. Freshness is the most important attribute when assessing the quality of seafood and is of great concern in the seafood sector [7]. The quality of seafood degrades after death due to the chemical reactions [changes in protein and lipid fractions, the formation of biogenic amines and hypoxanthine (Hx)] and microbiological spoilage. As a result of these events, sensory quality of seafood deteriorates [8–13]. Seafoods are rich in PUFAs, which are susceptible to lipid oxidation. It leads to the development of off flavor and off odors in edible oils and fat-containing foods called oxidative rancidity [14,15]. Because of their high degree of unsaturation, they are less resistant to oxidation than other animal or vegetable oils [14]. This chapter summarizes methods used for evaluation of freshness and spoilage of seafood. As it is well known, no single instrumental method is reliable for assessment of the freshness and spoilage of seafood. However, chemical, microbiological methods along with sensory methods have been applied by commercial seafood companies and many researchers to ensure that the seafood products meet expectations of consumers. The current regulation of the European Community (1996) establishes principles based on sensory, chemical, and microbiological analysis to control and certify the quality warranty in the seafood field (Council Regulation No.: 2406/96). The shelf life of fish is affected by many factors such as handling, storage condition from catch to the consumers, the kind of fishing gear, bleeding, gutting methods, season, catching ground, age, and life cycle of fish affecting the nutritional quality, freshness, and safety of seafood. Therefore, estimation of remaining shelf life of fish should be made with caution [7].
13.2
Sensory Methods
Sensory evaluation is the most important method in freshness assessments. Sensory evaluation is defined as the scientific discipline used to evoke, measure, analyze, and interpret reactions to characteristics of food as perceived through the senses of sight, smell, taste, touch, and hearing [16]. Sensory evaluation provides rapid measurements of freshness of seafood. There has been a trend to standardize sensory evaluation as an objective assessment of freshness. Sensory characteristics of
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whole fish are clearly visible to consumers, and sensory methods are still the most satisfactory way of assessing the freshness quality since they give the best idea of consumer acceptance [17]. Freshness declines as storage life progresses until the product is no longer acceptable to consumers. The most appropriate method to assess freshness is a sensory panel. There are many factors affecting the measurement of sensory quality, including the sample under investigation, the assessment method, and the judges [18]. There are two types of sensory methods, subjective and objective. Subjective assessments of fish have been used for acceptability. They are often estimated generally using adjectives such as like/dislike or good/bad, which require subjective decisions. Fish freshness is most commonly determined by objective scoring based on organoleptic changes that occur as fish storage time is extended [19]. Objective scoring schemes require trained, expert judges, but the advantage is that panels can be small. These assessors individually use their appropriate senses (sight, smell, taste, and touch) to determine the level of each sensory characteristic in the defined grade standard appropriate for the seafood examined [20]. Subjective assessment, where the response is based on the assessor’s preference for a product, can be applied in the fields like market research and product development where the reaction of consumers is needed. Assessment in quality control must be objective [16]. Assessors must be trained and have clear and descriptive guidelines and standards to get reliable results for sensory analyses [21]. Sensory methods are also fast and nondestructive unless fish is cooked.
13.2.1
The European Union Freshness Grading (EU or EC Scheme)
The EU Freshness Grading was introduced for the first time in the Council Regulation No. 103/76 (for fish) and 104/76 (for crustaceans) and updated by decision No. 2406/96 (for some fish, some crustaceans, and only one cephalopod, the cuttlefish). The EU scheme is commonly accepted in the EU countries for freshness grading to market fish within the Union and generally carried out by trained personnel in auctions. Whole and gutted fish are assessed in terms of appearance of skin, eyes, gills, surface slime, belly cavity, odor, and texture of fish. There are four quality levels in the EC scheme, E (extra), A (good quality), B (satisfactory quality), where E is the highest quality and below level B (called Unfit or C) is the level where fish is discarded or rejected for human consumption. However, there are still some disadvantages; trained and experienced persons are required, since the scheme uses only general parameters for iced fish [16,22,23]. It does not take differences between species into account. In addition, it does not give information on the remaining shelf life of fish. A suggestion for renewal of the EU scheme can be seen in the Multilingual Guide to EU Freshness Grades for Fishery Products [24], in which special schemes for some fish species (whitefish, dogfish, herring, and mackerel) were developed.
13.2.2 The Quality Index Method The quality index method (QIM) has been suggested as an alternative to the EU scheme. The QIM, originally developed by the Tasmanian Food Research Unit in Australia [25] and improved further, is considered to be rapid and reliable to measure the freshness of whole fish stored in ice [21,22]. This method is based on significant sensory parameters (skin, slime, eyes, belly, odor, gills, etc.) for raw fish [25,26], and the characteristics listed on the sheet are assessed and appropriate demerit point score is recorded (from 0 to 3). The scores for all characteristics are summed to give the overall sensory score. Quality index (QI) is close to 0 for very fresh fish, whereas higher scores are obtained as the fish deteriorates [16,26]. There is a linear correlation between the sensory
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quality expressed as a demerit score and storage life on ice, which makes it possible to predict remaining storage life on ice. During spoilage, a higher score can be given for a single parameter [27]. This method is considered to be a relatively fast, nondestructive method based on direct observation of sensory parameters of fish and can also be specific for species. In addition, the QIM is suitable for early stage of storage of fish where other instrumental methods are not accurate [28]. Hyldig [29] indicated that the QIM is expected to become the leading reference method for the assessment of fresh fish within the European community. QIM Eurofish published a manual [21] containing QIM schemes for 12 fish species and information about how to use the QIM schemes (QIM-Eurofish 2004). The advantages of QIM are that it requires short training, is rapid and easy to perform, and is nondestructive and can be used as a tool in production planning and quality warranty work [27]. Rapid PC-based QIM is also available on the Internet at http://www.dfu.min.dk/QIMRS/qim_0202.htm. QIM Rating system software was developed for cod, herring, saithe, and redfish by the Danish Institute for Fisheries Research. Recently developed QIM schemes were presented for raw gilthead sea bream (Sparus aurata) [30], farmed Atlantic salmon (Salmo salar) [31], fresh cod (Gadus morhua) [32], common octopus (Octopus vulgaris) [33], herring (Clupea harengus) (Table 13.1) [34], brill, haddock, plaice, pollock, redfish shrimp, sole, and turbot (Scopthalmus rhombus, Melanogrammus aelefigus, Pleuronectes platessa, Pollachius virens, Sebastes mentella marinus, Pandalus borealis, Solea vulgaris, and Scopthalmus maximus, respectively) [21].
13.2.3
The Torry Scheme
In contrast to the QIM, the Torry Scheme was developed at the Torry Research Station for use with expert and trained judges. The most comprehensive scoring scheme to assess fish is the Torry Scheme [36]. It has been widely used in its original or modified forms. The Torry Scheme, often referred to as the Torry scale, is a descriptive 10-point scale and has been developed for lean, medium fat, and fat fish species. In this scheme, panelists evaluate the odor and flavor of cooked fillets. The scores are given from 10 (very fresh) to 3 (spoiled) (Table 13.2). The average score of 5.5 may be used as the limit for consumption [21].
13.2.4
The Quantitative Descriptive Analysis
Quantitative descriptive analysis (QDA) is used by a trained sensory panel to analyze the sensory attributes of products such as texture, odor, and flavor. QDA provides a detailed description of all flavor characteristics in a qualitative and quantitative way. The method can also be used for texture. The trained panel is handed a broad selection of reference samples and use the samples for creating terminology that describes all aspects of the product [16]. Descriptive words should be carefully selected, and the panelists trained should agree with the terms. Objective terms should be used rather than subjective terms. In QDA, the words for describing the odor and flavor of the fish can be categorized into two groups, positive and negative sensory parameters based on whether fish are fresh fish or fish at the end of the storage period [37]. Objective sensory methods are essential for quality control and estimation of shelf life of seafood. However, sensory methods are time consuming, expensive, trained personnel required, and not always practical for large-scale commercial purposes. Therefore, instrumental methods are also needed to satisfy the need for quality measurements in fish industry.
Methods for Freshness Quality and Deterioration Table 13.1 QIM Scheme for Sensory Evaluation of Herring Quality Parameter Whole fish
Appearance of skin
Blood on gill cover
Texture on loin
Texture of belly
Odor
Eyes
Appearance
Shape
Gills
Color
Odor
Description
Score
Very shiny
0
Shiny
1
Matte
2
None
0
Very little (10%–30%)
1
Some (30%–50%)
2
Much (50%–100%)
3
Hard
0
Firm
1
Yielding
2
Soft
3
Firm
0
Soft
1
Burst
2
Fresh sea odor
0
Neutral
1
Slight off odor
2
Strong off odor
3
Bright
0
Somewhat lusterless
1
Convex
0
Flat
1
Sunken
2
Characteristic red
0
Somewhat pale, matte, brown
1
Fresh, seaweedy, metallic
0
Neutral
1
Some off odor
2
Strong off odor
3
Sources: Modified by Jónsdóttir, S., Quality Standards for Fish: Final Report Phase II, Nordic Industrial Fund (in Danish), pp. 37–59, 1992; developed by Nielsen, D. and Hyldig, G., Food Res. Int., 37, 975, 2004. With permission.
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Table 13.2 Torry Score Sheet for Freshness Evaluation of Cooked Cod Fillets Odor
Flavor
Score
Initially weak odor of sweet, boiled milk, starchy, followed by strengthening of these odors
Watery, metallic, starchy. Initially no sweetness but meaty flavors with slight sweetness may develop
10
Shellfish, seaweed, boiled meat
Sweet and meaty characteristic
9
Loss of odor, natural odor
Sweet and characteristic flavors but reduced in intensity
8
Wood shavings, wood sap, vanillin
Neutral
7
Condensed milk, boiled potato
Insipid
6
Milk jug odors, reminiscent of boiled clothes
Slight sourness, trace of “off” flavors
5
Lactic acid, sour milk, TMA
Slight bitterness, sour, “off” flavors, TMA
4
Lower fatty acids (e.g., acetic or butyric acids) decomposed grass, soapy, turnip, tallow
Strong bitterness, rubber, slight sulfide
3
Source: Shewan, J.M. et al., J. Sci. Food Agric., 4, 283, 1953. With permission.
13.3
Physical Methods
13.3.1 Texture Analysis Texture analyses for seafood are extremely important in research, quality control, and product development in the seafood industry [38]. Fish muscle may become soft or mushy as a result of autolytic degradation or tough as a result of frozen storage [16]. Fish muscle has higher levels of indigenous proteases, which immediately begin to break down the proteins after the harvesting, during processing, improper handling storage, and cooking [39,40]. Texture includes the most common characteristics such as hardness, springiness, and chewiness of food. Among textural attributes, hardness is the most important to the consumer, deciding the commercial value of the meat [41]. Numerous mechanical methods have been used to measure texture; however, there is little agreement on which is the best method [42].
13.3.2
The Torrymeter
The Torry fish freshness meter “Torrymeter” was developed at Torry Research Station in Aberdeen, Scotland. Dielectric properties of fish are used for determination of freshness. Dielectric properties of fish skin and muscle alter in a systematic way during spoilage as tissue components degrade. These changes occurring at microscopic level are related to alterations in appearance, odor, texture, and flavor during spoilage and have been used as quality indicators since the first commercial version of the Torrymeter in 1970 [43]. A linear relationship was found between Torrymeter readings and sensory attributes for cod, Baltic herring, hake, blue whiting, flounder, mackerel,
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whole, iced gilthead sea bream, and farmed Senegalese sole [43–49]. However, Gelman et al. [50] found that the Torrymeter readings obtained from six species of different origin were poorly correlated with sensory evaluation. Inácio et al. [51] also studied the effect of washing with tap and treated seawater on the quality of whole scad (Trachurus trachurus) and found that Torrymeter and RT-Freshmeter readings were significantly (P < 0.05) lower in fish washed with seawater than fish washed with tap water or unwashed. This could be explained by seawater containing ions, which interfere with the reading of both instruments as they are based on electrical properties of skin. The skin of fish could be affected by osmolarity and contact with electrically charged particles [51]. Fat also has an effect on the dielectric properties of fish and tends to make observed Torrymeter values more variable [47]. The loss of skin and muscle integrity and deterioration of the skin caused by bruising during harvesting and packing operations would result in more variable Torrymeter values.
13.3.3 The Intellectron Fischtester VI The basic principles of Torrymeter (the United Kingdom) and the Intellectron Fischtester VI (Germany) are similar, measuring the electric properties (resistance, conductivity, and capacitance) of the fish flesh [52]. The electric properties of fish can change after death of the fish due to disruption of the cell membranes by autolysis. The method is based on conduction through skin and, therefore, works only on whole fish and fillets with skin on. Mechanical abuse and freezing can affect the readings. The Intellectron Fischtester VI gives reliable information about the days in ice and left of iced stored fish. It has also reported that there is a linear correlation between the instrument readings obtained on the day of harvest/catch and the date of spoilage [53]. The Fischtester readings can be used as an objective criterion for the state of freshness/spoilage together with sensory data across the fish chain.
13.3.4
The RT-Freshtester
Like Torrymeter and the Intellectron Fischtester VI, RT-Freshtester reflects dielectrical properties of fish, and readings from all instruments decrease with storage time. RT-Freshtester, fast and nondestructive, allows automatic grading of 60–70 fish/min. However, these instruments need calibration depending on sample preparation, season, fishing grounds, and fish-handling procedures. They are unsuitable for frozen or thawed fish, partly frozen such as superchilled fish, and fish chilled in refrigerated seawater [54].
13.3.5
The Cosmos
The “Cosmos” instrument developed by Japanese is applied for the evaluation of fish quality by determination of smell intensity. Like other instruments, the “Cosmos” instrument is handheld, portable, as well as rapid and nondestructive. Therefore, it could be used for evaluation of fresh and chilled fish in the seafood industry and on fishing vessels. Gelman et al. [50] found strong correlation between the organoleptic and Cosmos results for six species of fish and concluded that application of the “Cosmos” instrument for objective quantitative evaluation of fresh and chilled fish quality by determination of smell intensity appears to be practicable.
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Electronic Nose
Odor, the main indicator of fish freshness, has been analyzed by sensory panel or gas chromatography (GC). Since these kinds of analyses are both time consuming and expensive, an electronic nose called FreshSense was developed and distributed by Element-Bodvaki in Iceland and has been found to be a rapid, nondestructive method to measure volatile compounds, indicating spoilage of odors in seafood. FreshSense is based on a closed, static sampling system and electrochemical gas sensors, which are sensitive to volatile compounds. The most important chemicals involved in fresh fish odors are long-chain alcohols and carbonyls, bromophenols, and N-cyclic compounds. However, short-chain alcohols and carbonyls, amines, sulfur compounds, and aromatic, N-cyclic, and acid compounds are produced by microbial activity and lipid oxidation during storage of fish [55,56]. The concentrations of these compounds are related to the degree of spoilage. Different electronic noses have been employed for measurement of fish freshness. These are metal-oxide semiconductor gas sensors, electrochemical sensors (CO, H2O, NO, SO2, and NH3), thickness shear mode quartz resonators, semiconductor dimethylamine (DMA) gas sensor, and prototype solid-state–based gas sensor called the FishNose [57–62]. Olafsdottir et al. [63] studied the freshness of iced redfish and found that there was a good correlation between the response of CO sensor and QIM method for both air and modified atmosphere storage of redfish. Trggvadottir and Olafsdottir [64] also found that the response of all electronic sensor (CO, H2O, NO, SO2, and NH3) results for haddock from different seasons showed a similar trend. Studies on cod fillets and heads also gave similar results, and it was found that CO sensor showed the highest response [65]. It has been indicated that a combination of electronic nose systems based on different sensor technologies improved the performances compared with the single technology for the codfish fillets [66]. Fish freshness has also been evaluated by a computer screen photoassisted technique (CSPT)based gas sensor array. This technique is based on the fact that a computer screen can be easily programmed to show millions of colors, combining wavelengths in the optical range [56]. Previous optics-based electronic noses relied on absorbance and fluorescence. However, CSPT evaluates both effects [56,67–71]. Data analysis is important in electronic nose measurements, which determines the relation between sensor output patterns and the properties of the sample being analyzed [72]. The most frequently used methods are artificial neural networks (ANNs), chemometric analysis such as principal component analysis (PCA), and partial least-square regression (PLS-R).
13.3.7 Near-Infrared Reflectance Spectroscopy Near-infrared reflectance spectroscopy has been used in various analytical applications. The technique is characterized by speed and simplicity; it has the ability to measure numerous samples within a short time; it can be operated on-/at-line; and it is nondestructive, easy to handle, and requires little training of operators [73]. This method has been applied for determination of fat, water, and protein content in fish [74–78], free fatty acid (FFA) in fish oils [79,80], water-holding capacity of thawed fish muscle [81], and quality assessment of frozen minced red hake [82], cod caught by long line and gillnet [73], and thawed, chilled modified atmosphere packed (MAP) cod fillets [83]. Fourier transform infrared (FT-IR) spectroscopy is another technology that is a rapid, nondestructive, online industrial production chain. On the other hand, it requires too much handling of samples, causing changes in protein and muscle structure. Compared with FT-IR, diff use reflectance infrared Fourier transform (DRIFT) spectroscopy has advantages; that is, it is fast,
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its use is simple, sensitive, cheap, and requires a small amount of sample. For the first time, this technique has been applied to sardine muscle during iced storage, and it has been indicated that this spectroscopic technique is useful in assessing the freshness and quality of sardine during iced storage [84]. Traceability is becoming a method of providing safer food supplies and of connecting producers and consumers. Traceability can be defined as the history of a product in terms of the direct properties of that product and/or properties that are associated with that product once these products have been subject to particular value-adding processes [85]. The traceability system can also be used for the determination of fish freshness, recording the product temperature from the moment of catch. This alternative method could be cost effective and definitely more reliable.
13.4 Chemical and Biochemical Methods Chemical and biochemical methods for the evaluation of seafood quality are more reliable and accurate, since they eliminate personal opinions on the product quality. These objective methods should correlate with sensory quality, and the chemical compound that is determined should increase or decrease as microbial spoilage or autolysis progresses [16]. Currently, the most used method to evaluate fish freshness is to combine several measurements obtained from different methods and correlate the findings with sensory analysis [59]. The most used procedures for the objective measurements of seafood quality are given in the next sections.
13.4.1 ATP and Its Breakdown Products Rigor mortis occurs in postmortem muscle tissue and is associated with stiffness of muscle or flesh. This process results from breakdown of adenosine triphosphate (ATP), which is the main energy source for metabolic activity. It has been indicated that there is a correlation between nucleotide catabolism and loss of freshness. Nucleotide breakdown reflects both action of autolytic enzymes and bacterial action [16]. The sequences of nucleotide catabolism proceed as shown in Figure 13.1. The initial stage of the reaction catalyzed by endogenous enzymes takes place quickly, leading to accumulation of adenosine diphosphate (ADP) and inosine monophosphate (IMP). The oxidation Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) Adenosine monophosphate (AMP) Inosine monophosphate (IMP) Inosine (Ino) Hypoxanthine (Hx) Xanthine (Xa) Uric acid (Uric)
Figure 13.1 shown.
In postmortem fish muscle, degradation of ATP proceeds according to the sequence
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of Hx to xanthine and uric acid is slower and is the result of endogenous enzyme activity or microbial activity [86]. The IMP is associated with fresh fish flavor, whereas inosine and Hx reflect poor quality [87]. The concentrations of ATP and its breakdown products have been used as indicators of freshness in many fish species [8,9,13,88–92]. The K value proposed by Saito et al. [93] is a biochemical index for fish quality assessment based on nucleotide degradation. The K value includes intermediate breakdown products, and it varies within species of fish [94,95]. Since adenosine nucleotides are almost converted to IMP within 24 h postmortem [96], Karube et al. [97] proposed the Ki value, which excludes ATP, ADP, and adenosine monophosphate (AMP). However, in some species ATP, ADP, and AMP remain even after 2 weeks [97]. With some species, the Ki value has been shown to increase very rapidly and then remain constant even though freshness quality continues to decrease greatly [98,99]. Therefore, the K value can be superior to the other values. The G value proposed by Burns et al. [100] was found to be superior to Ki value for iced Atlantic cod, although it was observed to decrease during the first 2 or 3 days of iced storage, before its subsequent increase. In addition, H values have been described by Luong et al. [101] as an index of freshness quality. The H value of iced Pacific cod was observed to increase steadily, indicating its superiority to Ki value [101]. Gill et al. [102] also proposed Fr value for yellow fin tuna. These results showed that measuring the concentration of single nucleotide degradation product to determine freshness quality of seafood is not appropriate, but measuring the concentration of ATP and its degradation products can be useful in determining freshness quality [20]. P value has been described by Shahidi et al. [103]. Determination of G and P values are useful with lean fish. However, it is difficult to obtain meaningful G and P values since fatty fish deteriorate due to rancidity [103]. It was reported that K and related values increased linearly (except Fr value) with storage time in turbot [91], European eel [13], and sea bream [104]. The rate of nucleotide degradation varies with species, body location (dark or white muscle), stress during capture, handling, season, and storage conditions [105,106]. Several methods have been proposed for the analysis of single or a combination of nucleotide catabolites, but the high-performance liquid chromatography (HPLC) method is the most reliable among them. The K, Ki, G, P, H, and Fr values are calculated by the procedures described by Saito et al. [93], Karube et al. [97], Burns et al. [100], Shahidi et al. [103], Luong et al. [101], and Gill et al. [102], respectively. The formulas are as follows: lno + Hx ⎡ ⎤ K (%) = ⎢ ⎥⎦ × 100 ATP + ADP + AMP + IMP + lno + Hx ⎣ lno + Hx ⎡ ⎤ K i (%) = ⎢ ⎥⎦ × 100 IMP + lno + Hx ⎣ lno + Hx ⎡ ⎤ G (%) = ⎢ × 100 ⎣ AMP + IMP + lno ⎥⎦ lno + Hx ⎡ ⎤ P (%) = ⎢ ⎥⎦ × 100 AMP + IMP + lno + Hx ⎣ Hx ⎡ ⎤ H (%) = ⎢ ⎥⎦ × 100 IMP + lno + Hx ⎣ IMP ⎡ ⎤ Fr (%) = ⎢ ⎥⎦ × 100 IMP lno Hx + + ⎣
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13.4.2 Biogenic Amines The concentration of biogenic amines has been reported to be a reliable method of measuring the quality of fish, depending on the species being examined [10,11,107,108]. The formation of biogenic amines results from microbial degradation during the later storage of fish, and the concentration of these increases with storage time [91,109,110]. Biogenic amines are generated by microbial decarboxylation of specific free amino acids in fish or shellfish tissue [111]. The most significant biogenic amines produced postmortem in fish and shellfish products are histamine, putrescine, cadaverine, tyramine, tryptamine, 2-phenylethylamine, spermine, spermidine, and agmatine. The importance of estimating the concentration of biogenic amines in fish and fish products is related to their impact on human health and food quality. Since the amines are produced by spoilage bacteria toward the end of shelf life of a fish, their levels are considered as indices of spoilage rather than freshness [112]. In addition, the disadvantages of using biogenic amines as an index of freshness quality are that their absence does not necessarily indicate a high-quality product [113]. Among the biogenic amines, histamine is potentially hazardous and the causative agent of histaminic intoxication [114]. The others especially putrescine and cadaverine have been reported to enhance the toxicity of histamine [115]. Consumption of seafood containing high amounts of these amines can have toxicological effects. These problems may be more severe in sensitive consumers who have a reduced mono- and diamine oxidase activity [116], the enzyme responsible for its detoxification. The hazardous concentrations of histamine are 5 mg/100 g and 20 mg/100 g fish—the legal limit for histamine set by the U.S. Food and Drug Administration [117] and the EU [118], respectively. The biogenic amine content of fish depends on fish species, free amino acid content [112], the presence of decarboxylase-positive microorganisms, the moment of capture, and stomach contents at death, since microbial flora vary seasonally [11]. By means of decarboxylation reactions, tyrosine produces tyramine, histidine yields histamine, and arginine leads to putrescine. Cadaverine is derived from lysine, tryptamine from tryptophan, and 2-phenylethylamine is derived from phenylalanine. Putrescine is also an intermediate of a metabolic pathway that leads to spermidine and spermine [119]. The QI and the biogenic amine index (BAI) were proposed by Mietz and Karmas [120] and Veciana-Nogues et al. [121] for determination of quality of fish, respectively. The formulas used were as follows: QI = (histamine + putrescine + cadaverine)/1 + (spermidine + spermine) BAI = (histamine + putrescine + cadaverine + tyramine) QI is based on the increases in putrescine, cadaverine, and histamine and decreases in spermine and spermidine during storage of fish, whereas BAI is based on increases in histamine, putrescine, cadaverine, and tyramine. There are various analytical techniques used to determine the concentration of biogenic amines, including thin-layer chromatography (TLC) [122,123], HPLC [120,124,125], GC [126,127], capillary zone electrophoresis (CZE) [128,129], and use of a biosensor [130–132]. Among these techniques, HPLC is mostly performed because of its sensitivity, reliability, and reproducibility.
13.4.3
pH
The pH is also an important parameter to show depletion in tissue and quality of flesh during storage. Process technology is influenced by rigor development, postmortem temperature, and pH [133]. Postmortem pH varies from 5.5.0 to 7.1 depending on season, species, and other factors
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[134,135]. Low pH is used as an indicator of stress at the time of slaughtering of many animals. Low initial pH is associated with higher stress before slaughtering [13,136–139]. Th is is caused by the depletion of energy reserves, mainly glycogen, with the production of lactate. Since the activity of enzymes depends on pH, it affects reactions taking place during storage of fish. A relatively low pH may cause a decrease in water binding to the myofibrils, affecting light scattering and the appearance of fish. Low pH also promotes oxidation of myoglobin and lipids [134].
13.4.4 Total Volatile Basic Nitrogen In seafood, total volatile basic nitrogen (TVB-N) primarily includes trimethylamine (TMA, produced by spoilage bacteria), ammonia (produced by deamination of amino acids and nucleotide catabolites), and DMA (produced by autolytic enzymes during frozen storage). The analyses of these indicators are considered unreliable because they reflect later stages of spoilage rather than freshness [140]. The European Commission (Council Regulation No. 95/149/EEC of March 1995) on fish hygiene specifies that if the organoleptic examination indicates any doubt as to the freshness of the fish, TVB-N should be used as a chemical check. The level of TVB-N in freshly caught fish is generally between 5 and 20 mg N/100 g muscle. However, the levels of 30–35 mg N/100 g muscle are considered the limit of acceptability for icestored cold-water fish [17,141]. Based on the results obtained from the literature, TVB-N level correlated with fish quality, as shown in a variety of fish such as European hake [142], Atlantic cod [143], sardine [12, 144], and European eel [13]. However, the level of TVB-N was not correlated with the time of storage of some fish species, such as frozen eel [145], turbot [92], pike perch [146], farmed gilthead sea bream [147], and hake [148]. Therefore, it could not be regarded as a good indicator of fish freshness and proved to be better as a spoilage index. It is well known that determination of TVB-N differs systematically according to the procedures used. The EC reference method for TVB-N determination, involving preliminary deproteinization with perchloric acid, was compared with two routine methods. The first one includes direct distillation of fish after adding magnesium oxide, whereas the second one includes the use of trichloroacetic acid instead of perchloric acid [149]. It was found that there was a good correlation between three methods, and direct distillation methods have been recommended as a rapid routine method.
13.4.5
Trimethylamine
The one type of spoilage caused by microorganisms often detected as a fishy odor is due to the decomposition of trimethylamine oxide (TMAO) via the enzyme TMAOase demethylase, as shown below: Following death of fish, bacteria act upon TMAO to produce TMA, which is considered to be the main cause of off odors in fish products [58,59]. Therefore, TMA is produced by the decomposition of TMAO due to bacterial spoilage and enzymatic activity [150,151], and it has been used as an indicator of marine fish spoilage: CH3 CH3 – N=O CH3 TMAO
CH3 CH3 – N CH3 TMA
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TMAO appears to be part of the system used for osmoregulation. The TMAO content of seafood varies with species, age, fish size, time of year, and environmental factors [152]. Seawater fish have 1–100 mg TMAO in every 100 g muscular tissue, whereas freshwater fish generally contain only 5–20 mg% [153]. TMA can be used as a spoilage indicator and not as an index of freshness. However, its usefulness depends on time of year, location of catching, stage of spoilage, type of storage and processing, and methods employed for analysis. TMA is not produced in a significant amount during the early stages of chilled storage of fish, but it appears after 3 or 4 days, after which the rate of production of TMA parallels the bacterial proliferation pattern [154]. Fresh fish has a very low amount of TMA with values less than 1.5 mg TMA/100 g in fresh cod, but values increase during spoilage. The fish is considered stale when the rate of TMA production is higher than 30 mg/100 g cod [155]. Several assays have been described for the determination of TMAOase activity in fish muscle [151,156,157]. Many analytical methods have been developed for the measurements of TMA, DMA, or TVB-N contents, including steam distillation [158], Conway microdiff usion and titration [159], colorimetric method [160], photometry [161], HPLC method [162], GC method [163, 164], a capillary electrophoresis method [165], semiconducting metal–oxide array [166], flowinjection-gas diff usion method [167], biosensor using flavin-containing monooxygenase type-3 [168], and solid-state sensors based on bromocresol green [169].
13.4.6
Dimethylamine
As mentioned earlier, fish contain TMAO, which is converted to TMA by bacteria in iced fish. During chilled or frozen storage of fish, when bacterial growth is inhibited, this reaction is replaced by a slow conversion by an enzyme to DMA and formaldehyde [16,150]. The formation of these products may cause severe quality changes or spoilage during prolonged frozen storage. The amount of DMA produced depends on species (except gadoid species, other species do not develop adequate amounts of DMA), the storage temperature, and time. DMA can be used as a spoilage index during frozen storage of some species such as frozen hake [170].
13.4.7
Formaldehyde
The formaldehyde content in seafood products is generally considered as nontoxic, but it can react with a number of chemical compounds such as amino acid residues, terminal amino groups, and low-molecular weight compounds, causing denaturation and cross-linking of proteins [171]. This reduces the solubility of myofibrillar proteins [172]. The formaldehyde content of frozen seafood is generally used as a spoilage index, especially in gadoid fish.
13.4.8
Lipid Oxidation Indicators
During processing and storage, enzymatic and nonenzymatic lipid oxidation occurs. A close relationship has been found between lipid damage and quality of the final product [173]. Fresh fish has a limited shelf life and is prone to deterioration, whereas fish can be stored in a frozen state for several months without severe changes in quality. The limiting factor of frozen storage in lean fish species is denaturation of proteins, which results in a dry and firm texture of the fish muscle [174]. However, lipid oxidation is the limiting factor in fatty fish species, resulting in rancidity.
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Handbook of Seafood and Seafood Products Analysis Initiation: Initiators (heat, light, trace of heavy metals) R+H
RH Propagation: O2 R
RO2
RO2 + RH
ROOH + R
ROOH
RO + OH
2ROOH
ROO + ROO + H2O
Termination: R+R
RR
R + ROO
ROOH
ROO + ROO
ROOH + O2
Figure 13.2 The autoxidation of fatty acids. (From Hamilton, R.J., in Rancidity in Foods, 3rd edn., Allen, J.C. and Hamilton, R.C. (Eds.), Chapman & Hall, London, U.K., 1994, pp. 1–22.)
Off taste and off odor are usually defined as rancidity. Seafood has highly unsaturated lipid content. Under chilled/frozen conditions, lipid oxidation compounds interact with proteins, leading to protein denaturation, nutritional losses, and modification of electrophoretic profiles of proteins [172,175–177]. Many factors affect the onset and development of rancidity (oxidative and hydrolytic degradation of lipids), including the degree of unsaturation of the oil, the type and concentrations of antioxidants, pro-oxidants, moisture content, oxygen availability, temperature, and degree of exposure to light [178–180]. Several chemical and physical techniques applied alone or together have been used to determine the degree of oxidation and hydrolytic degradation of lipids in edible oils. There are three steps in autoxidation of unsaturated fatty acids; initiation, propagation, and termination (Figure 13.2). Initiators (such as light, heat) convert RH to free radicals (initiation phase), and free radicals react with oxygen to produce peroxide radicals (ROO). The peroxide radical can attack another lipid molecule RH, resulting in peroxide (ROOH) and new free radical (propagation phase). Peroxides are not stable compounds, and they break down to aldehydes, ketones, and alcohols, which are the volatile products causing off flavor in products. The amount of reactive compounds increases gradually, and then the quantity of radicals and peroxides decreases, forming stable deterioration products (termination phase) [181,182]. Excess free radicals and peroxides in foods cause destruction of essential fatty acids and vitamins A, C, E, B6, thiamine, and pantothenic acid. Free radicals from oxidizing lipids can polymerize with proteins and destroy certain amino acids. Peroxides can also react with proteins and result in a decrease in their nutritional value. They also destroy pigments, produce toxins, and cause off flavor/odors [183]. Fish oil contains about 20% of their total fatty acids as long-chain PUFA, consequently, fish and fish oils are highly susceptible to the development of oxidative rancidity, unpleasant odors, off flavors, and taints [179,180]. The amount of hydroperoxides can be used as a measure of the extent of oxidation in the early stages. The hydroperoxide value is generally shortened to peroxide value (PV). The major chemical indicators for the determination of the extent of oxidative rancidity
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are anisidine value (AV), PV, TOTOX (2VP + AV), and thiobarbituric acid (TBA). PV measures primary products of lipid oxidation, which break down to secondary products of oxidation or react with proteins. Increase in the PV is most useful as an index of the earlier stages of oxidation; as oxidation proceeds the PV can start to fall. AV and TBA values measure the secondary products of lipid oxidation. During prolonged storage of seafood, PV, AV, and TBA values may increase, reach a peak, and decline [184,185]. Many methods have been employed for the measurements of lipid oxidation in foods as a means of determining the degree of damage [20,186]. However, there are some difficulties with common methods when quality has to be assessed, since oxidation products are unstable and react with biological amino constituents, such as proteins, peptides, free amino acids, and phospholipids, causing production of interaction products [187]. Analysis of these interaction products by fluorescence detection as a quality assessment index for frozen-stored sardine was studied by Aubourg et al. [188] and it was found that fluorescence detection of interaction compounds can provide an accurate method to assess quality differences during frozen storage of sardine. Cozzolino et al. [80] also reported that PLS-R and near-infrared (NIR) spectroscopy to monitor both oxidation and hydrolytic degradation of lipids in fish oil can be successfully employed.
13.4.9
Lipid Hydrolysis
Hydrolysis leads to hydrolytic rancidity and involves hydrothermal or enzymic (lipase) hydrolysis to FFA and other products. FFAs and their oxidation products would have an effect on muscle texture and functionality, since they interact with myofibrillar proteins and promote protein aggregation [189]. A gradual increase in FFA formation was obtained for all kinds of samples as a result of the frozen storage time for fatty fish such as tuna, sardine, European eel, horse mackerel [13,188,190,191], and lean fish such as blue whiting, haddock, cod [192,193], and also freshwater fish [194].
13.5
Microbiological Methods
Numbers and types of microbes present in foods are important indicators of safety and quality. Microbiological analyses of seafood involve testing for presence or absence of pathogens such as salmonellas and determination of numbers of colony-forming units (CFU) named “total viable counts (TVC)” or “aerobic plate count (APC),” or numbers of CFU of indicator organisms such as Enterobacteriaceae, coliforms, or enterococci [195]. Microbial assessments have been carried out to monitor the numbers of various groups of microorganisms during the production process as part of food safety objectives and also hazard analysis critical control point (HACCP) systems [196]. Spoilage of fish and fish products is a result of the production of off odors and flavors mainly caused by bacterial metabolites [197]. The numbers of specific spoilage organisms (SSOs) and the concentration of their metabolites can be used as objective quality indicators for determination of shelf life of seafood. It is possible to predict shelf life of seafood based on knowledge of initial numbers and growth of SSO. Microbial growth models can be used to determine the effect of various time/temperature combinations on shelf life of fish in production and distribution chain. It was indicated that the main requirements for shelf life predictions are to collect information about SSO, spoilage domain such as the range of environmental conditions over which a particular SSO is responsible for spoilage and spoilage level [198]. Mathematics models have been well established for the growth of spoilage bacteria such as Photobacterium phosphoreum, Shewanella putrefaciens
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[198], Brochothrix thermosphacta [199], Listeria monocytogenes [200], and Clostridium perfringens [201], which were shown to correlate with remaining shelf life of product and also correlated better than classical TVC measurements. Prediction of the remaining shelf life of seafood requires reliable estimates of the initial population of SSO, because it varies from batch to batch due to season, feeding, catching method, handling, and storage after catch [202]. Among the microbiological methods for determination of bacterial counts in a short time, impedance is the most promising [203]. Mathematical models along with impedance technique may provide reliable information on shelf life of seafood within 24 h. The change in electrical properties (impedance, conductance, and capacitance) due to the growth of microorganisms in the culture media has been used for the rapid estimation of total bacterial counts [204], coliforms [205], and Salmonella spp. [206]. The principle of the impedance measurement is based on the phenomenon that at a time point (i.e., detection time—DT) at which bacteria have grown to a population of approximately 107 CFU/mL or higher, an accelerating change in impedance (or conductance) will occur in the growth media. The decrease in impedance (or increase in conductance) is due to the breakdown of the substrate molecules in the media to smaller molecules (e.g., acids), which have more charges than the substrate itself [207]. Current microbiological culture methods rely on growth in culture media, followed by isolation, and biochemical and serological identification. These methods are laborious and time consuming, requiring a minimum of 1 or 5 days to recognize. These methods are also not appropriate for online processing of seafood. However, modern microbiological techniques [such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR)], antibody techniques [such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunomagnetic chemiluminescence (ELIMCL)], and oligonucleotide probes give results in 1 day or even less [209–213]. On the other hand, these methods have limitations in performing quantitative analyses, also lack in sensitivity, and are costly.
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200. Carrasco, E., Valero, A., Pérez-Rodríguez, F., García-Gimeno, R.M., and Zurera, G., Management of microbiological safety of ready-to-eat meat products by mathematical modelling: Listeria monocytogenes as an example. Int. J. Food Microbiol., 114, 221–226, 2007. 201. Juneja, V.K., Huang, L., and Thippareddi, H.H., Predictive model for growth of Clostridium perfringens in cooked cured pork. Int. J. Food Microbiol., 110, 85–92, 2006. 202. Koutsoumanis, K.P., Taoukis, P., Drosinos, E.H., and Nychas, G.-J.E., Applicability of an Arrhenius model for the combined effect of temperature and CO2 packaging on the spoilage microflora of fish. Appl. Environ. Microbiol., 66, 3528–3534, 2000. 203. Koutsoumanis, K., Lambropoulou, K., and Nychas, G.-J.E., A predictive model for the non-thermal inactivation of Salmonella enteritidis in a food model system supplemented with a natural antimicrobial. Int. J. Food Microbiol., 49(1–2), 63–74, 1999. 204. Ogden, I.D., Use of conductance methods to predict bacterial counts in fish. J. Appl. Bacteriol., 61, 263–268, 1986. 205. Firstenberg-Eden, R. and Klein, C.S., Evaluation of a rapid impedimetric procedure for the quantitative estimation of coliforms. J. Food Sci. 48, 1307–1311, 1983. 206. Bullock, R.D. and Frodsham, D., Rapid impedance detection of salmonella in confectionery using modified LICNR broth. J. Appl. Bacteriol., 66, 385–391, 1989. 207. Wu, J.J., Huang, A.H., Dai, J.H., and Chang, T.C., Rapid detection of oxacillin-resistant staphylococcus aureus in blood cultures by an impedance method. J. Clin. Microbiol., 1460–1464, June 1997. 208. Amagliani, G., Giammarini, C., Omiccioli, E., Brandi, G., and Magnani, M., Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control, 18, 1137–1142, 2007. 209. Landete, J.M., Rivas, B. de las, Marcobal, A., and Muñoz, R., Molecular methods for the detection of biogenic amine-producing bacteria on foods. Int. J. Food Microbiol., 117, 258–269, 2007. 210. Nowak, B., Müffling, T.V., Chaunchom, S., and Hartung, J., Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique. Int. J. Food Microbiol., 115, 259–267, 2007. 211. Lee, C.Y., Panicker, G., and Bej, A.K., Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLinkk NH microwell plate sandwich hybridization. J. Microbiol. Methods, 53, 199–209, 2003. 212. Gehring, A.G., Irwin, P.L., Reed, S.A., Tu, S., Andreotti, P.E., Akhavan-Tafti, H., and Handley, R.S., Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7. J. Immunol. Methods, 293, 97–106, 2004. 213. Lida, K., Abe, A., Matsui, H., Danbara, H., Wakayama, S., and Kawahara, K., Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization, FEMS Microbiol. Lett., 114(2), 167–172, 1993.
Chapter 14
Analytical Methods to Differentiate Farmed from Wild Seafood Iciar Martínez, Inger Beate Standal, Marit Aursand, Yumiko Yamashita, and Michiaki Yamashita Contents 14.1 Introduction ..................................................................................................................216 14.2 Morphological Examination..........................................................................................216 14.3 Genetic Analysis ............................................................................................................217 14.3.1 Sample Preservation and DNA Extraction Methods ........................................217 14.3.2 DNA Markers ..................................................................................................218 14.4 Analysis of Proteins .......................................................................................................218 14.4.1 Sample Preservation......................................................................................... 219 14.4.2 Protein Extraction ........................................................................................... 220 14.4.3 Analysis of Proteins ......................................................................................... 220 14.5 Analysis of the Lipid Content ........................................................................................221 14.5.1 Sample Preservation ........................................................................................ 222 14.5.2 Lipid Extraction and Gas Chromatography .................................................... 222 14.5.3 1H NMR and 13C NMR Analyses ................................................................... 222 14.6 Stable Isotopes .............................................................................................................. 224 14.6.1 Sample Preservation ........................................................................................ 225 14.6.2 SNIF–NMR and IRMS .................................................................................. 225
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14.7 Trace Element Fingerprint............................................................................................ 226 14.7.1 Sample Preservation ........................................................................................ 226 14.7.2 ICP-MS ........................................................................................................... 227 14.8 Other Methods ............................................................................................................ 227 Acknowledgments ................................................................................................................... 227 References ............................................................................................................................... 228
14.1 Introduction The implementation of analytical methods to differentiate farmed from wild-produced seafood is important to ensure correct consumer information and avoid fraud: Information about the production method of seafood is obligatory in the EU (CR EC No 2065/2001 of October 22, 2001 laying down detailed rules for the application of CR EC No 104/2000 regarding informing consumers about fishery and aquaculture products) and similar laws apply in Japan (Law on Standardization and Proper Labeling of Agricultural and Forestry Products, JAS Law, of 1999), and the United States (The Federal Food, Drug, and Cosmetic Act and The Fair Packaging and Labeling). Correct information about the production method of seafood is also important, because farmed and wild organisms carry different hazards and are therefore submitted to different regulations and analytical controls. For example, commercially farmed specimens may contain residues of veterinary drugs whose presence is unlikely in wild seafood, but wild specimens may contain parasites harmful to humans, and these are seldom present in farmed seafood. The production method is also part of the information essential to fulfill the traceability of a product and, therefore, analytical methods should be made available to confirm it. Several methods have been successfully applied to differentiate farmed from wild seafood, including morphological examination, genetic analyses, analysis of the protein and lipid contents, as well as examination of the stable isotope and trace element profiles. Standards for organic farming are still under development in many countries. Although in this work no special mention is made to organic farming, the set of technologies to apply are basically the same as those described here. In particular, stable isotope analyses combined with fatty acid (FA) profiles have proven particularly useful when tested.1
14.2 Morphological Examination There are few publications and no official guidelines for the morphological differentiation of farmed and wild aquatic organisms. In small Salmo salar, 100% discrimination between farmed (AquaGen strain) and wild parr was achieved by examining the body form, shape of the head, size of the eyes and mouth, and length of the pectoral fin.2 Also in an earlier study,3 it was shown that the morphology of the head, fins, and caudal peduncles could be used for a total correct classification of wild, farmed, and sea-ranched S. salar parr. The later study showed that the environmentally induced phenotypic divergence increased with age and with the numbers of generations under domestication. In cod, the most prominent differences are the higher condition factor, larger liver, and smaller head4 as well as backbone malformations in farmed specimens. Farmed cod often present unattractive black lines consisting of layers of melanin-filled cells associated with blood vessels due to overabundance of copper in commercial feeds.5 The flesh of farmed cod sometimes presents
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a translucent grayish aspect, in contrast to the white opaque color of the wild, and the liver in farmed cod is much bigger than the liver of wild fish.6 However, classification based on morphological criteria demands the presence of the morphologic diagnostic characters, which are usually absent in many intermediate products as well as in the ready-to-eat dish, thus limiting its application.
14.3 Genetic Analysis Doyle et al.7 proposed that the genetic diversity of aquacultured stocks of fish should be maintained and their genetic impact on wild stocks minimized by using breeding programs designed to generate genetic diversity. If this policy had been followed, it would be relatively difficult to find markers for wild and farmed fish, since diversity would be one of the selected traits in the farmed fish. However, in most breeding programs the fish are indeed selected based on commercially interesting traits such as growth performance,8,9 resistance to diseases or to stress,10 and optimal adaptation to different environments.9 Genetic analyses have allowed the differentiation of wild from farmed fish populations in a variety of species,11–13 and a loss of rare alleles has usually been observed in the farmed populations.11,14–16 Hayes et al.17 suggested that it was possible to assign accurately a fish sampled from the market place to either the farmed population or the wild using either microsatellite or single nucleotide polymorphism (SNP) markers. Genomics analyses are dealt with in more detail in Chapter 4 of this handbook.
14.3.1 Sample Preservation and DNA Extraction Methods The sample should be extracted as soon as possible after sampling, in particular if it has a high enzymatic activity (for example if it contains the hepatopancreas in a crustacean). Delays and the use of preservations methods will diminish the quality and the yield of DNA. If the sample must be preserved, the best method is to freeze it in liquid nitrogen or in a biofreezer. Depending on the type of sample and its use, a normal freezer (−20°C) may also be used. For very long periods, we recommend preservation in 96% ethanol. To extract frozen samples we recommend to start the procedure before the sample is completely thawed, since enzymatic activity also takes place at subzero temperatures. Samples fi xed in ethanol must be allowed to dry completely, so that all the ethanol is evaporated, and then be rehydrated in water or in the extraction buffer. One requisite condition for any genetic analysis is the obtention of good quality DNA suitable for PCR amplification, which is the most common analysis. Many commercial kits, such as Qiagen, Dynal (Invitrogen), Nucleospin (Clontech), Amersham Biosciences (GE Healthcare), Wizard (Promega), GeneRelease (Bio Venture Inc.), E.Z.N.A Stool DNA Isolation Kit (United Bioinformatica Inc.), and others, give satisfactory results. Each kit is provided with a detailed description of how to use it. The basic steps in all DNA extraction methods include the inactivation of nucleases, for example by chelating divalent cations using EDTA and EGTA. Th is step is not necessary in samples preserved in ethanol, because the enzymes are inactivated by the fi xation. Then, the cells are opened (by heat treatment, sonication, or by the use of Proteinase K) and proteins are removed usually by incubation with Proteinase K. The DNA is then separated from the contaminating cellular components by salt precipitation, chloroform extraction, treatment with Chelex,18 or gel filtration, and then recovered by ethanol or isopropanol precipitation. The DNA
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pellet is usually washed at least once with 70% ethanol, the alcohol is allowed to evaporate, and the DNA is reconstituted in double-distilled sterile water or in a slightly alkaline buffer (50 mm Tris–EDTA, pH 8.0). Three more methods that have reputedly produced good quality DNA suitable for amplification, and also for forensic studies, from food matrices include the use of hexadecyl–trimethyl ammonium bromide (CTAB),19 the Chelex method,18 and the salt extraction method.20,21 When using the salt extraction method with heavily degraded samples, we have found it helpful to leave the tubes after the first precipitation of DNA with isopropanol in a freezer at −20°C or at −80°C for a few hours before centrifugation (Marian Martinez de Pancorbo, University of the Basque Country, Spain, personal communication). In these samples the pellet may be practically invisible, so the next step, which is washing the pellet with 50% ethanol, must be performed very carefully not to lose the sample. On other occasions, however, it is possible to amplify the DNA of a sample by simply dehydrating it and placing a small amount directly into the PCR amplification mixture. This method has been successfully used by the authors of this paper (unpublished results) and by Bucklin and Kochert22 with whole individuals of Calanus.
14.3.2
DNA Markers
In recent years, several countries, such as Norway, the United States, China, India, Japan, and others, have started programs to map the whole genome of some species, including oysters, salmonids (salmon, trout, Arctic charr), cod, tilapia, catfish, shrimp, and bass. The outcome of these programs is already producing lists of genetic markers linked to traits of interest, which may be used to identify the strains of the farmed individuals that display an increased frequency of the desired traits.23 In principle, any marker, whether microsatellites, SNPs, genomic, or mitochondrial, has the potential to be useful to differentiate farmed from wild specimens of a given species.11,13,15,17 However, which markers and how many of them are necessary to differentiate a wild from a farmed specimen are completely dependant on the species and the breeding stock and need to be examined on an individual basis. It is worth mentioning, however, that the Norwegian company GenoMar has patented a method to trace back farmed individual Atlantic salmon, tilapia, Atlantic halibut, cod, and sea bass, by using a series of SNP and microsatellite polymorphisms by PCR and by oligonucleotide ligation assay (OLA).24 The method requires that all parent fish of the brood stock are DNA typed as well as all the individuals under examination. In the future, DNA analyses may be performed using chips that permit the determination in one fast step of many characteristics simultaneously, including the species, traits, breeding stock, and production method. An additional advantage is that it is possible to use robots for many of the steps (DNA preparation, sample preparation, etc.), which further increases the amount of samples that can be processed.
14.4 Analysis of Proteins No clear protein marker has been identified to discriminate farmed from wild seafood. However, protein markers commonly used for genetic analyses have the potential to be used as markers for farmed or wild, since some alleles are more frequent in one group than in the other.14 In addition, differences in the protein pattern of liver25 and muscle26,27 tissues between farmed and wild salmon and cod have been reported.
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Industrial fish farming is a relatively new activity compared with farming of land animals. Although feeds and breeding conditions need to be developed and optimized for each species, it is common to apply directly to new species those conditions that have proven successful for other species, and then modify them depending on the results. Thus, neither feeds nor breeding conditions may be optimal for farming, and this may induce stress in the farmed animals, which is reflected in the composition of their organs.25–27 In addition, the depletion of the wild stocks of pelagic fish and the high price of feeds based on fish meal and oil, which would be a natural diet, have prompted the development of feed formulations based on vegetable oils and proteins.28–31 The source of protein in teleost fish is very important, and they require high levels of dietary protein (30%–60%), which they use as their preferred energy source.32 Unfortunately, the amino acid profiles of plant proteins do not meet the essential amino acid requirements of fish, and feed diets based on plant protein require supplementation with synthetic amino acids.33 Moreover, it has been shown that components in fish feeds may contain very high levels of metals (Cu, Zn, Fe, Mg, and Ca)34 and that vegetable meals may contain antinutritional factors (protease inhibitors, lectins, antigenic proteins, etc.)29 that may have adverse effects on fish.33,35,36 Martin et al.25 attributed the alteration in the protein expression in the liver of rainbow trout to the presence of antinutritional factors in feeds containing soy protein. Using proteomic analysis, these authors identified 33 differentially expressed proteins, including heat shock proteins, several enzymes, and structural and FA-binding proteins. The authors noted a downregulation of some structural proteins in fish-fed soy proteins, attributed to the fish’s increased requirement for energy metabolism. In addition, several enzymes involved in anabolic metabolism were downregulated in fish fed the diet rich in soybean meal, indicating increased emphases on catabolism relative to anabolism in the fish fed this diet. Interestingly, Olsson et al.,27 also registered the altered expression of five enzymes implicated in the glycolytic pathway and citric acid cycle in farmed cod. Texture is an important quality attribute of the fish flesh. Soft texture, usually considered negative, is more common in stressed and in farmed than in wild fish.37 Martinez et al.26 examined the protein expression in skeletal muscle of farmed and wild cod by high-resolution twodimensional electrophoresis and found differences between the two, which were attributed to increased proteolytic activity in the muscle of the farmed compared with the wild cod. Johnston et al.38 found that the reason for the softening in this species did not seem to be the faster growth of the farmed fish, and they hypothesized that the greater concentration of insoluble collagen present in wild salmon may contribute to their firmer texture. Some enzymatic systems that may be responsible for the muscle softening are metalloproteases and collagenases, lysosomal cathepsins, neutral calcium-activated calpains, and the proteasome. However, no study has identified yet the main system/s responsible for the soft texture in farmed fish or the spots that may be used as markers to discriminate farmed from wild fish. Proteins and proteomic analyses are dealt with in more detail in a different chapter of this handbook.
14.4.1 Sample Preservation The optimal case would be when the extraction of proteins can take place on the sample immediately after the experimental treatment, that is, with no preservation at all. When this is not possible, a preservation procedure that minimizes the modifications (denaturation, aggregation, loss of functional groups, and proteolysis) of the proteins in the sample should be chosen. Optimal methods include fast freezing and frozen storage using temperatures as low as possible. Thus, optimal freezing would be achieved immediately after excision by submersion in liquid nitrogen and storage at −80°C or by freezing and storing directly at −80°C. For short periods of time, −20°C
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may be acceptable. It should be noted that any preservation procedure will alter the protein profile in the sample, and, therefore, one should be very careful when comparing samples preserved and stored under different conditions.
14.4.2
Protein Extraction
There are many methods for extraction of proteins, depending on the proteins one wishes to examine. Since current studies are still trying to identify markers, we focus on the use of techniques with the potential to identify such markers. Proteomic techniques have a clear advantage in this field. The first step in proteomic analyses is to extract as many proteins as possible from the sample. However, due to the great diversity and properties of the proteins contained in the edible tissues of seafood, as well as the different degrees of processing to which the sample may have been submitted (freezing, cooking, etc.), the optimal extraction procedure for any given sample must be determined empirically. It is common to use several buffers with increasing concentration of chaotropic agents (urea, thiourea), detergents (CHAPS, Triton X-100, SDS), and reducing agents (b-mercaptoethanol, dithiothreitol (DTT), tributylphosphine) to solubilize the widest possible spectrum of proteins. Some authors have claimed that the use of DNase I and RNase in the extraction buffer increases the number of spots in the gels. In our experience, this may be because some commercial preparations of these enzymes are contaminated with proteases. We therefore recommend not to use such enzymes. In addition to published works,25–27 BioRad39 and GE Healthcare Amersham have some excellent manuals about protein extraction and analysis. The use of protease inhibitors should always be considered: use of some inhibitors and cocktails may help to preserve the sample during the extraction procedure, but they will hamper the study of protease activities that may be relevant in some other works. Their use should be evaluated for each particular study.
14.4.3 Analysis of Proteins As already mentioned, there are many methods suitable for protein analyses. The choice of method depends on the protein and the property one wishes to examine, and there are special protocols for each application. Proteomics permits the separation of many proteins (often thousands) from a complex protein mixture in one step, and, therefore, its application is widespread in many fields. The proteins are separated first according to their pI in 3% polyacrylamide gels in which a pH gradient is created using a mixture of ampholytes. The optimal pH range to choose depends on the sample, but 3–10 are commonly used for wide screenings. Afterward, the strip containing the proteins separated by their pI is loaded on top of the second-dimension SDS-PAGE gel, usually 8%–20% or 12% PAGE, for a wide screening. Both first and second-dimension gels can be purchased as precast, ready to use gels from several companies. After separation, the gels can be stained by Coomassie Blue (low sensitivity but compatible with mass spectrometry (MS) analysis necessary for subsequent peptide fingerprinting and sequencing), silver (high sensitivity, but not all protocols are compatible with MS), and fluorescent labeling or staining (of intermediate sensibility and also compatible with MS). The pictures of the gels containing similar samples of wild and farmed specimens obtained after scanning are compared using adequate software (such as Bionumerics or PDQuest) to identify differentially expressed spots that are then excised from the gels, destained, reduced, alkylated, and digested, usually with trypsin. The tryptic fragments are cleaned from contaminants, and peptide mass fingerprinting of the digests is then
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usually performed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. The proteins are afterward identified by searching in databases (National Centre for Biotechnology Information, NCBI) using suitable software (MASCOT, ProteinLynx Global SERVER), which uses MS data to identify proteins from primary sequence databases. The whole procedure is described in detail by Martin et al.25 and reviewed by Granvogl et al.41 The workload can be reduced by using precast gels and automated procedures with suitable software and robotic stations (sample and gel handling and staining, identification of diagnostic spots, spot cutters, etc.). However, due to frequent errors in the automated spot identification procedure (because of imperfect spot separation and identification caused by overlapping of spots, very different staining intensities, etc.), the final identification and assignment of the spots in the gels must be performed visually by trained personnel. This is probably the most time consuming step of the whole procedure. Proteomic analysis is a complicated procedure necessary to identify the biological markers. Once the diagnostic proteins are identified, however, the whole process can be greatly simplified by targeting only the biomarkers: raising or synthesizing antibodies targeting those proteins in order to use them in several formats. For example, lateral flow strip tests permit in situ easy and fast screening of seafood samples; and ELISA format will permit the routine analyses of many samples, for example, in control laboratories. Development of protein chips will facilitate the simultaneous screening of many targets and samples.
14.5 Analysis of the Lipid Content The analysis of the triglyceride (TG) fraction, in particular when combined with stable isotope composition (see the following paragraph), has often given correct classification of farmed and wild specimens.42,43 The changes in the FA composition of the TG fraction following changes in the composition of the diet have been explained using a dilution model.44 Frequently, the total amount of TGs alone may be used as a criterion to differentiate farmed from wild fish, because farmed fish usually have a much higher content than wild ones.45–48 In addition, the FA profile of TGs reflects that of the feed,44 and this FA fingerprint has often been successfully used49–52 as a diagnostic to identify the production method. The FA profile of vegetable oils (such as corn, cottonseed, linseed, olive, palm, rapeseed, soybean, and sunflower) used as partial substitutes for marine oils in fish feeds53–57 have in common a very low or undetectable amount of the long-chain omega-3 polyunsaturated fatty acids (PUFAs) C20:5n3 (EPA) and C22:6n3 (DHA) characteristic from fish oils, and often a single FA may account for about 50% of the total FA content in these oils. The FA composition of fish oils is more complex, that is, there are more FAs present in detectable amounts, and it is seldom that only one of them makes more than 25% of the total. As in vegetable oils, which FA is more abundant is species dependant. C16:0 and C18:1n9 are relatively abundant in all fish oils, but C22:1 (several isomers) is relatively more abundant in Coho salmon, capelin, herring, and sand eel and C22:6n3 is more abundant (over 10%) in Atlantic and Coho salmon, sand eel, and sardines than in capelin, herring, or menhaden.53,55–58 Specific FAs are selectively retained or used. In Atlantic salmon for example, it has been shown that there was selective deposition and retention of C22:6n3, so that the concentrations in flesh were higher than in the diet, whereas C18:1n9, C22:1n11, C18:2n6, and C18:3n3 were selectively metabolized.59 Other studies in the same species showed that the flesh had higher levels of C18:1n9 and C22:6n3 and less C20:5n3 than the feeds.60 The FAs C18:1n9 and 18:2n6 may act as markers
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for vegetable oils, and in particular, the latter seems to be the most persistent after a dietary switch to fish oil diet, and even after the levels of C20:5n3 and C22:6n3 were restored to the original high levels, the ratio n3/n6 was not fully restored.52,61 As indicated by Refsgaard et al.,62 one must always take into account the very wide variation in the concentrations of lipid components that can be found in apparently homogeneous populations of farmed salmon, which, together with the special feed formulations used for organic farming and the fact that escaped farmed fish and wild fish eating around farms may display intermediate lipid profiles,63,64 may contribute to the difficulty of performing correct classifications as wild/ farmed based only on the FA composition.
14.5.1 Sample Preservation Due to the high levels of PUFAs, it is particularly important to exercise care when working with marine lipids: it is recommended to use low temperature (work in ice or in a cold room) and avoid or minimize exposition to air and light in order to prevent lipid hydrolysis, oxidation, and polymerization. Fresh samples should be kept wrapped in air- and light-tight containers and stored at low temperatures. If freezing is required, it is best to use as low a temperature as possible, that is, −80°C, and an inert atmosphere.
14.5.2 Lipid Extraction and Gas Chromatography Procedures for lipid extraction are described in another book chapter of this series. For detailed descriptions of the analysis of fish samples, the reader is directed to several publications46,50,52 that give detailed descriptions of the procedure.
14.5.3
1
H NMR and 13C NMR Analyses
High-resolution nuclear magnetic resonance spectroscopy (HR-NMR) has emerged as a popular technique in the analysis of foodstuff, including fish and fish products. NMR spectroscopy exploits the magnetic properties of certain nuclei: nuclei that contain odd numbers of protons or neutrons have an intrinsic magnetic moment and angular momentum. The most commonly measured nucleus is 1H (the most receptive isotope at natural abundance), but NMR is applicable to any nucleus possessing spin (e.g., 2H, 13C, 15N, 14N, 19F, 31P, 17O, 29Si, 10B, 11B, 23Na, 35Cl, 195Pt). NMR spectroscopy can be used to identify functional groups, since in a one-dimensional spectrum each peak is produced by those nuclei placed in an identical local chemical environment. The spectrum is often used to obtain information about the number and type of molecules in a mixture. HR-NMR has been particularly valuable in the study of marine lipids, because it provides multicomponent information and can be applied nondestructively.65 NMR gives a fingerprint of the sample analyzed, which may be used as a rapid profiling technique. The most commonly used HR-NMR techniques in wild/farmed classification are 1H NMR and 13C NMR, both of which are able to detect a range of metabolites in a nontargeted way. The major advantage of 1H NMR spectroscopy compared with 13C NMR is the higher sensitivity and thereby shorter acquisition times per experiment. On the other hand, 13C NMR has a greater range of chemical shifts, which leads to less overlapping of signals, and is the preferred tool in lipid analysis when interpretation of
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spectra is the goal. Multivariate methods are frequently applied to study differences among NMR spectra.66,67 1H NMR has been used to perform quantitative measurements of total n-3 FAs and of the levels of DHA.68,69 This analysis can be carried out with a high degree of automation and gives a rapid fingerprint (2–5 min) of the lipid profile. 1H NMR has also been applied to differentiate between wild and farmed salmon and sea bream of different origins.70,71 13C NMR gives information about FA composition of fish72 and the positional distribution of PUFAs in triacylglycerols and phospholipids,73 which is of value for authentication purposes. Both HR 1H and 13C NMR, in conjunction with chemometrics, have allowed the differentiation between wild and farmed salmon74 and cod51 of different origins. The most commonly used solvent in the analysis of neutral lipids is deuterated chloroform (i.e., 99.8% CDCl3), which is easily evaporated, leaving the sample ready for analysis. Typically, a sample size of 50–100 mg of lipid in 0.5–0.8 mL solvent is used, although the optimal sample size depends on the instrument. Tetramethylsilane (TMS) is usually added as a chemical shift and intensity reference. Standardized procedures should be followed to ensure repeatability and comparability,75 and it is important that all the samples contain the same volume. 13C NMR and 1H NMR spectra are fi rst obtained by Fourier transformation of the resulting free-induction decay (FID) function after applying a prospective line-broadening function. Normally, phasing and baseline correction are applied but no zero fi lling, because it may interfere with the multivariate data analysis. Typically, the chemical shift scale is referred to the shift of TMS or indirectly to TMS by the peaks from chloroform at 7.28 ppm for 1H NMR and by the triplet of CDCl3 at 77.0 ppm for 13C NMR. Factors that affect the exact chemical shift of NMR signals include the type of solvent used, pH, interactions with metal ions, hydrogen bondings, and other intermolecular interactions.76 In some studies, a semiquantitative 13C NMR approach has been chosen, due to the fact that quantitative measurements require a considerable longer experimental time. However, even though the signal intensities within each spectrum are not quantitative, the relative intensities for corresponding signals across different spectra are comparable. The assignment of spectral resonances gives information about the chemical composition of the samples, but it is not necessary for classification purposes. The application of multivariate statistics to NMR spectral data increases the potential of the technique considerably. Both the area/ intensities of peaks and full spectra can be input for multivariate analysis. When full spectra are used, they are normally converted to ASCII or JCAMP file formats. Regions without signals or unwanted signals are removed before multivariate analysis.66 Potential problems about inconsistencies in ppm values between samples in the data analyses should be solved by manual alignment or data pretreatment methods.77 Regarding reproducibility issues, the whole procedure from sample preparation to analysis by a data exploration technique can be affected by factors unrelated to the sample characteristic of interest.75 Small differences in experimental conditions, such as instabilities in apparatus, temperature variations, inhomogeneities in the applied magnetic field, or differences in relative concentrations of the samples analyzed, may lead to erroneous classification.77 It is advisable to check that all spectra have acceptable linewidth and lineshape after the NMR analysis. It is expected that in the future the use of flow injection systems, ideally for screening many samples with short acquisition time, will increase the sample throughput significantly, although this approach has still not been widely used for authentication purposes.71 Another technique that in the future may be used more often is the analysis of intact tissue by high-resolution magic angle spinning (HR-MAS).78
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14.6 Stable Isotopes The variation in the abundance of the stable isotopes of carbon, nitrogen, and oxygen has been proposed as a method suitable for food authentication.79 Carbon exists as two stable isotopes: 12C (abundance 98.89%) and 13C (1.11%); nitrogen as two: 14N (99.63) and 15N (0.37%); and oxygen as three: 16O (99.759%), 17O (0.037%), and 18O (0.204%). Since a molecule containing heavier isotopic forms has stronger chemical bonds, the abundance of stable isotopes varies among different compounds. In addition, a kinetic fractionation occurs, because the enzymatic reaction rates on substrates that contain the lighter isotopic forms are faster than in reactions involving the heavier isotopic forms. Thus, the abundances of the stable isotopes differ between substrate and product. Moreover, since the physical properties of molecules containing heavier isotopic forms are different, equilibrium reactions also lead to a fractionation of the isotopic forms. A significant kinetic fractionation is already found in the initial fi xation of carbon dioxide in photosynthesis: the isotopic signature of C3 plants (plants that form a three-carbon compound as the first stable intermediate in the incorporation of CO2, mostly broadleaf plants and plants in the temperate zones) shows a higher degree of 13C depletion than the C4 plants (where the CO2 is converted first into a four-carbon organic acid: these plants are mostly found in warm sunny regions, typically tropical grasses, such as maize, although many broadleaf plants are also C4). For example, while typical d13C mean values of C3 plants may be −26/−28‰, C4 plants may have d13C mean values of −12/−14‰.80 The natural isotopic abundance largely varies depending on the chemical forms. Some atmospheric gases, such as CO2, N2, and O2, exhibit limited variation. In contrast, N2O and CH4 exhibit wide isotopic variation, and they reflect both significant isotopic fractionation by microbes and the different biological substrates producing these gases. The isotopic abundances in animal tissues and animal food products are the summation of the feeds ingested throughout all their life, plus the kinetic fractionations occurring in animal metabolism. For example, the 13C/12C ratio for both milk fat and cheese protein give information on the type of forage fed to the cows.81 This is because the 13C/12C ratio depends almost exclusively on the photosynthetic mechanism used by the plants for CO2 fi xation. Differences in the 15N/14N ratio also result essentially from diet. Usually animal products become enriched in the heavier isotope (15N and 13C), depending on their diet and their position in the trophic chain: the higher its position in the trophic chain, the higher the proportion of the heavier isotope.82 Dempson and Power83 examined the potential of using stable isotopes of carbon and nitrogen 13 (d C and d15N) by isotope ratio mass spectrometry (IRMS) to identify escaped farmed Atlantic salmon. Samples of muscle tissue of wild salmon were significantly more enriched in nitrogen (d15N: mean = 12.75; SD ± 0.38‰) but depleted in lipid-corrected carbon (d13C: mean = −20.51; SD ± 0.23‰) than the aquaculture specimens, resulting in a complete separation of the two groups. Aursand et al.50 were also able to correctly classify Atlantic salmon according to their geographic origin and production method by using four FA compositions (C16:0, C16:1n-9, C18:1n-9, and C22:1n-9) together with the overall isotope ratio 2H/1H of the fish oils and three deuterium molar fractions obtained by site-specific natural isotope fraction studied by NMR (SNIF–NMR). Using the d15N of choline and the d18O of total oil, Thomas et al.52 were able to classify correctly according to the production method, 171 Atlantic salmon specimens originating from three continents and 15 different geographic regions. Introducing the percentage of C18:2n6 as a third variable in their model, they were also able to correctly classify the fish according to their geographic origin. Bell et al.46 were equally successful classifying sea bass using the FA profile, d13C of individual FA,
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d13C and d18O of total muscle oil, and d15N of the glycerol choline fraction of flesh phospholipids. Molkeltin et al.1 were able to differentiate wild, organic, and commercially farmed Atlantic salmon measuring d13C and d15N by IRMS in raw fillets. The research of Sweeting et al.84 has helped to understand the nitrogen isotopic variations in fishes, since these authors assessed the effects of body size, experimental duration, and environmental conditions on fish tissue. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue but not for liver. Interestingly, the d15N values of heart and liver were also affected by environmental temperature, probably reflecting the metabolic functions of these tissues and their associated turnover rates.
14.6.1 Sample Preservation It is very important not to contaminate the sample during handling. For example, it must not be washed in the laboratory after collection (which may alter the O and H profile of the sample). The C and O isotopic profiles of fish tissues may be altered if CO is used for stunning or killing, and so on. Usually, the collected tissue samples are dried at a constant temperature of approximately 50°C for 48 h, pulverized to a fine powder using a ball mill grinder, and stored in glass desiccation vials until analyzed.
14.6.2 SNIF–NMR and IRMS Two methods are used to assess stable isotopes: SNIF–NMR and IRMS.48 NMR techniques have been described previously. An advantage of SNIF–NMR over IRMS is that it produces a distinct isotopic fingerprint giving information on the frequency of each isotope in a given molecule and the position of the isotope in the molecule, whereas IRMS gives only an average value of the isotopic forms in the molecule. However, SNIF–NMR can only be applied to the few isotopomers possessing spin, whereas IRMS can be applied to all except 12 elements. The light elements, such as carbon, nitrogen, and oxygen isotopes, are typically determined with a gas isotope rationing mass spectrometer. The instrument consists of an ionizing source, a flight tube with a magnet, and a detector to measure the different isotopic species. The element is converted to a gaseous form to be analyzed by the mass spectrometer, thus hydrogen is introduced as H2, carbon as CO, nitrogen as N2, and oxygen as CO2. The gas is introduced in the mass spectrometer and is ionized by removal of an electron in the ion source. The ionized gas is then introduced in the flight tube under vacuum or carried by helium; the paths of isotopic species are deflected by the magnet by an angle that is a direct function of their mass over charge ratio, the ions are finally detected at the detector, and the abundance ratios of the heavy and light isotopic species are then calculated. Approximately 1 mg of dried, ground tissue is used in the simultaneous analysis of stable C and N isotopes.85,86 To facilitate comparisons between specimens with differing lipid contents, d13C values are normalized for lipid content following techniques developed by McConnaughey and McRoy87 and validated by Kline et al.88 Stable isotope ratios are expressed in delta (d) notation with measurements consisting of parts per thousand difference (‰) between the isotopic ratio of a sample relative to an international standard, as follows: d = (R sample/R standard − 1) × 1000‰, where R is the heavy:light isotopic ratio of the sample or standard, respectively (for carbon 13C:12C, for nitrogen 15N:14N). Enriched samples contain relatively more of the heavier isotopes. All international standards are
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set at 0‰ by convention. Carbonate rock from the Pee Dee Belemnite formation89 and nitrogen gas in the atmosphere90 are used as the standards for carbon and nitrogen, respectively.
14.7
Trace Element Fingerprint
Sometimes, farmed and wild specimens of the same species have different geographic distributions, and often the geographic origin of both farmed and wild seafood may be of relevance for its safety, quality, and price. Multivariate trace elemental analysis is increasingly used as a technique to differentiate the geographic origins of foodstuff.91 In the case of fish, otolith chemistry is used as a recorder of time and environmental conditions.92–94 Otolith chemistry is useful for identifying the natal origin and assessing the relative contribution of different nursery areas to mixed adult stocks. Thus, in addition to DNA-based species identification techniques, multivariate trace elemental analysis is expected to be helpful in determining whether the fish was farmed and its geographic distribution. Biochemical analytical techniques using multiple elemental analysis, as well as vitamin K and its metabolites, have been used to differentiate the geographical distribution of origin of farmed Japanese eel. Recently, false labeling problems were encountered in which imported live Japanese eels from Taiwan were illegally sold as being of Japanese origin. The origins of farmed and wild eel collected from different regions in Japan, Taiwan, and China were compared by analyzing the trace and heavy metal contents in the muscles to determine the differences among the fish farms for cultured eels and also to identify the river where wild eels had been caught.95 By using ICP-MS analysis the sensitivity in the determination of rare trace elements can be increased from the nM to pM level. Rare trace elements taken from the environment, such as uranium, lead, cadmium, and vanadium, were shown to be of relevance to determine the origin of eels. The same research group (Yamashita et al., unpublished data) examined the trace element composition of the muscle and shell of littleneck clams collected in Japan, Korea, and China, and they found distinct patterns for each of the three origins. Thirteen elements were shown to be the most diagnostic. Multivariate analysis showed that differences in elemental composition in the muscle between Japanese and imported clams were mainly due to two factors: Factor 1, attributable to cobalt, copper, and strontium levels and Factor 3, attributable to manganese and vanadium levels. In addition, cadmium and arsenic levels in the muscles of clams from China and Korea were higher than those of clams from Japan, with the exception that clams from Miyagi had high arsenic content. Therefore, multiple elemental analysis could also be used in this case to identify imported clams from China and Korea.
14.7.1 Sample Preservation As for stable isotope analysis, it is very important to avoid contaminating the sample during sampling, handling, and analysis, in particular since the analysis may detect contaminants at the pM level. All implements and containers should be cleaned with 0.5 M nitric acid and rinsed with Milli-Q ultrapure deionized water. Each sample should be separated from the tissues using ceramic knives and scissors and Teflon-coated tweezers to avoid contamination of metals, and it must be accurately weighed with a microbalance. The sample may be stored in a centrifuge tube at a temperature of −40°C or lower until analyzed. The first step in the analysis is the digestion of the sample: 0.1–1 g of tissue samples are placed into 50 mL Teflon tubes and 8–16 sample volumes of a mixture of concentrated trace-metal-grade nitric acid/hydroperoxide mixture (5:3) is added. The
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digestion may be carried out by placing the tubes in a microwave oven (for example, Multiwave 3000 Microwave Oven, Perkin-Elmer), and the resulting digest is a clear liquid with a yellow tint. Afterwards, an internal standard mixture is added, the samples are diluted to a final volume of 50 mL in Digitube (SCP Science, Canada), and stored at room temperature until use.
14.7.2 ICP-MS Multielement determination of trace elements is usually measured by inductively coupled plasma mass spectroscopy (ICP-MS).96 Samples digested as described above are introduced by pneumatic nebulization into a radio frequency plasma, where energy transfer processes cause desolvation, atomization, and ionization. The ions are extracted from the plasma through a differentially pumped vacuum interface and are separated on the basis of mass-to-charge ratio by a quadrupole mass spectrometer that has a minimum resolution capability of 1 atomic mass unit (amu) peak width at 5% peak height. Ions transmitted through the quadrupole are detected by continuous dynode electron multiplier assembly, and the ion information is processed by a data handling system. To initiate the proper operating configuration of the instrument and data system, the mass calibration and resolution are checked using diluted metal solutions as standards. For internal standardization, five internal standards are used: Sc, Y, In, Tb, and Bi. To verify that the instrument is properly calibrated on a continuous basis, a calibration blank and calibration standards are used as surrogate test samples after every 10 analyses. If the measured concentration deviates from the true concentration by more than 10%, the instrument is recalibrated, and the last 10 samples are analyzed again. For the determination of mercury, which suffers from severe memory effects, the total mercury concentration is determined by cold vapor atomic absorption spectrometry,97 using an automatic mercury analyzer (Hiranuma HG-200, Japan). Each solution (5 mL) of the microwave samples is applied to the atomic absorption spectrophotometer.
14.8
Other Methods
Depending on the species, there might be specific requirements that may be targeted to identify the production method. In the farming of salmon for example, the use of carotenoids is allowed, but although the diet of wild salmon contains astaxanthin, most artificial feeds contain a mixture of canthaxanthin and astaxanthins of different origins (both natural and synthetic). Analysis by chiral chromatography can be used to identify a chiral form (the meso form 3R,3′S) that does not occur naturally and can therefore be used as a marker for farmed salmon.42,98 However, much of the astaxanthin used in fish feed nowadays is produced from cultured microalgae or from krill,98 making this approach more unreliable than it used to be.
Acknowledgments This work was carried out with the financial support of the EU-STREP Project Sigma Chain: “Developing a Stakeholders’ Guide on the Vulnerability of Food and Feed Chains to Dangerous Agents and Substances” Contract No FOOD-CT-2004-506359, the Norwegian Research
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Council, SINTEF Fisheries and Aquaculture, and by grants from the Fisheries Research Agency and the Ministry of Agriculture, Forestry and Fisheries of Japan.
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22. Bucklin, A. and Kochert, T.D., Source regions for recruitment of Calanus finmarchicus to Georges Bank: Evidence from molecular population genetic analysis of mtDNA, Deep Sea Res. II, 43, 1665, 1996. 23. Delghandi, M., Mortensen, A., and Westgaard, J.I., Simultaneous analysis of six microsatellite markers in Atlantic cod (Gadus morhua): A novel multiplex assay system for use in selective breeding studies, Mar. Biotechnol., 5,141, 2003. 24. Lie, O. et al., Verification of food origin based on nucleic acid pattern recognition, Genomar, Asa (NO), EP1472366, http://www.freepatentsonline.com/EP1472366A2.html, 2004. 25. Martin, S.A.M. et al., Proteomic sensitivity to dietary manipulations in rainbow trout, Biochim. Biophys. Acta, 651, 17, 2003. 26. Martinez, I., Slizyte, R., and Dauksas, E., High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish, Food Chem., 102, 504, 2007. 27. Olsson, G.B. et al., Metabolic disorders in muscle of farmed Atlantic cod (Gadus morhua), Aquacult. Res., 38, 1223, 2007. 28. Gomes, E.F. et al., Replacement of fish meal by plant-proteins in diets for rainbow trout (Oncorhynchus mykiss)-effect of the quality of the fish-meal based control diets on digestibility and nutrient balance, Water Sci. Technol., 31, 205, 1995. 29. Kaushik, S.J. et al., Partial or total replacement of fish meal by soybean protein on growth, protein utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss, Aquaculture, 133, 257, 1995. 30. Burel, C. et al., Potential o f plant-protein sources as fish meal substitutes in diets for turbot (Psetta maxima): Growth, nutrient utilisation and thyroid status, Aquaculture, 188, 363, 2000. 31. Carter, C.G. and Hauler, R.C., Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo salar L., Aquaculture, 185, 299, 2000. 32. Cowey, C.B., Protein and amino acid requirements: A critique of methods, J. Appl. Ichthyol., 11, 199, 1995. 33. Krogdahl, A., Lea, T.B., and Olli, J.L. Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss), Comp. Biochem. Physiol., 107A, 215, 1994. 34. Olsson, G.B. et al., Gelatinolytic activity in muscle of farmed and wild Atlantic cod (Gadus morhua) related to muscle softening, in Seafood from Fish to Dish, Quality, Safety and Processing of Wild and Farmed Fish, Luten, J.B. et al., Eds., Wageningen Academic Publishers. the Netherlands, 2006, 161. 35. Vielma, J. et al., Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load, Aquaculture, 183, 349, 2000. 36. Francis, G., Makkar, H.P.S., and Becker, K., Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish, Aquaculture, 199, 197, 2001. 37. Roth, B., Slinde, E., and Arildsen, J., Pre or post mortem muscle activity in Atlantic salmon (Salmo salar). The effect on rigor mortis and the physical properties of flesh, Aquaculture, 257, 504, 2006. 38. Johnston, I.A. et al., Fast growth was not associated with an increased incidence of soft flesh and gaping in two strains of Atlantic salmon (Salmo salar) grown under different environmental conditions, Aquaculture, 265, 148, 2007. 39. Bio-Rad. 2-D Electrophoresis for Proteomics: A Methods and Product Manual, Garfin, D. and Heerdet, L., Eds., http://www.biorad.com/LifeScience/pdf/Bulletin_2651.pdf, 2001. 40. GE Healthcare. 2-D Electrophoresis. Principles and Methods. Handbook 80-6429-60AC. (http://www1. gelifesciences.com/aptrix/upp00919.nsf/Content/2A3643B6787 885E0C 12570BE000DC671/$file/ 80642960.pdf, 2004. 41. Granvogl, B., Ploscher, M., and Eichacker, L.A., Sample preparation by in-gel digestion for mass spectrometry-based proteomics, Anal. Bioanal. Chem., 389, 991, 2007.
230 ◾ Handbook of Seafood and Seafood Products Analysis 42. Martinez, I., Revision of analytical methodologies to verify the production method of fish, in Seafood from Fish to Dish, Quality, Safety and Processing of Wild and Farmed Fish, Luten, J.B. et al., Eds., Wageningen Academic Publishers, the Netherlands, 2006, p. 541. 43. Martinez, I., Evaluation of the profile of lipids as a tool to discriminate wild from farmed salmon, 52 p. SEAFOODplus report 6.3.8. ISBN 978-87-7075-001-1, 2006. 44. Jobling, M., Are modifications in tissue fatty acid profiles following a change in diet the results of dilution? Test of a simple dilution model, Aquaculture, 232, 551, 2004. 45. Olsson, G.B. et al., Seasonal variations in chemical and sensory characteristics of farmed and wild Atlantic halibut (Hippoglossus hippoglossus), Aquaculture, 217, 191, 2003. 46. Bell, J.G. et al., Discrimination of wild and cultured European sea bass (Dicentrarchus labrax) using chemical and isotopic analyses, J. Agric. Food Chem., 55, 5934, 2007. 47. Grigorakis, K., Compositional and organoleptic quality of farmed and wild gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) and factors affecting it: A review, Aquaculture, 272, 55, 2007. 48. Martinez, I., Authenticity assessment based on other principles: Analysis of lipids, stable isotopes and trace elements, in Fishery Products: Quality, Safety and Authenticity, Oehlenschläger, J., and Rehbein, H., Eds., Blackwell Publishing, p. 388, 2009. 49. Chen, I. et al., Differentiation of cultured and wild sturgeon based on fatty acid composition, J. Food Sci., 60, 631, 1995. 50. Aursand, M., Mabon, F., and Martin, G.J., Characterization of farmed and wild salmon (Salmo salar), JAOCS, 77, 659, 2000. 51. Standal, I.B. et al., Discrimination of cod liver oil according to wild/farmed and geographical origins by GC and 13C NMR, JAOCS, 85, 105, 2008. 52. Thomas, F. et al., Determination of origin of Atlantic salmon (Salmo salar): The use of multiprobe and multielement isotopic analyses in combination with fatty acid composition to assess wild or farmed origin, J. Agric. Food Chem., 56, 989, 2008. 53. Gunstone, F.D., The Lipid Handbook, 2nd ed., Gunstone, F.D., Harwood, J.L., and Padley, F.B., Eds., Chapman & Hall, London, U.K., 1994. 54. Henderson, R.J., Bell, J.G., and Park, M.T., Polyunsaturated fatty acid composition of the salmon (Salmo salar L.) pineal organ: Modification by diet and effect on prostaglandin production, Biochim. Biophys. Acta, 1299, 289, 1996. 55. Dosanjh, B.S. et al., Influence of dietary blends of menhaden oil and canola oil on growth, muscle lipid composition, and thyroidal status of Atlantic salmon (Salmo salar) in sea water, Fish Physiol. Biochem., 19, 123, 1998. 56. Grisdale-Helland, B. et al., Influence of high contents of dietary soybean oil on growth, feed utilization, tissue fatty acid composition, heart histology and standard oxygen consumption of Atlantic salmon (Salmo salar) raised at two temperatures, Aquaculture, 207, 311, 2002. 57. Torstensen, B.E., Froyland, L., and Lie, Ø., Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil—Effects on Atlantic salmon (Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities, Aquacult. Nutr., 10, 175, 2004. 58. Aursand, M., Standal, I.B., and Axelson, D.E., High-resolution C-13 nuclear magnetic resonance spectroscopy pattern recognition of fish oil capsules, J. Agric. Food Chem., 55, 38, 2007. 59. Bell., J.G. et al., Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism, J. Nutr., 131, 1535, 2001. 60. Nichols, P.D., Mooney, B.D., and Elliot, N.G., Nutritional Value of Australian Seafood II, CSIRO Marine Research and FRDC, Hobart, Tasmania, Australia, 198pp. ISBN 1-876-996-07-2, 2002. 61. Bell, J.G. et al., Rapeseed oil as an alternative to marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): Changes in flesh fatty acid composition and effectiveness of subsequent fish oil “wash out,” Aquaculture, 218, 515, 2003.
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62. Refsgaard, H.H.F., Brockhoff, P.B., and Jensen, B., Biological variation of lipid constituents and distribution of tocopherols and astaxanthin in farmed Atlantic salmon (Salmo salar), J. Agric. Food Chem., 46, 808, 1998. 63. Skog, T.E. et al., Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L., Aquaculture Res., 34, 999, 2003. 64. Fernandez-Jover, D. et al., Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms, Mar. Environ. Res., 63, 1, 2007. 65. Gribbestad, I.S., Aursand, M., and Martinez, I., High resolution 1H magnetic resonance spectroscopy of whole fish, fillets and extracts of farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses, Aquaculture, 250, 445, 2005. 66. Lindon, J. C., Holmes, E., and Nicholson, J. K., Pattern recognition methods and applications in biomedical magnetic resonance, Prog. Nucl. Magn. Reson. Spectrosc., 39, 1, 2001. 67. Alam, T. M. and Alam, M. K. Chemometric analysis of NMR spectroscopy data: A review, Annu Rep. NMR Spectrosc., 54, 41, 2005. 68. Aursand, M., Rainuzzo, J., and Grasdalen, H., Quantitative high-resolution 13C and 1H nuclear magnetic resonance of fatty acids from white muscle of Atlantic salmon (Salmo salar), J. Am. Oil Chem. Soc., 70, 971, 1993. 69. Sacchi, R. et al., Proton nuclear magnetic resonance rapid and structure-specific determination of w-3 polyunsaturated fatty acids in fish lipids, J. Am. Oil Chem. Soc., 70, 225, 1993. 70. Masoum, S. et al., Application of support vector machines to 1H NMR data of fish oils: Methodology for the confirmation of wild and farmed salmon and their origins, Anal. Bioanal. Chem., 387, 1499, 2007. 71. Rezzi, S. et al., Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis, J. Agric. Food Chem., 55, 9963, 2007. 72. Aursand, M. and Grasdalen, H., Interpretation of the 13C NMR spectra of omega-3 fatty acids and lipid extracted from the white muscle of Atlantic salmon (Salmo salar), Chem. Phys. Lipids, 62, 239, 1992. 73. Aursand, M., Jørgensen, L., and Grasdalen, H., Positional distribution of n-3 fatty acids in marine lipid triacylglycerols by high-resolution 13C nuclear magnetic resonance spectroscopy, J. Am. Oil Chem. Soc., 72, 293, 1995. 74. Aursand, M. and Axelson, D.E., Origin recognition of wild and farmed salmon (Norway and Scotland) using 13C NMR spectroscopy in combination with pattern recognition techniques, in Magnetic Resonance in Food Science: A View to the Future, Webb, G.A. et al., Eds., RSC Books, London, U.K., 2001, p. 227. 75. Defernez, M. and Colquhoun I.J., Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra, Phytochemistry, 62, 1009, 2003. 76. Fan, T.W.M., Metabolite profiling by one-and two-dimensional NMR analysis of complex mixtures, Prog. Nucl. Magn. Reson. Spectrosc., 28, 161, 1996. 77. Forshed, J., Schuppe-Koistinen, I., and Jacobsson, S.P., Peak alignment of NMR signals by means of a genetic algorithm, Anal, Chim. Acta 487, 189, 2003. 78. Aursand, M., Gribbestad, I.S., and Martinez, I. Omega-3 fatty acid content of intact muscle of farmed Atlantic salmon (Salmo salar) examined by 1H MAS NMR spectroscopy, in Handbook of Modern Magnetic Resonance Modern Magnetic Resonance, Part 1: Applications in Chemistry, Biological and Marine Sciences. Webb, G.A., Ed., Springer, Amsterdam, the Netherlands, 2006, 931. 79. Dennis, M., Recent developments in food authentication, Analyst, 123, 151R, 1998. 80. De Niro, M.J. and Epstein, S., Carbon isotopic evidence for different feeding patterns in two hyrax species occupying the same habitat, Science, 201, 906, 1978. 81. Camin, F. et al., Application of multielement stable isotope ratio analysis to the characterization of French, Italian, and Spanish cheese, J. Agric. Food Chem., 52, 6592, 2004.
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82. Franke, B.M. et al., Geographic origin of meat - elements of an analytical approach to its authentication, Eur. Food Res. Technol., 221, 493, 2005. 83. Dempson, J.B. and Power, M., Use of stable isotopes to distinguish farmed from wild Atlantic salmon, Salmo salar, Ecol. Freshwater Fish, 13, 176, 2004. 84. Sweeting, C.J. et al., Effects of body size and environment on diet-tissue d15N fractionation in fishes, J. Exp. Mar. Biol. Ecol., 340, 1, 2007. 85. Doucett, R.R. et al., Evidence for anadromy in a southern relict population of Arctic charr from North America, J. Fish Biol., 55, 84, 1999. 86. Guiguer, K.R.R.A. et al., Using stable isotopes to confirm the trophic ecology of Arctic charr morphotypes from Lake Hazen, Nunavut, Canada, J. Fish Biol., 60, 348, 2002. 87. McConnaughey, T. and McRoy, C.P., Food-web structure and the fractionation of carbon isotopes in the Bering Sea, Mar. Biol., 53, 257, 1979. 88. Kline, T.C., Wilson, W.J., and Goering, J.J., Natural isotope indicators of fish migration at Prudhoe Bay, Alaska, Can. J. Fish. Aquat. Sci., 55, 1494. 1998. 89. Craig, H., Isotopic standards for carbon and oxygen and correction factors for mass spectrometric analysis of carbon dioxide, Geochim. Cosmochim. Acta, 12, 133, 1957. 90. Mariotti, A., Atmospheric nitrogen is a reliable standard for natural 15N abundance measurements, Nature 303, 685, 1983. 91. Ghidini, S. et al., Stable isotopes determination in food authentication: A review, Ann. Fac. Medic. Vet. Di Parma, XXVI, 193, 2006. 92. Campana, S.E., Chemistry and composition of fish otoliths: Pathways, mechanisms and applications, Mar. Ecol. Prog. Ser., 188, 263, 1999. 93. Rooker, J.R. et al., Identification of northern bluefin tuna stocks from putative nurseries in the Mediterranean Sea and western Atlantic Ocean using otolith chemistry, Fish. Oceanogr., 12, 75, 2003. 94. Thorrold, S.R. et al., Trace element signatures in otoliths record natal river of juvenile American shad (Alosa sapidissima), Limnol. Oceanogr., 43, 1826,1998. 95. Yamashita, Y., Omura, Y., and Okazaki, E., Distinct regional profiles of trace element content in muscle of Japanese eel Anguilla japonica from Japan, Taiwan, and China, Fish. Sci., 72, 1109, 2006. 96. AOAC Official Method 993.14. Trace elements in waters and wastewaters. Inductively coupled plasma-mass spectrometric method. First action, 1993. 97. Ihnat, M., Committee on residues and related topics—Metals and other elements, J. AOAC Int., 89, 290, 2006. 98. Albert, R. et al., Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin, J. AOAC Int., 80, 622, 1997.
Chapter 15
Smoke Flavoring Technology in Seafood Vincent Varlet, Thierry Serot, and Carole Prost Contents 15.1 Introduction ................................................................................................................. 234 15.2 Smoke Flavoring Process .............................................................................................. 234 15.2.1 Liquid Smokes................................................................................................. 236 15.2.2 Smoke Oils ...................................................................................................... 237 15.2.3 Smoke Powders ............................................................................................... 237 15.2.4 Smoke By-Products ......................................................................................... 237 15.3 Use of Smoke Flavorings .............................................................................................. 238 15.4 Chemical Composition of Liquid Smokes .................................................................... 239 15.5 Role of SF Process Parameters in Volatile Compounds Generation .............................. 240 15.6 Organoleptic Roles of Volatile Compounds of SF .........................................................241 15.6.1 Role of Volatile Compounds of SF in the Odor ............................................... 241 15.6.2 Role of Volatile Compounds of SF in the Flavor .............................................. 245 15.6.3 Role of Volatile Compounds of SF in the Texture ............................................ 246 15.6.4 Role of Volatile Compounds of SF in the Aspect and Color ............................ 246 15.7 Role of Volatile Compounds of SF in Preservation ........................................................247 15.8 Polycyclic Aromatic Hydrocarbons............................................................................... 248 15.8.1 Properties and Toxicology ............................................................................... 248 15.8.2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF ...................................................................................................249 15.9 Legislative Aspects.........................................................................................................249 15.9.1 European Regulations on PAH Found in SF ....................................................249 233
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15.9.2 European Regulations on PAH Concentration in Food Treated by SF ....................................................................................250 15.10 Conclusion ..................................................................................................................250 References ................................................................................................................................251
15.1
Introduction
Smoking is the oldest food preservation technique. Coupled to salting and drying steps, it allows decreasing microorganism activity. Moreover, wood smoke phenolic components are known to be antioxidants. Simultaneously, wood smoke imparts desired organoleptic characteristics such as smoky flavor. However, the smoking process has two main inconveniences: the production of carcinogenic contaminants—polycyclic aromatic hydrocarbons (PAHs)—during the incomplete pyrolysis of wood used to produce smoke and the release of smokes in the atmosphere. In United States where 75% of smoked foods are treated by liquid smokes, liquid smokes are commercialized since the end of the nineteenth century. This kind of smoke flavoring (SFs) appears as an alternative to the smoking process as it is carried out in Europe. Indeed, the use of liquid smokes avoids the release of smokes, allows a better control of PAH in the final product, and provides a higher diversity of smoked food [1]. Today, between 20% and 30% of European smoked food is treated by liquid smokes. SFs are widely used in the meat industry, and their uses in the seafood industry are increasing, especially thanks to the industrial benefits brought about by their use. By comparison with the smoking technology, the use of SF allows an easier storage (SF bottles versus wood logs), a better preservation of the combustible, and reduced risk of accidents due to fire. This industrial process leads to more homogenous products smoked with a repeatable intensity and provides an easier cleaning of the smokehouse. However, the legislation and the organoleptic quality of SF and products treated by SF constitute critical points that show the necessity of better improvement and harmonization of this technology.
15.2 Smoke Flavoring Process The first liquid SF was developed and patented by the Kansas pharmacist Wright in the late of nineteenth century [2]. SFs are obtained by the condensation of wood smoke and can be further fractionated, purified, or concentrated. The main woods used for smoke production are oak, beech, hickory, and marple, that is, mainly hard woods. Indeed, the chemical composition of soft woods is responsible for the generation of higher quantities of contaminants as PAH. The fractionation of smoke condensates allows obtaining a high diversity of SFs (powders, oils, aqueous solutions, etc.) with a wide range of organoleptic qualities, which give rise to new perspectives in the food industry [3]. It also allows the reduction of the PAH final concentrations. A simplified version of SF processes is presented in Figure 15.1. Wood sawdust is pyrolyzed in a furnace with low oxygen content. The smoke is filtered to eliminate particules and condensed. The gaseous smoke can be cooled down by water or by organic solvents. The combustible gases are recycled and directed to the furnace. After condensation, the crude smoke condensates are separated in three phases: a water insoluble heavy oil by-products phase, a water-soluble phase, and a water insoluble tar phase. The heavy oil by-products, obtained after a settling out time (several days) of the smoke condensates in the settling tank, are recycled and directed also to
Recycled combustible gases
Recycled heavy oil by-products
Settling tank
Figure 15.1 Diagram of fabrication of SFs.
Wood dust by-products
Patented furnace
Condensing tower
Filter stage one
Filter stage two
Dryer/blender
Oil exchange system
Further processing
Smoke powders
Smoke oils
Aqueous smokes
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Smoke extracts
Primary products
Liquid smokes
Smoke distillates
Smoke oils
Aqueous flavours
Soluble aqueous flavours Concentrates of liquid smoke
Buffered aqueous flavours
Smoke powders
Figure 15.2 SFs from primary products.
the furnace because they cannot be used for human consumption. However, a purified extract of the high-density water insoluble tar phase can be used for the production of SFs and is called primary tar fraction (PTF). The water-soluble phase leads to primary smoke condensates (PSC). Therefore, smoke condensates obtained from PTF and PSC are named primary smoke products (PP). Different SFs (liquid smokes, smoke oils, smoke powders, or smoke by-products) can be obtained after different steps of filtration, separation, or drying/blending of these PP. They are presented in Figure 15.2.
15.2.1
Liquid Smokes
Different kinds of liquid smokes are available: aqueous flavors, concentrates of liquid smokes, soluble aqueous flavors, and buffered aqueous flavors. Aqueous flavors, due to their low pH, can be used directly or diluted for applications requiring lower concentrations [4]. They can be employed in sauces or marinades of seafood products. This form of SF is especially used for the smoky taste that it confers to the food. Concentrates of liquid smokes consist of concentrated versions of aqueous flavors and require lower usage quantities. They are especially used when the final water rate in the treated food must be low. They are employed to confer smoky organoleptic qualities (tastes, flavors) and also the characteristic aspect and color of smoked food. Soluble aqueous flavors are aqueous flavors that contain an emulsifier such as polysorbates, allowing better water solubility. They are used when intermediate product dispersion is required as in brine. Finally, buffered aqueous flavors are partially neutralized or buffered aqueous flavors. These products have a pH greater than 4 and can also be added to the brine.
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◾
237
Smoke Oils
Smoke oils are made by blending liquid smokes with vegetable oils, most frequently in 90:10 (v/v) proportions. They are less acidic than aqueous flavors and allow to exhibit more complex smoky tastes. However, these SFs are not used much in seafood industry, because smoke oils are especially employed in food preparations such as emulsions. As seafood emulsions are not very common, smoke oils can be only used in preparations such as taramas, fish sauces, or fish oils.
15.2.3 Smoke Powders Smoke powders are obtained by blending liquid smokes and dry powder carriers such as maltodextrines or barley and corn flours and drying them [5]. These smoke powders can be added to salt used for salting steps or to dehydrated sauces or soups elaborated from seafood products. Smoke powders can also be rehydrated and used in brine as liquid smokes. The final composition of smoke powders must be known in order to avoid the presence of allergens or other nonrequired additives such as nitrited salts. Nitrited salts, generally forbidden in seafood industry according to the countries, can be added to the smoke powders used in the meat industry to improve simultaneously the storage of food and to confer smoky characteristics to the final product. A reaction between the phenolic compounds of SFs and these nitrited salts or powders can lead to a nitrosation and to nitrophenols. These molecules can increase the generation of carcinogenic nitrosamines, consequent to the reaction between amino acids and nitrite. Therefore, smoke powders used in the meat industry should be different from those used in seafood industry in order to avoid nitrited salts in seafood treated with smoke powders. Therefore, it is very important to consider the salting step made with common salt mainly authorized for seafood and the curing technology made with a salt treated by nitrite and nitrate authorized for meat. We must distinguish dry salting and wet salting. Dry salting (or dry curing if nitrited salts are used) is made with dry salt deposited directly on food. Wet salting (or curing if nitrited salts are used) is made with brines spread on food or in which the food is dipped. Therefore, in seafood industry, the liquid smokes and smoke powders can be added to salt or in brine but not smoke oils. As smoke oils, smoke powders are mainly used to confer smoky tastes to the final product, whereas liquid smokes are used for the characteristic smoky odor and color of smoked products.
15.2.4 Smoke By-Products Smoke by-products are constituted by smoke extracts and smoke distillates [6]. Smoke extracts are produced by way of more or less selective extraction of smoke constituents directly from the smoke aerosol (by countercurrent circulation of water or organic solvents) or from the PP. Smoke distillates are obtained by the fractionating distillation of PP. The distillation is commonly performed with steam water at atmospheric pressure. Smoke by-products constitute more complex SFs. Consequently, their uses are really characteristic of a product and cannot be employed for a wide variety of food due to their typical organoleptic qualities. Consequently, hundreds of smoke by-products are available, but their uses are specific to a food: smoke aromatic preparations can be produced to treat certain kinds of meat and cannot be used for fishes for example. Indeed, the organoleptic qualities can vary in a high range changing the food matrix. Today, smoke manufacturers can control their products and can create smoke by-products whose uses are recommended for a kind of fish. SFs for herring, salmon, and so forth, are present in the market.
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The development of synthetic SFs must be also noticed. The progress made during the last decades in elucidating the chemical composition of wood smoke gave rise to attempts aiming at producing SF, composed entirely of synthetic compounds or partly from a liquid smoke base [7]. However, the synthetic SFs created are not sufficiently similar to real wood smoke or to SFs. Besides, SFs are so easy to produce that it would not be profitable to create synthetic SFs when natural ones are available at a cheap price.
15.3 Use of Smoke Flavorings There are four techniques to incorporate or deposit SFs in or on seafood products: showering, drenching/soaking, direct addition, and atomization. Showering is a technique currently used in North America, especially used for meat products, but it is also employed in the seafood industry. Water-based composed SFs such as soluble aqueous flavors or buffered aqueous flavors are commonly used in this technique, because to guarantee the homogeneity of the SFs during the treatment and to prevent the settling out of smoke condensates in water, an emulsifier must be added in the SFs. The final organoleptic qualities (color, taste, etc.) are dependant of the dilution of SFs in water according to proportions varying between 20% and 25% for SFs and 75% and 80% for water. Diluted SFs fall by gravity through perforated plates on the hung products. Liquid smoke solution is therefore recycled and filtered and the concentration is readjusted. Drenching/soaking is the opposite of showering technique, because the products are immersed in the SFs solution instead of pouring the SFs solution on the products. Products are dipped in SFs solution for short periods (from 5 to 60 s). Soluble aqueous flavors or buffered aqueous flavors are mainly used. They provide a better water solubility and prevent the heterogeneity of layer formation on the product surface or the product separation during storage. Direct addition consists in the incorporation of SFs during the fabrication of the food products. SFs can be incorporated directly with the ingredients during the formulation or through injection needles when the shape of the product cannot be modified. According to the final product, there are different carriers of SFs. Smoke powders are preferred when water use is impossible as in dehydrated mixes. Smoke oils are preferred for lipidic emulsions or lipidic sauces, but aqueous liquid smokes are the most used SFs in this technique. Indeed, liquid smokes are also employed in the curing brine, which can be injected into the product as for the salting step. Finally, atomization of SFs consists in the vaporization of liquid smokes, mainly liquid smoke concentrates on the products in a smokehouse. SF is sprayed with air under pressure through special nozzles and forms a wood smoke mist in the cell of smoking. The mist obtained is constituted of small droplets with a similar size as in real wood smoke, that is, between 15 and 20 mm. Therefore, from the granulometry point of view, vaporized liquid smoke is similar to real wood smoke, and this technique appears as an alternative to the smoking process. However, wood smoke is composed by a gaseous phase formed by the most volatile compounds, which carries a particulate or dispersed phase [8]. The mist generated is composed only by small droplets and there is no gaseous phase. From a physical point of view, the composition of liquid smoke mist is not similar to real wood smoke. This difference constitutes a critical point in the liquid smoking status, especially in the labeling of the smoked products. Indeed, in numerous European countries, products treated by liquid smoke atomization are considered as flavored and not smoked. In France, meat treated by this process is considered as smoked but « smoked by liquid smoke » must appear on the package. Other devices have been optimized in order to generate a similar physical composition of
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wood smoke with liquid smoke atomization. The SF is sprayed on a surface at high temperature, which favors the vaporization of SF [9]. Therefore, it creates a gaseous phase, but the optimization of the parameters to have a similar particulate or dispersed phase is not easy. In seafood industry, liquid smoke atomization is the most used technique of SF. The volume of liquid smoke mist is controlled by the number of nozzles and the smokehouse size. The adjustments are carried out on the flow of liquid smoke from the tank owing to a temporization on the liquid admission and on the flow of air under pressure. The smokehouse must be hermetically closed during atomization. The moisture control is essential, first to control the drying of the product and second, to favor the deposition of smoke components. In fish smoking, the drying step is necessary to prepare the surface of the fillets. This step must take into account the initial water rate of the raw material and the composition of the final product. The surface must present a beginning of protein coagulation, which confers a subtle glossy and sticky aspect. Important moisture favors the smoke penetration and strong smoky organoleptic characteristics, whereas weak moisture gives to the product a good color but a weaker smoky taste. According to the liquid smoke used, ventilation must be planned in order to reduce the moisture. Therefore, the methods of production and the possibilities of applications of SF are very wide. A high knowledge of the biochemical composition of the wood used and the parameters of the combustion are essential to generate SF. Similarly, a good knowledge of the food matrix to be treated is required to apply SF in the best conditions and to reach the expected organoleptic qualities controlled by SF chemical composition.
15.4
Chemical Composition of Liquid Smokes
The chemical composition of SF depends on the composition of the wood raw material used and especially the relative amounts and structure of its main components: two polysaccharides namely cellulose and hemicellulose and lignin. The SF composition can be complexified by the addition of spices and aromatic herbs [10]. The role of pyrolysis parameters as pyrolysis temperature, wood moisture, airflow, and air moisture are also essential in the SF final composition. The pyrolysis of cellulose initiates the hydrolysis of glucose followed by dehydration to 1,6-anhydroglucose (betaglucosan) and finally to acetic acid and its homologues, water, and sometimes small quantities of furans and phenolic compounds. The compounds generated from hemicellulose pyrolysis depend on the nature of the wood. Indeed, hemicellulose in hardwood (nonconiferous woods) is mainly constituted by pentosans whereas hemicellulose in softwood (coniferous woods) is mainly composed by hexosans. Hemicellulose pyrolysis leads to furan and its derivatives and aliphatic carboxylic acids. The thermal decomposition of pentosans provides a higher amount of furans than hexosans, which decompose to form alpha cellulose and provide a higher amount of PAH, hence the limitation of the use of softwoods for smoking. The wood polysaccharides lead to methanol, methanal, formic acid, acetaldehyde, hydroxyacetaldehyde, acetic acid, furfural and homologues, furanones, and various anhydroglucopyranoses (mostly levoglucosans) [11]. Glucuronic acids decompose to carboxylic acids, hence the high acidity of liquid smokes. Finally, lignin thermal decomposition provides compounds considered as most important for the smoke flavor, such as alkyl phenolic compounds and derivatives like phenolic ethers with methoxy groups in ortho position (guaiacol derivatives, predominant in softwoods) and in para position (syringol derivatives predominant in hardwoods). The main characteristics that permit the differentiation of hardwoods and softwoods are the guaiacol:syringol (G/S) and guaiacol:phenol (G/P) ratios. Hardwoods lead to G/S and G/P ratios, respectively, of 1.5 and 2. The pyrolysis of lignin can also lead to alkyls aryls
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ethers from lignans, lignin dimers, and trimers [12], but they have a weak impact on the smoky flavor of SF and food processed with SF [13]. Therefore, the main organoleptically active volatile compounds generated during the pyrolysis process can be sorted in three groups of molecules: the phenolic compounds, known as the smoky skeleton of SF, the furannic derivatives, and the enolones derivatives [14].
15.5
Role of SF Process Parameters in Volatile Compounds Generation
Except the wood type that influences the smoke quality strongly [15], the wood granulometry and moisture, the pyrolysis temperature, the velocity, and humidity of air constitute key parameters of SF composition. The generation of volatile compounds is dependent on the wood pyrolysis temperature [16]. After the water release (close to 120°C–150°C), exothermic reactions of pyrolysis of wood components occur between 200°C and 250°C for hemicellulose, between 280°C and 320°C for cellulose, and 400°C for lignin [17]. According to the pyrolysis process, different groups of compounds are formed. The rate of carbonyl compounds increases gradually with the temperature from 200°C to 600°C. The rate of acids is higher for temperature lower than 300°C and decreases after 300°C with the increase in temperature. From 200°C to 600°C, the quantity of phenolic compounds increases with a maximum close to 500°C and decreases after 500°C. However, differences can be observed depending on the molecules. For example, phenol amount is multiplied by two between 450°C and 650°C, whereas syringol quantity is tripled. A temperature of 450°C–500°C was reported to lead to the best composition for the creation of carbonyls, furannic compounds, and phenolic compounds [10,18,19]. PAH must also be surveyed, because their contents in smoke or in food increase from 400°C to 1000°C. As the best pyrolysis temperature to obtain the required volatile compounds are between 380°C and 500°C, it seems difficult to generate the desired organoleptic volatile compounds without PAH contaminants. A step of filtration is almost obligatory to avoid these contaminants. Indeed, steps of SF purification through filters or apolar solvent washes are often required to decrease the PAH levels. The wood moisture appears as the second important parameter [20]. The high moisture allows to reduce the wood combustion efficiency. Therefore, a lower temperature is reached and allows increasing the generation of smoke volatile compounds and minimizing PAH formation. The use of hardwood, with a lower water rate than that in softwood, is recommended because it burns slower. An optimal moisture is planned in the industry between 17% and 20%, whereas a rate between 20% and 30% has been reported as optimal to reduce the emission of particules [11]. The air velocity indirectly influences the SF composition by the modification of pyrolysis temperature or smoke temperature [21,22]. A slow combustion is reached with weak air velocity. Air moisture is also very important and must be set in adequation with air velocity to keep the water rate constant in the air during the combustion. Lower concentrations of oxygenated compounds have been found to be caused by an oxygen depletion during combustion [20]. Wood granulometry can also influence SF composition, because it plays a role in the pyrolysis temperature. The combustion is faster when the granulometry of the wood raw material is important [23,24]. Then, the diversity of settings of pyrolysis parameters can explain the diversity of organoleptic volatile compounds and the diversity of qualities of SF. The manufacturer can choose SF according to the required result on the organoleptic characteristics of the final product. Due to their
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chemical composition, the diversity of SF causes diverse consequences on the texture, odor, taste, color, and preservation of the product.
15.6
Organoleptic Roles of Volatile Compounds of SF
15.6.1 Role of Volatile Compounds of SF in the Odor Even if the concentrations of odorant volatile compounds in SF can be various, two main classes of odor-impact molecules can be defined: phenolic compounds and carbonyl compounds, which gather furannic and enolone derivatives. Phenolic compounds are known to constitute the odorant “smoky” skeleton of wood smoke and smoked fish. They are the major compounds in SF with a wide range of odorant thresholds (Table 15.1) [14,28,29]. Some of them have very low odorant thresholds, making them odorant at low concentrations. Many studies have indicated that phenolic compounds present in the vapor phase of smoke may be odor-active compounds [30–32]. Phenolic compounds of medium volatility have been considered as the most important odorant molecules. The medium-boiling fraction (91°C–132°C) composed of isoeugenols, syringol, and methylsyringol has a pure and characteristic smoky flavor [10]. These observations have been recently corrected [14,33,34]. Phenolic compounds of low-boiling fraction (60°C–90°C) composed mainly of phenol, cresols, guaiacol, and alkylguaiacol may also contribute to imparting a smoky flavor to smoked fishes [13,34] (Table 15.2). The role of syringol is important, but it may not be the main contributor to wood smoke flavor. Similarly, phenolic compounds are not sufficient to explain the SF role in smoked fish odors. A much more complex mixture of compounds is responsible for the characteristic aroma of smoked fishes [35]. Table 15.1 Odorant Thresholds of Various Phenolic Compounds Phenolic Compounds
Odorant Thresholds in Water (μg/L)
References
Phenol
5900
[25]
o-Cresol
650
[25]
m-Cresol
680
[25]
p-Cresol
55
[26]
Guaiacol
3–21
[25]
4-Methylguaiacol
90
[25]
4-Ethylguaiacol
50
[27]
4-Vinylguaiacol
3
[26]
Vanilline
20–200
[25]
Syringol
1850
[25]
Eugenol
6–30
[26]
Ethylvanilline
100
[25]
LRI (DB5)
859
865
875
890
904
920
925
970
992
1036
1052
1068
1093
1110
1130
Compounds
Furfural
4-Methylpyridine
Furfuryl alcohol
2,6-Dimethylpyridine
2,4-Hexadienal
2-Methyl-2-cyclopenten1-one
2-Acetylfuran
5-Methylfurfural
Phenol
2-Hydroxy-3-methyl-2cyclopenten-1-one
2,3-Dimethyl-2cyclopentenone
o-Cresol
p-Cresol
Guaiacol
2,6-Dimethylphenol
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI
MS, LRI
MS, LRI, STD
MS, LRI
MS, LRI, STD
Means of Identificationa
Chemical, burnt, spicy/ woody
Smoked, vanilla, ink
Animal, spicy, burnt
Chemical, spicy, burnt,
Spicy, wood fire, roasty
Cooked, spicy
Marine, metallic, chemical, mushroom
Cooked, earthy, green
Cooked vegetable, potato
Cooked potato, green
Cooked vegetable, fatty
Roasty, green, milk
Cooked/soup, chemical
Green, milk
Smoke, green
Odorant Attributes Given by the Judgesb
(3)
8
8
8
6
7
7
(4)
7
6
(5)
6
7
Number of Judgesc
(2)
7
6
5
3
4
4
(2)
6
5
(2)
4
4
Average Intensity d
(1.27 ± 0.75)
360.45 ± 172.07
74.18 ± 37.53
49.74 ± 27.62
17.48 ± 8.94
23.64 ± 18.44
65.55 ± 39.97
(24.63 ± 13.50)
20.22 ± 9.34
8.37 ± 3.98
(1.33 ± 1.53)
4.27 ± 2.90
42.17 ± 26.25
16.55 ± 9.12
124.24 ± 63.04
Mean ± SDe
Table 15.2 Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke
242 ◾ Handbook of Seafood and Seafood Products Analysis
1132
1140
1147
1160–1180
1192
1247
1266
1282
1287
1307
1330
1330
1365
1370
1382
1400
2,3,4Trimethylcyclopenten-1one
3-Ethyl-2-hydroxy-2cyclopentenone
1,2-Dimethoxybenzene
2,4- and 2,5Dimethylphenol/ (E)-2-nonenal
4-Methylguaiacol
2,3-Dimethoxytoluene
(E)-2-Decenal
3,5-Dimethoxytoluene
4-Ethylguaiacol
Indanone
4-Vinylguaiacol
(E,E)-2,4-Decadienal
Syringol
Eugenol
4-Propylguaiacol
1,2,3-Trimethoxy-5methylbenzene
MS, LRI
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI, STD
MS, LRI
MS, LRI, STD
MS, LRI
MS, LRI
MS, LRI
MS, LRI, STD
MS, LRI, STD
MS, LRI
MS
MS
Cooked, earthy
Green, spicy, vanilla
Spicy, smoke, clove
Burnt rubber, spicy
Oily, green, fatty
Smoke, green, spicy
Sawdust, rotten, burnt
Green, smoke, vanilla, clove
Burnt, green, chemical
Spicy, green, milk
Cooked vegetable, fatty, green
Candy, spicy, smoked
Cucumber, violet, spicy, smoked
Ashes, green
Solvent, medicinal
Cooked, green, spicy
(5)
8
8
8
7
(3)
7
8
(5)
6
7
7
8
6
7
6
(2)
5
5
5
5
(2)
4
6
(3)
3
4
5
6
3
4
5
◾
(continued)
(2.15 ± 1.15)
15.21 ± 7.86
36.51 ± 18.17
44.61 ± 22.91
8.82 ± 6.72
(3.24 ± 1.95)
2.87 ± 1.71
86.85 ± 40.97
(6.25 ± 4.15)
4.26 ± 1.79
6.62 ± 4.12
482.15 ± 243.13
18.96 ± 10,46
11.16 ± 5.50
10.49 ± 6.36
17.25 ± 10.35
Smoke Flavoring Technology in Seafood 243
1423
1473
1527
1615
1680
(Z)-Isoeugenol
(E)-Isoeugenol
2,3,5-Trimethoxytoluene
4-Allylsyringol
8-Heptadecene
MS, LRI
MS, LRI
MS, LRI
MS, LRI, STD
MS, LRI, STD
Means of Identificationa
Animal, roasty, chemical
Smoke, rotten
Spicy, woody
Clove, green, roasty
Burnt rubber, spicy
Odorant Attributes Given by the Judgesb
6
7
(4)
7
6
Number of Judgesc
4
4
(2)
4
3
Average Intensityd
6.87 ± 2.68
1.23 ± 0.39
(20.55 ± 8.48)
24.81 ± 11.35
7.40 ± 3.77
Mean ± SDe
e
d
c
b
a
Means of identification: MS, mass spectrum (identified using the mass spectra of the compounds), LRI, linear retention index (when the LRI of the identified compound corresponds to the LRI in the literature), STD, standard (when the retention time, spectrum, and odor description of an identified compound correspond to the retention time, spectrum, and odor description of the injected standard of this compound). When only MS is available for identification, it must be considered as an attempt of identification. The odor given corresponds to the odor detected by the judges during olfactometric analysis for its retention time but not surely due to the compound that we try to identify. Number of judges (out of eight) who have detected an odor. Average intensity of the eight judges is rounded to the nearest whole number. An intensity between 5 and 5.5 is rounded to 5 and an intensity between 5.5 and 6 is rounded to 6 (1 = very weak odor, 9 = very strong odor intensity). In micrograms equivalents of dodecane per 100 g of smoked salmon. Means of three fillets.
Note: Frequency of detection, odor intensity, and quantities of odor-active compounds detected by fewer than six judges are indicated in parenthesis.
Source: Varlet, V. et al., J. Agric. Food Chem., 55, 4518, 2007.
LRI (DB5)
Compounds
Table 15.2 (continued) Odorant Characteristics and Concentrations of the Most Potent Odorant Volatile Compounds in Salmon Fillets Treated by Liquid Smoke
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Carbonyl compounds have also been reported as contributors to the smoky aroma of wood smoke. A polyfunctional carbonyl subfraction was isolated from wood smoke and possessed a caramellic/burnt sugar aromatic note [36]. Two categories of carbonyl compounds can be differentiated: furannic compounds and enolone derivatives. Furfural and homologues exhibit cooked/roasty aromatic notes. Furannic compounds were thought to contribute to soften the heavy smoky aromas associated with phenolic compounds [37,38]. More recently, furannic compounds were found to play a role in cold smoke odors of liquid smoke or fishes treated by liquid smoke [14,34]. Enolone derivatives are compounds derived from cyclopentenone. They were isolated early from wood smoke and described as grassy, sometimes cooked, and seem to contribute little to overall aroma, because they are not the compounds mainly detected in SF and seafood treated by SF odors by sensory analysis [37]. However, as the other minor odor-active compounds, if they do not have a strong individual influence, they may contribute in mixture to the overall odor. The determination of the role of SF components in the final product odor is complex due to the odorant interactions that can occur between the odor-active compounds. Synergic or masking effects are possible and make the final odor complex. Furthermore, reactions between liquid smoke compounds and the components of the matrix can occur through Maillard and Strecker reactions. The amino acids from the seafood matrix and the carbonyl compounds from the SF can generate furannic compounds and nitrogen-containing compounds with roasty/smoky aromatic notes [19,39]. Moreover, the physical state of SF can also influence the aroma, and the odorant contribution of odor-active compounds cannot be the same if the SF is in the form of powders, liquids, or oils. The oils used in smoke oils can soften the smoke aroma.
15.6.2 Role of Volatile Compounds of SF in the Flavor Phenolic compounds were shown as the major contributors of the smoky flavor [35]. The taste thresholds of some phenolic compounds were determined [40] and showed a high diversity between the molecules, but taste was not investigated as much as odor. For several decades, it was commonly admitted that syringol derivatives impart a smoky odor and guaiacol derivatives contribute to a smoky flavor. Recently, guaiacol derivatives and more generally the phenolic compounds of low-boiling fraction molecules (60°C–90°C) have been shown to cause the odor. As for the assessment of the odor, the determination of the effects of compounds of SF on the flavor is complex, and very little information is available. However, early works performed on individual phenolic compounds have identified the impact of guaiacol on the smoky flavor, whereas syringol and 4-methylguaiacol showed the same but lower effect than guaiacol [40]. Concerning the bitter taste, 4-methylguaiacol perception was superior to that of syringol and guaiacol. The high-boiling fraction of phenolic compounds (133°C–200°C) was described with an acid and chemical property that was judged of poor quality. Then, the same compounds responsible for the odor should be involved in the flavor that SF confers to seafood. Sensory analysis performed on standards confirmed the importance of guaiacol and o-cresol in the smoky flavor and dimethylphenol, 4-methylguaiacol, and isoeugenol in spicy/sweet flavor [41]. However, phenolic compounds are not the only flavor-active compounds. The fractionation of a commercial liquid smoke preparation evaluated by a sensory panel concluded that the phenol fraction was essential but not complete from a sensory standpoint [42]. The results of this fractionation are given in Table 15.3.
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Sensory Taste Intensities of Liquid Smoke Fractions Fractionb
Taste Property
a b
a
1
2
3
4
5
6
Smoke taste intensity
6
7
3
11
4
10
Tarry taste intensity
3
1
2
0
6
1
Chemical taste intensity
1
1
3
0
1
0
Acidulous taste intensity
1
2
3
0
0
0
Intensity scale: 0 = below threshold; 11 = highest value. 1, distilled at 67°C–90°C; 2, distilled at 91°C–132°C; 3, distilled at 133°C– 200°C; 4, phenolic subfraction; 5, terpene subfraction; 6, whole liquid smoke.
Other compounds such as enolone derivatives could also play a role in the SF flavor. Studies on standards have shown that cyclotene was a flavor-active compound [41]. According to their concentrations found in the different SF, furannic compounds could also have an effect on the flavor [43].
15.6.3 Role of Volatile Compounds of SF in the Texture The texture of smoked products is due to coagulation of proteins. Formaldehyde, which has been reported in wood smoke and smoked meat [41] but not in smoked fishes until now, can react with proteins. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side chains of arginine, cysteine, histidine, and lysine residues [44]. Formaldehyde seems to be involved in the texture of smoked fishes and to be responsible for the layer at the dried surface of fishes [45]. The acidic aqueous SF can also increase the coagulation of proteins and act on the texture. SFs under powder or oil forms do not act on the surface texture, because they are added in the product during its fabrication. However, they could play a role in the inner texture of the product.
15.6.4 Role of Volatile Compounds of SF in the Aspect and Color The color of seafood treated by SF can derive from physical and chemical reactions. Indeed, smoke condensates are colored mainly due to phenolic compounds, which have brown/yellow characteristic color. Their physical deposition of SF on seafood can confer its color to the product, which can vary from golden yellow to dark brown according to the nature of the wood, the eventual dilutions of SF, and the intensity of the process. However, Maillard and Strecker compounds can also be responsible of the color of the smoked product [45]. In the liquid smoking process, the product must also be placed in a dry and hot ambient atmosphere for short periods in order to favor color formation. Thus, the deposition of Maillard compounds leads to a darker color of fish flesh [46]. A brief drying after smoke absorption can cause a higher level of dehydration and lead to higher amounts of Maillard products. After scission and dehydration, melanoidines could be created by polymerization through aldolic condensations. These
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compounds give to the final product a brown color, but no information is available concerning this pathway [47]. Carbonyl-amino reactions as Maillard reaction could play a main role in smoked food. Protein-bound lysine, the most prevalent essential amino acid in fish, because of its terminal amino group, is considered as a major source of the amino components in such reactions, but a loss in arginine and histidine is also observed. Glycolic aldehyde, methylglyoxal, and 2-oxopropanal are considered to be important color precursors [6,24]. A part of the fi nal color could derive from phenolic compounds with aldehyde function. Coniferaldehyde and syringaldehyde are considered to be irreversibly bound to proteins and to contribute orange tints to the products [24]. Finally, the glossy aspect noticeable on certain smoked products is the result of reactions between phenolic compounds and aldehydes [48]. They lead to resinous substances (phenoplasts). The polymerization is favored by the heat, and the degrees of reticulation of the molecule vary as a function of time [49].
15.7
Role of Volatile Compounds of SF in Preservation
Smoking process is the oldest preservation technique because of the antimicrobial and antioxidants properties of wood smoke. Food industries are working to develop new applications of smoke condensates, which could contribute to product safety by controlling the growth of foodborne pathogens. Studies on the antimicrobial activity of some smoke condensates have revealed very variable effects on the growth of microorganisms [50]. The antioxidant compounds of wood smoke condensates are those with an active phenolic function. The antioxidant behavior is increasing with the temperature of the boiling point of the phenolic compounds [44]. The most active compounds are polyhydroxyphenolic compounds such as pyrogallol and resorcinol. Among monohydroxyphenolic compounds, the antioxidant properties depend on the radical located in the para position from the hydroxy group as in 4-methylguaiacol, 4-vinylguaiacol, or 4-propenylsyringol. The antioxidant activity of guaiacol, syringol, 4-methylsyringol, and 4-vinylsyringol is lower. An oxidant molecule acts by electronic capture and can trouble the preservation of the product by the initiation of lipid oxidation. The phenolic compounds can give an electron to stabilize the oxidant molecule and with their ringlike structure and mesomeric forms, phenolic compounds can easily support the lack of electrons. A synergic effect has been shown between high-boiling point phenolic compounds and oxidized phenolic compounds and it prolongs the antioxidant action [44]. However, there is a critical concentration that must not be overcome to avoid an inversion of antioxidant effect that can become prooxidant. Concerning the antimicrobial effect of wood smoke condensates, it seems that phenolic compounds and carboxylic acids play an inhibitory role, especially against bacteria [51]. Carbonyl compounds and esters are nearly not implied, and hydrocarbons are not influential. As in odor and flavor, the activity of compounds must take into account the synergic or antagonist effects in mixture; that is why some researchers have concluded the absence of relation between the inhibitory effect of essential oils and their phenolic content. Therefore, phenolic compounds and carboxylic acids, alone or in synergy, could be responsible for most of the antimicrobial properties.
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15.8
Polycyclic Aromatic Hydrocarbons
15.8.1
Properties and Toxicology
PAHs are well known as being food contaminants and carcinogens [52]. As they can be absorbed by animals, they are considered as environmental pollutants and can contaminate the human feed raw material [53,54]. However, home cooking and industrial food processes represent the major source of human contamination [55]. PAHs are considered as carcinogenic contaminants of processed food [56], particularly smoked food [57,58]. These compounds have been studied for several years, especially benzo[a]pyrene (B[a]P) [59]. B[a]P is the first PAH whose toxicity and carcinogenicity was assessed from the observations of Sir Percival Plott in 1775 at St Bartholomew hospital of London about cancer of the scrotum of the chimney sweepers. Therefore, it is used as the leading substance to illustrate PAH contamination. PAHs are formed by the incomplete burning of carbon-containing material. In SF, PAHs are generated during smoke production by wood pyrolysis. PAHs comprise fused aromatic rings made up of carbon and hydrogen atoms: up to four fused benzene rings, they are considered as heavy PAHs; more toxic than light PAHs, which are constituted by less than four benzene rings. Hundreds of individual PAHs may be formed and released during the process of incomplete burning of the wood. Owing to their lipophilic properties (log Kow between 4 and 7), PAHs can cross the biological membranes and accumulate in tissues. They are considered as carcinogenic contaminants, because their catabolism leads to poly-hydroxy-epoxy-PAH suitable for binding to DNA adducts, hence their toxicity (Figure 15.3). Therefore, the uses of SF in food industrial processes must be ruled out in order to guarantee food
O DNA adducts OH OH B[a]P 7,8-dihydrodiol-9,10-epoxyde
Benzo[a]pyrene:B[a]P
Glutathion S OH OH OH
O
OH OH B[a]P 7,8-catechol
OH
OH B[a]P 7,8-dihydrodiol
O B[a]P 7,8 epoxyde
OH
S
B[a]P glutathion conjugate
COOH O O OH
O
O
OH
OH
OH OH
B[a]P sulfo-conjugate
Detoxification products
Figure 15.3 Benzo[a]pyrene metabolization.
B[a]P glucuronide
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safety avoiding PAH contamination. In the 1980s, the U.S. Environmental Protection Agency (US-EPA) identified a list of 16 PAHs as the most frequently found [60]. Among them, eight light PAHs were considered as environmental contaminants, with a weak toxicity but high concentrations in the samples analyzed. The eight heavy PAHs left were shown as being carcinogenic or mutagenic contaminants and gave rise to serious health concern. Indeed, even if they were found in weak quantities, these PAHs were considered as toxic at low levels.
15.8.2 Extraction and Analysis Methods of PAH in SF and Seafood Treated by SF The quantification of PAHs in SF and seafood treated by SFs is performed in two steps: an extraction step and the analysis step, which combines a separation step and a detection step. The extraction step must integrate the composition of the matrix. In the case of liquid matrices as liquid smoke, a liquid–liquid solvent extraction is often used [61–63]. Apolar solvents or mixes of apolar and semipolar solvents are used to extract the maximum of PAHs. In the case of solid seafood treated by liquid smoke, solid–liquid extraction can be carried out. However, PAHs are often coextracted with fat matter, which can disturb the extraction, cause chromatographic coelutions, and lead to mistakes in the identification. Therefore, purification and/or delipidation steps such as saponification are often applied to reduce the fat matter rate of samples [64]. Purification was especially performed on an alumina or silica column, but solid-phase extraction (SPE) cartridges are now more frequently employed. The nature of the SPE cartridge phase is linked to the extraction method and the biochemical composition of the initial matrix. Other extraction devices have been developed to investigate PAH in smoked food such as accelerated solvent extraction (ASE) [65], supercritical fluid extraction (SFE) [66] or solid-phase microextraction (SPME) [67], and stir bar sorptive extraction (SBSE) [68] but not on liquid smokes or seafood treated by SF. Although all steps are important, the analysis step is the most critical point. For the separation of the PAHs extracted from SF or seafood treated by SF, gas chromatography and liquid chromatography are the most used techniques [55]. Gas chromatography is coupled to mass spectrometry [58,69], flame ionization detector (FID) [60], or tandem mass spectrometry [70], and liquid chromatography is coupled to ultraviolet or fluorimetric detector [59–63]. Parameters of the chromatographic separation and detection must be adjusted to avoid coelutions with interferences from lipids. Moreover, the chromatography must be sufficiently efficient to separate isomers of PAH, because they do not have the same toxicity. Thus, it is essential to quantify only the toxictargeted compounds. Several devices are therefore developed to optimize the analysis, such as bidimensional chromatography at the gaseous phase (GC/GC) [71] or liquid phase (LC/LC) [72], but, to our knowledge, they have not been applied to SF or seafood treated by SF.
15.9
Legislative Aspects
15.9.1 European Regulations on PAH Found in SF In 2003, a European regulation set the maximum contents of PAH in the primary products (PP) of smoke condensates used for the production of SF, that is, PSC and PTF. In both condensates, the concentrations of benzo[a]anthracene (B[a]A) and benzo[a]pyrene (B[a]P) must not exceed 20 and 10 mg/kg of liquid smoke, respectively [73]. This harmonization was necessary to homogenize the legislation about SFs. For example, in Italy, the maximum levels of B[a]A and B[a]P were set at 20 and 10 mg/kg of liquid smoke, respectively [74].
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However, the 2003 maximum values must be reviewed again, because these values were set for PP and not SF, whereas food is treated by SF and not directly by PP. The high maximum values authorized in PP do not seem well adjusted with the weak final PAH contamination of SF. Indeed, the PAH contamination reached in SF is largely below the values authorized in PP [60,69,75]. However, according to the origin of SF and industrial manufacturers, important differences in PAH concentrations are noticeable [57,63] which justifies controls and regulations. Moreover, the toxicity of other heavy PAHs was recently demonstrated and the monitoring of these PAHs was recommended by a European regulation published in 2005 [76]. Therefore, it is legitimate to wonder if the exclusive monitoring of the B[a]A and B[a]P in PP is adequate to illustrate the PAH contamination of SF.
15.9.2 European Regulations on PAH Concentration in Food Treated by SF In 1988, a European regulation set the maximum content of B[a]P in foodstuffs treated by SF at 0.03 mg/kg. As for SF, the PAH contamination was only set for B[a]P [77]. This value is very low compared to those authorized in PP. Th is fact can be understood by the use of smoke condensates in flavoring quantities, that is, very small amounts. However, the vaporization of SF in a smokehouse causes a loophole in the legislation. In certain countries such as France, for meat industry, atomization of liquid smoke in a smokehouse is considered as a smoking process but the maximum level of PAH must not overcome that of flavoring legislation, that is, 0.03 mg/kg. Indeed, this process can also be considered as a flavoring of the surface of the product, as drenching or showering. Therefore, it is paradoxical to apply flavoring regulations to the smoking process. Indeed, the smoking regulations set a maximum B[a]P value of 5 mg/kg of smoked fishery products and smoked crustaceans, excluding bivalve molluscs, brown meat of crab, and head and thorax meat of lobster and similar large crustaceans [76,78]. This value is the result of the necessary harmonization between the national laws of European countries [79]. Thus, atomization of liquid smoke must be lower than 0.03 mg/kg and leads often to noncompliant smoked products. Indeed, SFs are used in higher quantities than those employed in flavoring processes. However, if it is considered as smoking technique, the respective legal B[a]P amount is 5 mg/kg. In this case, atomization of liquid smoke would constitute the smoking technique, leading to less PAH contaminated food by comparison with the traditional smoking techniques [80].
15.10
Conclusion
A wide range of SFs and uses of SFs are now available to flavor seafood products. Moreover, SFs appear as a safe alternative to smoking techniques. The content of PAH in SFs and in the final product can be better controlled than during traditional smoking. Moreover, it leads to lower PAH contents. Finally, the liquid smoking process decreases the emissions of PAH compounds to the environment. All these benefits could help to reconsider the status of atomization of liquid smoke and the maximum PAH contents related. Nevertheless, it can lead to problems of labeling, but it could also initiate an international consideration of labeling of smoked and flavored food. The main criticism that can be formulated against SF is the lack of control of the final organoleptic qualities of such processed food. Therefore, it is necessary to better control the composition of SFs and to improve knowledge about the influence of the pyrolysis parameters (wood nature,
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wood size, temperature, moisture, etc.). However, the optimization of SFs effects on food products must be done avoiding PAH generation. Finally, the traceability of SF must be improved, which could contribute to give to the SF a less processed characteristic. Indeed, in France, SFs are forbidden for the smoking of organic products from aquaculture, whereas SFs are produced from natural wood. The problem can come from the emulsifiers that are sometimes added in SFs. Besides, according to allergic people and religious groups, the processed food cannot be consumed, and today no information is available.
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20. Guillén, M.D. and Ibargoitia, M.L., Influence of the moisture content on the composition of the liquid moke produced in the pyrolysis process of Fagus sylvatica L. wood, J. Agric. Food Chem., 47, 4126, 1999. 21. Lantz, A.W. and Vaisey, M., Flavor effects of different woods on whitefish smoked in a kiln with controlled temperature, humidity, and air velocity, J. Fish. Res. Board Can., 27(7), 1201, 1970. 22. Chan, W.S. and Toledo, R.T., Effect of smokehouse temperature, humidity and air flow on smoke penetration into fish muscle, J. Food Sci., 40, 240, 1975. 23. Rusz, J. and Miler, K.B.M., Physical and chemical processes involved in the production and application of smoke, Pure Appl. Chem., 49, 1639, 1977. 24. Clifford, M.N., Tang, S.L., and Eyo, A.A., Smoking of foods, Process Biochem., June/July, 8, 1980. 25. Fazzalari, F.A., Compilation of odor and taste threshold values data, ASTM Data Series DS 48A, American Society for Testing and Materials, Philadelphia, PA, 1978. 26. Swan, J.S. and Burtles, S.M., The development of flavour in potable spirits, Chem. Soc. Rev., 7(2), 201, 1978. 27. Buttery, B.G., Turnbaugh, J.G., and Ling, L.C., Contribution of volatiles to rice aroma, J. Agric. Food Chem., 36(5) 1006, 1988. 28. Ojeda, M. et al., Chemical references in sensory analysis of smoke flavourings, Food Chem., 78(4), 433, 2002. 29. Sérot, T. et al., Effect of smoking processes on the contents of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus), Food Chem., 85, 111, 2004. 30. Bratzler, L.J. et al., Smoke flavor as related to phenol, carbonyl and acid content of bologna, J. Food Sci., 34, 146, 1969. 31. Baryłko-Pikielna, N., Contribution of smoke compounds to sensory, bacteriostatic and antioxidative effects in smoked foods, Pure Appl. Chem., 49, 1667, 1977. 32. Hamm, R., Analysis of smoke and smoke products, Pure Appl. Chem., 49, 1655, 1977. 33. Cardinal, M. et al., Effects of the smoking process on odour characteristics of smoked herring (Cuplea harengus) and relationships with phenolic compound content, Food Chem., 96, 137, 2006. 34. Varlet, V., Caractérisation des composes volatils responsables des qualities odorants du saumon fume (Salmo salar) et evaluation des contaminants du fumage (Hydrocarbures Aromatiques Polycycliques), Thesis, University of Sciences of Nantes, 2007. 35. Daun, H., Sensory properties of phenolic compounds isolated from curing smoke as influenced by its generation parameters, Lebensm.-Wiss. U.-Technol., 5(3), 102, 1972. 36. Fiddler, W., Wasserman, A.E., and Doerr, R.C., A “smoke” flavor fraction of a liquid smoke solution, J. Agric. Food Chem., 18(5), 934, 1970. 37. Kim, K., Kurata, T., and Fujimaki, M., Identification of flavour constituents in carbonyl, noncarbonyl neutral and basic fractions of aqueous smoke condensates, Agric. Biol. Chem., 38, 53, 1974. 38. Radecki, A. et al., Isolation and identification of some components of the lower-boiling fraction of commercial smoke flavourings, Acta Aliment. Pol., 3, 203, 1977. 39. Guillén, M.D., Manzanos, M.J., and Ibargoitia, M.L., Carbohydrate and nitrogenated compounds in liquid smoke flavorings, J. Agric. Food Chem., 49, 2395, 2001. 40. Wasserman, A.E., Organoleptic evaluation of three phenols present in wood smoke, J. Food Sci., 31, 1005, 1966. 41. Toth, L. and Potthast, K., Chemical aspects of the smoking of meat and meat products, Adv. Food Res., 29, 87, 1984. 42. Olsen, C.Z., Chemical composition and application of smoke flavor, Proc. Eur. Meet. Meat Res. Workers, 22, F 7:1, 1976. 43. Burdock, G.A., Feranoli’s Handbook of flavor Ingredients, CRC Press LLC, Boca Raton, FL, 2002. 44. Metz, B. et al., Identification of formaldehyde-induced modifications in proteins: reactions with model peptides, J. Biol. Chem., 279, 6235, 2004.
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45. Sainclivier, M., L’industrie alimentaire halieutique. Des techniques ancestrales à leurs réalisations contemporaines: salage, séchage, fumage, marinage, hydrolysats, Bulletin scientifique et technique de l’Ecole Nationale Supérieure Agronomique Centre de Recherches de Rennes, 219, 1985. 46. Tilgner, D.J., The phenomena of quality in the smoke curing process. Pure Appl. Chem., 49, 1629, 1977. 47. Müller, W.D., Curing and smoking. Fleischwirtsch, 71(1), 61, 1991. 48. Girard, J.P., La fumaison, in Technologie de la viande et des produits carnés, Lavoisier, Paris, 171, 1988. 49. Varlet, V., Prost, C., and Sérot, T., Volatile aldehydes in smoked fishes: Analysis methods, occurrence and mechanisms of formation, Food Chem., 105, 1536, 2007. 50. Suñen, E., Aristimuño, C., and Fernandez-Galian, B., Activity of smoke wood condensates against Aeromonas hydrophila and Listeria monocytogenes in vacuum-packaged, cold-smoked rainbow trout stored at 4°C, Food Res. Int., 36, 111, 2003. 51. Suñen, E., Fernandez-Galian, B., and Aristimuño, C., Antibacterial activity of smoke wood condensates against Aeromonas hydrophila, Yersinia enterocolitica, and Listeria monocytogenes at low temperature, Food Microbiol., 18,387, 2001. 52. Baird, W.M., Hooven, L.A., and Mahadevan, B., Carcinogenic polycyclic aromatic hydrocarbonDNA adducts and mechanism of action, Environ. Mol. Mutagen., 45, 106, 2005. 53. Nyman, P.J. et al., Comparison of two clean-up methodologies for the gas chromatographic/ma ss spectrometric determination of low nanogram/gram levels of polynuclear aromatic hydrocarbons in seafood, Food Addit. Contam., 10(5), 489, 1993. 54. Scientific Committee on Food (SCF), Opinion of the Scientific Committee on Food on the risks to human health of polycyclic aromatic hydrocarbons in food (expressed on 4 December 2002), SCF/ CS/CNTM/PAH/29 final, 2002. 55. Stołyhwo, A. and Sikorski, Z.E., Polycyclic aromatic hydrocarbons in smoked fish—A critical review, Food Chem., 91(2), 303, 2005. 56. Mottier, P., Parisod, V., and Turesky, R.J., Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry, J. Agric. Food Chem., 48, 1160, 2000. 57. Chen, B.H., Wang, C.Y., and Chiu, C.P., Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography, J. Agric. Food Chem., 44, 2244, 1996. 58. Jira, W., A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes, Eur. Food Res. Technol., 218, 208, 2004. 59. Šimko, P., Changes of benzo(a)pyrene contents in smoked fish during storage, Food Chem., 40, 293, 1991. 60. Simon, R., Palme, S., and Anklam, E., Validation (in-house and collaborative) of a method based on liquid chromatography for the quantitation of 15 European-priority polycyclic aromatic hydrocarbons in smoke flavourings: HPLC-method validation for 15 EU priority PAH in smoke condensates, Food Chem., 104(2), 876, 2007. 61. Guillén, M.D., Sopelana, P., and Partearroyo, M.A., Study of several aspects of a general method for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings by gas chromatography-mass spectrometry, Food Addit. Contam., 17(1), 27, 2000. 62. Guillén, M.D., Sopelana, P., and Partearroyo, M.A., Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavorings of different compositions by gas chromatography–mass spectrometry, J. Agric. Food Chem., 48, 126, 2000. 63. Simon, R., Palme, S., and Anklam, E., Single-laboratory validation of a gas chromatography–mass spectrometry method for quantitation of 15 European priority polycyclic aromatic hydrocarbons in spiked smoke flavourings, J. Chromatogr. A, 1103, 307, 2006. 64. Šimko, P., Determination of polycyclic aromatic hydrocarbons in smoked meat products and smoke flavourings additives. J. Chromatogr. B, 770, 3, 2002.
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65. Wang, G. et al., Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples, J. Agric. Food Chem., 47, 1062, 1999. 66. Järvenpää, E., Huopalahti, R., and Tapanainen, P., Use of supercritical fluid extraction-high performance chromatography in the determination of polynuclear aromatic hydrocarbons from smoked and broiled fish, J. Liq. Chromatogr. Relat. Technol., 19(9), 1473, 1996. 67. Popp, P. et al., Determination of polycyclic aromatic hydrocarbons in wastewater by off-line coupling of solid-phase microextraction with column liquid chromatography, J. Chromatogr. A, 897, 1–2, 153, 2003. 68. King, A.J., Readman, W., and Zhou, J.L., Determination of polycyclic aromatic hydrocarbons in water by solid-phase microextraction–gas chromatography–mass spectrometry, Anal. Chim. Acta, 523(2), 259, 2004. 69. Pimenta, A.S. et al., Evaluation of acute toxicity and genotoxicity of liquid products from pyrolysis of Eucalyptus grandis wood, Arch. Environ. Contam. Toxicol., 38, 169, 2000. 70. Varlet et al., Determination of PAH profiles by GC-MS/MS in salmon muscle processed according to four different smoking techniques, Food Addit. Contam., 24(7), 744, 2007. 71. Purcaro, G. et al., Determination of polycyclic aromatic hydrocarbons in vegetable oils using solidphase microextraction—Comprehensive two-dimensional gas chromatography coupled with time-offlight mass spectrometry, J. Chromatogr. A, 1161, 284, 2007. 72. Moret, S., Conte, L., and Dean, D., Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method, J. Agric. Food Chem., 47, 1367, 1999. 73. EC 2065/2003, Regulation (EC) No 2065/2003 of the European Parliament and of the Council of 10 November 2003 on smoke flavourings used or intended for use in or on foods, Off. J. Eur. Union, L. 309, 1, 2003. 74. Decreto Legislativo N°107 del 25/01/1992, attuazione delle direttive 88/388/CEE e 91/71/CEE relative agli aromi destinati ad essere impiegati nei prodotti alimentari ed ai materiali di base pere la loro preparazione, Italian Law Decree, Allegato III. 75. Dos Santos Barbosa, J.M., Ré-Poppi, N., and Santiago-Silva, M., Polycyclic aromatic hydrocarbons from wood pyrolysis in charcoal production furnaces. Environ. Res., 101, 304, 2006. 76. EC 2005/108, Commission Recommendation of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods, Off. J. Eur. Union, L 34: 43, 2005. 77. EC 88/388, Council Directive of 22 June 1988 on the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials for their production, Off. J. Eur. Union, L. 184, 1, 1988. 78. EC 1881/2006, Commission Regulation of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs, Off. J. Eur. Union, L. 364: 5, 2006. 79. Wenzl, T. et al., Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union, Trends Anal. Chem., 25(7), 716, 2006. 80. Hattula, T. et al., Use of liquid smoke flavouring as an alternative to traditional flue gas smoking of rainbow trout fillets (Oncorhyncus mykiss). Lebensm.-Wiss. U.-Technol., 34, 521, 2001.
NUTRITIONAL QUALITY
III
Chapter 16
Composition and Calories Eva Falch, Ingrid Overrein, Christel Solberg, and Rasa Slizyte Contents 16.1 Introduction ................................................................................................................. 258 16.2 Nondestructive Analysis of Total Proximate Composition............................................. 258 16.3 Lipids ............................................................................................................................267 16.3.1 Nutritional Aspects ..........................................................................................267 16.3.2 Methods for Determination of Total Lipids .....................................................267 16.3.3 Nondestructive Methods ................................................................................. 268 16.3.4 Comparison of Methods ................................................................................. 269 16.4 Proteins ........................................................................................................................ 269 16.4.1 Nutritional Aspects ......................................................................................... 269 16.4.2 Methods for Protein Determination .................................................................270 16.4.3 Determination of Total Nitrogen .....................................................................270 16.4.4 Direct Methods for Soluble Protein Determination .........................................270 16.4.5 Nondestructive Analysis of Proteins ................................................................ 273 16.5 Determination of Carbohydrate Content ..................................................................... 273 16.6 Determination of Water Content ..................................................................................274 16.7 Calories .........................................................................................................................274 16.7.1 Direct Measurement of Energy ........................................................................274 16.7.2 Indirect Measurements of Energy.....................................................................275 16.7.3 Food Composition Tables and Databases .........................................................276 References ................................................................................................................................276
257
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16.1
Introduction
The proximate composition in most fish and shellfish is primarily water, proteins, and lipids. In fish meat these constituents make up about 98% of the total mass, and the other minor constituents include carbohydrates, vitamins, and minerals [1]. Proximate data on different fish species are collected in databases such as the FishBase (www.fishbase.org); however, the chemical composition of fish generally varies due to seasons, geographical locations, stages of maturity, and sizes, and so on. Therefore, to ensure obtaining data on the exact proximate composition, analysis should be performed on the specific samples. There are several methods available to analyze the major components in seafood and the main methods along with their advantages and limitations are presented in Table 16.1 and further discussed in the text below. Section 16.7 deals with the different methods to determine and calculate calories in fish and shellfish.
16.2 Nondestructive Analysis of Total Proximate Composition Analysis of each nutrient separately is time-consuming and requires a diverse set of equipments. Methods for simultaneous determination of the major components are therefore valuable. Nearinfrared spectroscopy (NIR) is the most common method for such analysis and is therefore comprehensively presented in this chapter. NIR has been found to be a reliable, rapid, and easy to perform nondestructive analysis for simultaneous determination of the major components in fish. The development of NIR in food analysis started with the development of analysis of cereal grains and oilseeds in Canada [2]. As well as increased efficiency of the Canadian wheat segregation program, the adoption of NIR testing resulted in a total cost saving of CAN$ 2.5 m per year and a saving for the environment by replacing the Kjeldahl system, which involves concentrated sulfuric acid and heavy metal catalysts, by the chemical-free NIR method. When Williams was running the program for the Canadian Grain Commission, 600,000 Kjeldahl analyses were conducted per year and incidentally producing 47 ton of caustic waste in the process. The first instruments on the market were filter instruments measuring in reflectance mode. During the 1980s monochromator instruments were developed, making it possible to measure over the whole NIR spectrum and not only on a small number of selected filters. The NIR spectrum is defined between the wavelength 800 and 2500 nm, but the available detectors cover a smaller range; the silicon detector covers the range 400–1100 nm, an indium gallium arsenic covers the range 800–1700 nm, and a lead sulfide, the range 1100–2500 nm. The NIR radiation interacting with a sample may be absorbed, transmitted, or reflected depending on the interaction with NIR wavelength and physical status of the sample as transparent or nontransparent. Diff use transmittance measurements are usually carried out in the 800–1100 nm region of the spectrum, where the weak absorptions enable useful data to be obtained using sample thickness of 1–2 cm of samples such as meat, cheese, or whole grain. However, in this spectral region the spectrum of the transmitted light is very compact and no single peaks are visible, making it difficult to use this spectral range before the development of multivariate calibration technology, such as the introduction of partial-least squares (PLS) by Martens in 1982 [3]. The spectral data will be reduced by principal component analysis, and then one can perform a linear regression on the principal components. The end result is a calibration equation from which the constituent of interest is calculated from a linear combination of spectral data.
Reflection, transflection, or transmission of nearinfrared light (850–1700 nm)
Measurement of ultrasonic velocity
Samples are placed in an electromagnetic field and electric conductivity is measured
Ultrasound
Total body electrical conductivity (TOBEC)
Principle
NIR/NIT
Total Proximate Composition
Methods
May obtain data on water, lipids, and protein content noninvasively, nondestructive, can be used on live fish
Rapid, nondestructive, precise, fully automated, and can be performed online
Rapid method simultaneously analyzing fat, water, and protein. Nondestructive and can be used on live fish.
Advantages
Need more research
Few articles on fish composition
Specific for different species, physiological and physical states can affect values of conductivity, can be nonsensitive, expensive
Ultrasonic properties of tissue depend on composition and temperature
For reflectance instruments (surface analysis) some drawbacks such as interference by starch and lipids, displacement of reflectance spectrum by moisture content, and disturbance by particle size in samples
Calibrations need to be made against reference methods. Different calibrations for different species and organs. Calibrations require skilled personnel. The equipments are relatively expensive.
Drawbacks
(continued)
[21,172–174]
[17–20,173]
[11,133]
[4–6,8]
Selected References
Table 16.1 Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products
Composition and Calories ◾ 259
Extraction of minced samples generally using chloroform and methanol as solvent Gravimetric determination
Extraction of minced samples by solvents in automatic systems (Soxhlet, (SoxTech, fexICA), Fosslet, etc.)
The sample is dried and from the water content found, the fat content can be calculated theoretically by the formula Fat% = 80 − water %
Manual solvent extraction
Automatic solvent extraction
Microwave drying
Chemical Extractions:
Total Lipid Determination
Nuclei of atoms in a sample provide spectra when the sample is exposed to a magnetic field
Principle
A simple and inexpensive method
No use of chemicals
No laboratory facilities are required
Automatic, less exposure to chemicals (compared with manual solvent extraction)
Possibilities to further characterize the lipids extracted
Provides high total lipid yield
Rapid, nondestructive (See under NMR below)
Advantages
Precision level may be dependent on sample (maturity stages of the fish, lipid content, location of lipids, processing)
Physical and chemical changes might occur during examination
Requires laboratory facilities
May discriminate structured fat (such as phospholipids)
Requires well-trained laboratory personnel
Destructive technique
Use of health hazard chemicals
Time-consuming
Requires laboratory facilities
Excellent for determination of fat and water content or even distinguish lipid classes and water properties. Need of sample specific calibrations
Weaknesses due to quantification of proteins without combining with destructive methods
Drawbacks
[46]
[43]
[26–31]
[13–15,22,55,57]
Selected References
Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products
◾
Nuclear magnetic resonance (NMR)
Methods
Table 16.1 (continued)
260 Handbook of Seafood and Seafood Products Analysis
Transmitted or transflected Near Infrared light (800–1700 nm).
See NMR above
NIR/NIT
Low-field NMR
NMR mouse
Determination of water by analyzing the dielectric properties using a microwave strip (calculation of lipids as for the drying method).
Fat meters
Nondestructive Methods:
Nondestructive and rapid. The NMR mouse is rapid, portable (small size), and nondestructive and allows in vivo measurements
Allows in vivo measurements
Some portable instruments are available
Broad range of applications, may also provide other nutrient data in the same analysis
Nondestructive and rapid
Allows in vivo measurements
Relatively inexpensive, rapid, easy, nondestructive, and portable
No laboratory facilities are required
Traditional low-field instruments require withdrawal of homogeneous samples for analysis (invasive)
Expensive, requires specific calibrations
See NIR/NIT above
Most suitable for neutral lipid determination
Needs to be calibrated for the individual species
Precision level may be dependent on sample (maturity stages of the fish, lipid content, location of lipids, processing)
(continued)
[14–16,22,50,55]
[4–6,8]
[46,51–52]
Composition and Calories ◾ 261
High temperature combustion and detection of N by thermal conductivity detector
Dumas combustion method
Direct Protein Determination on Soluble Proteins
Sample digestion followed by neutralization, distillation, trapping of ammonia, and titration with acid
Kjeldahl
Total Nitrogen Determination
Proteins
Principle
Rapid, inexpensive to use and sensitive to low concentrations of proteins
Rapid, easy to perform, inexpensive to use, safe (no chemical exposure), and environmentally friendly
Widely used internationally, standard method for comparison, high precision and good reproducibility, independent of physical state of sample
Advantages
Most samples must undergo steps of sample preparation before they can be analyzed. Absorbance depends on the type of protein analyzed
Limited to soluble proteins
High initial costs. Difficult to obtain the accurate protein concentration
Does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein, interference by nonprotein nitrogen compounds, time-consuming, low sensitivity, hazardous, potentially toxic chemicals are used
Drawbacks
[53, 74,133]
[53,67,120,133]
Selected References
Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products
◾
Protein Determination
Methods
Table 16.1 (continued)
262 Handbook of Seafood and Seafood Products Analysis
A violet-purplish color is produced when copper(II) ions interact with peptide bonds under alkaline conditions. Absorbance at 540 nm
Copper(II) ions in alkaline solution react with protein to form complexes, which react with the Folinphenol reagent, and reaction products are detected between 500 and 750 nm
The protein and dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorption is proportional to the protein present
Measurement of UV absorption (280 nm)
Biuret method (Alkaline copper reagent test)
Lowry protein assay
Dye-binding (Bradford) method
Near-UV absorption
Rapid, nondestructive, no addition of reagents required
Rapid, easy to perform, high sensitivity, and internationally accepted
High sensitivity and easy to perform
Negligible interference from materials that absorb at lower wavelengths, technique is less sensitive to protein type: it utilizes absorption involving peptide bonds that are common to all proteins, easy to perform
Low sensitivity, interference by UV-absorbing compounds (nucleic acids and nucleotides), depends on amino acid composition
Color formation and binding depend on proteins present, protein-dye complex adsorbs on glass surface, interference from common laboratory chemicals, variation of binding capacity for different batches of commercial grade dyes
Standard curve is nonlinear. Unstable reagents are used. Other compounds can interfere, color development depends on amino acid composition
Relatively low sensitivity compared with other UV-visible methods. High amounts of endogenous proteases may cause errors, interference from ammonia, buffers salts, detergents
[133]
(continued)
[68,131,133,176,177]
[67,133]
[133,175]
Composition and Calories ◾ 263
Hydrolysis, derivatization, chromatographic separation, and detection of amino acids with UV absorbance, fluorescence
Highperformance chromatography (HPLC)
Absorption at 780–2500 nm
Transmitted or transflected Near-infrared light (800–1700 nm)
Infrared absorption
NIR/NIT
Nondestructive Determination
Measurement of UV absorption
Principle
See NIR/NIT above
Rapid, nondestructive, multicomponent analysis
Faster than ion exchange chromatography, quantifies amino acids, value for net protein, option to quantify free amino acids, sensitive
Rapid, nondestructive, no addition of reagents required, high sensitivity, low dependency of signal response on amino acid composition, low interference from nucleic acids and nucleotides
Advantages
See NIR/NIT above
Strong interference by water, influence by lipids and sample particle size, complex calibration
Most methods do not include all amino acids, hydrolysis destroys some of the amino acids. Derivatization agents: OPA: no derivatization with secondary amino acids. FMOC: less soluble, might interfere
Interference by oxygen and UV-absorbing compounds (buffer, salts)
Drawbacks
[133]
[90–92,96,97, 108,178]
[132,133]
Selected References
Overview of the Most Common Methods for Analysis of Proximate Composition in Fish and Fish Products
◾
Far-UV absorption
Methods
Table 16.1 (continued)
264 Handbook of Seafood and Seafood Products Analysis
The sample is dried until constant weight (e.g., 12 or 24 h) and water evaporated is determined.
Drying by irradiation
Volumetric analysis of water after boiling in toluene
See NIR/NIT above
See NMR above
Air or vacuum drying
Infrared drying Microwave drying
Dean and Stark method
NIR/NIT
NMR
Water Determination
Possible to distinguish between free and bounded water
See NIR/NIT above
Faster than oven drying methods
Shorter analysis time compared with air and vacuum drying. For the microwave method it is possible to analyze many samples simultaneously
Simple to use and inexpensive equipments required
Calibrations are needed and knowledge on chemometry is an advantage
See NIR/NIT above
Uses health hazard chemicals (toluene)
Requires laboratory facilities
Risk of overheating
Long analysis time. Air drying (101°C) may lead to thermal damage, Vacuum drying: may be difficult to keep uniform temperature distribution in the oven
[152,178]
[151]
[40]
[151]
Composition and Calories ◾ 265
266
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Near-infrared transmittance (NIT) instruments are particularly suitable to the analysis of fish. Generally, the sample has to be minced, and it is usually possible to run several subsamples. The results are averaged to obtain more representative spectral data from the sample. The spectral data are then used to perform multivariate calibrations against the chemical or physical data. The same spectral data will be used against the different selected variables, so one can simultaneously predict, for example, water, fat, and protein content from the same spectral data as accurately as the traditional “wet” chemical methods [4]. To analyze directly on a fillet one needs an interactance probe; this involves illumination and detection at laterally separated points on the sample’s surface. It is normally accomplished using a fiber-optic probe in which one set of fiber-optic bundles carries the incident radiation and another carries the reflected radiation. Due to the striped structure of fish muscle, it is necessary to have a large interactance probe, usually two times 2 cm. With this type of probe it is possible to make analysis directly on the fillet, without previous mincing, but with a slightly lower accuracy [4–6]. Portable instruments are now available [7], and successful results are also obtained for whole fish [5] and for live fish [8]. Instead of a conventional monochromator, instruments are now also made with diode arrays, making it possible to measure the whole spectrum at the same time and in that way reducing the time for measurement, making online analysis possible [8]. NIR absorption will change with temperature and calibration, and NIR measurements must therefore be made on samples with approximately the same temperature [9]. Moreover, the measurements are affected by texture and whether the sample has been frozen and thawed [10,11]. Due to the requirement of extensive sample specific calibrations, the analysis should be performed by skilled personnel [12]; however, once calibrated the analysis is easy to perform. Nuclear magnetic resonance (NMR) is another nondestructive technique that enables determination of fat and water, and recent studies have shown that it might be possible to also gain data on protein levels in dried samples [13]. The low-field NMR instruments commonly in use require withdrawal of cylindrical samples of 10–40 mm diameter for analysis [14,15]. The method is fast, accurate, and easy to use when the calibrations are performed. A new handheld portable NMR instrument (NMR mouse) has recently been developed [16,14], and it enables an analysis time of less than 20 s and can even be used in vivo on living fish [14]. Less common methods for nondestructive analysis of proximate composition in fi sh are ultrasound techniques [17–20], the total body electrical conductivity (TOBEC) technique [21], and magnetic resonance imaging (MRI) [22]. The ultrasound method is rapid, automated, and can be used online, and empirical equations have been developed to relate the ultrasonic velocity to composition [17]. A weakness in this method is the variations in ultrasonic properties of fi sh tissue due to temperature [17]. For nonfatty fi sh, the solid nonfat content can be determined from a single measurement; however at least two temperatures are suggested during analysis of fat and solid nonfat in fatty tissue [17]. In the TOBEC method the live fish is placed in a low-frequency electromagnetic field, and the distinct electrical characteristics of body fat and fat free tissue provide the proximate data [21]. MRI can provide valuable information on proximate composition and distribution of chemical constituents in fish samples [14]; however, these imaging instruments are expensive and are used primarily in certain research laboratories. Calculation of fat content by measuring the water content is possible with cheap, robust instruments (see below), but they can be used only when the protein content is stable.
Composition and Calories
16.3
◾
267
Lipids
16.3.1 Nutritional Aspects Marine lipids contain the omega-3 fatty acids such as C20:5n-3 (EPA) and C22:6n-3 (DHA) with well-documented beneficial health effects [23–25]. These fatty acids are found in all parts of the fish and are constituents of different lipid classes such as phospholipids, triacylglycerols, lysophospholipids, partial glycerides, esters, and free fatty acids. Marine lipids are the only source of EPA and DHA, and extraction and utilization of these fatty acids is a major industry. The market shares for higher value applications such as food ingredients, health care products, and medicine are increasing owing to the supply to aquaculture business.
16.3.2 Methods for Determination of Total Lipids The lipid content in fish can be determined by several different methods varying in efficiency, total lipid yield, accuracy, skill requirement, and cost. The main methods are shown in Table 16.1 ranging from organic solvent extraction, microwave drying, to nondestructive techniques. Fish lipids are generally composed of polar and neutral lipid compounds. Although the triacylglycerols dominate in the lipid classes of fatty fish such as the pelagic species, the phospholipids are the main lipid class in lean white fish species. In addition, other derivatives of fatty acids (partial glycerides, free fatty acids, esters etc.), sterols, fat-soluble vitamins, and carotenoids are found in fish and comprise the large group called total lipids. Chemical methods: Traditional methods for determination of total lipids are generally based on solvent extraction followed by gravimetric determination. The lipid yield obtained is highly dependent on the solvent system, and using a combination of polar and nonpolar solvents it is possible to extract the total lipids and not only the free lipids such as triacylglycerols. Differences in lipid yield among the methods are claimed to correlate with the extraction efficiency of the more tightly bounded polar lipids such as phospholipids [26]. A combination of chloroform, methanol, and water is most often used for manual extraction of total lipids in fish [27,28]. The methanol penetrates the tissue while the chloroform dissolves the fat. The samples are first homogenized and after several extraction steps, followed by evaporation of solvents, the total lipids are gravimetrically determined. The Bligh & Dyer method (B&D) was originally used on fish muscle and less solvent volumes were used compared with the Folch method. A comparison between the Folch and B&D method has previously shown that the B&D method underestimates the lipid yield when the lipid content in fish muscle is above 2%, whereas no significant differences are found at lower levels [29]. Modifications of the B&D method are widely reported in the literature [30,31], although these specific modifications are rarely described in detail [29]. One recent study demonstrated that a modified B&D method using NaCl and electrolyzed cathode water gave higher lipid yield compared with the conventional method [32]. Generally, the crude lipids extracted by B&D compose a broad range of lipid classes, and the method demonstrates a high efficiency in extracting both polar and neutral lipids. However, parameters such as solvent ratio, order of solvent addition, and number of extraction steps are important parameters that affect the lipid yield and might be individually suited for specific sample material differing in lipid class composition. An example is the increased lipid yield obtained when using higher amounts of methanol, which was explained by a better extraction of phospholipids in a study by Smedes and Askland [31].
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Due to the high lipid yield generally obtained by the B&D method, it has been widely used as a reference to test the efficiency of other methods, and it is particularly used in research laboratories. Additionally, this extraction allows the successive characterization of lipids such as lipid classes (tri-, di-, and monoacylglycerols, free fatty acids, phospholipids etc.), lipid oxidation products, and fatty acid composition. Hence, manual extraction is relatively time-consuming, requires laboratory facilities, and the solvents used are toxic to humans and environment. Less toxic solvents are used in some studies [31,33–37] without achieving the same lipid yield as that obtained by using the traditional solvents. Solvent extraction of animal tissues in general and procedures for preparation of samples are comprehensively discussed by Christie [38] and by the same author in the Lipid Library Website (http://www.lipidlibrary.co.uk/topics/extract2/index.htm). Another commonly used method for solvent extraction of fatty fish species is the ethylacetate method [39] without the use of expensive equipment. The method even specifies what part of the fish should be included in the analysis. Ethylacetate has replaced the health-harmful benzene that was used in the early extractions. Among the automatic solvent extraction techniques, the Soxhlet method [40] and modifications of this method have been most widely used for determination of total lipids in fish. The sample is lyophilized before solvent extractions, removal of solvents, and gravimetric determination [41]. Petroleum ether and diethyl ether are the most common solvent used but the use of hexane and acetone are also reported in some studies [41,26]. The original Soxhlet method was developed by Soxhlet in 1879. This was originally a time-consuming method (16 h); however, today, there are more rapid methods available based on the same principle with commercial instrumentation such as the SoxTec equipment. New developments in this field are continuously reducing the analysis time, and a new microwave-integrated Soxhlet may run samples in less than an hour [42]. Lipid content can also be determined without the use of chemicals such as in the microwave drying method. This is a simple and inexpensive method that indirectly calculates the lipid content from the water content analyzed [43]. The principle behind this method is a reported reverse intercorrelation between water and lipid content in clupeid fish [43–45] calculated from the following formula: Fat content% = 80% − water content % [43]. Limitations in this method lie particularly in the lack of fitness of the intercorrelation between water and lipids during different maturity stages for the fish [46] and also variations between different locations in the fish [46–50]. Furthermore, this intercorrelation is affected by processing, particularly heat treatment, that might reduce the water content.
16.3.3 Nondestructive Methods The intercorrelation between water and lipids in fish is also applied as the principle for the nondestructive portable Fat Meters developed by Kent [44,51–52]. The sample is irradiated by microwaves with a microwave strip, the water is measured by the dielectric properties, and the lipid content is then calculated. These instruments (Fish Fat Meters and Torry Fat Meters) are calibrated for a range of fish species [45], and they are simple to use. However, these methods share some of the same limitations as those in the microwave drying method such as the lack of fitness during spawning, and additionally, the accuracy of the Fat Meters has also been reported to be dependent on the lipid content in the fish [46]. Although the Fat Meter is limited to determining fat and water content, methods such as NIR spectroscopy may simultaneously determine the content of lipids, proteins, and water from the surface of the sample in a few seconds [4,53]. The NMR technique has particularly been applied
Composition and Calories
◾
269
in quantification of lipids in fish [15,46,54–56], and the low-field NMR can distinguish between different lipid classes [57]. When increasing the field strength to high-resolution NMR, a range of different lipid constituents can be detected [58,59]. The ultrasound velocity technique has provided data that enable classification of salmon muscle into low, medium, and high fat [20]. See earlier section in this chapter for further information on these methods.
16.3.4 Comparison of Methods Nondestructive and rapid techniques are of particular importance for fatty fish such as herring, mackerel, and some farmed fish species. The lipid content in these species usually shows large variation, and analysis results are valuable on board the fishing vessel or processing plant for sorting into groups based on their lipid content. Vogt et al. [43] who compared the lipid yield obtained by Torry Fat Meter, NIR, the microwave method and a modified Soxhlet, found that the NIR- and microwave methods were closest to the reference solvent extraction (R 2 = 0.90). A high correlation (R 2 = 0.96) has been found between ethyl acetate extraction and NIT analysis of whole minced capelin [60], and another study [46] demonstrated a good correlation between NIR and solvent extraction in specific locations of the fish (middle part of fish and fi llet skin side) (R 2 = 0.80–0.93). NMR measurements, in the same study, showed a good correlation with the solvent extraction when the analysis was performed on minced samples. Generally, the solvent extraction techniques obtain the higher yield, which might be explained by the contribution of other lipid classes than triacylglycerols, such as polar lipids and sterols that are not always included in the rapid analyses. However, readings from the Fat Meter have been reported to show higher yield than reference values in samples of herring [61], which might be explained by the variation in the intercorrelation between water and lipids. Th is same study demonstrated a bigger difference between the methods at higher lipid content in the samples. Higher variation between methods are reported when analyzing lean fish compared with fatty fish high in unpolar lipids [26]. The statement of what is the most suitable method for lipid determination is highly dependent on the applicability and what criteria are the most important for the analysis such as accuracy, robustness, time of analysis, use of solvents, and portability, and so on.
16.4
Proteins
16.4.1 Nutritional Aspects Due to its favorable content and balance of essential and nonessential amino acids, fish protein is regarded to be of high nutritive value. Seafood proteins are also highly digestible, which adds to the understanding that digestibility of raw fish meat is in the range 90%–98% and that of shellfish about 85% [62]. Protein and amino acid requirements vary through life and are generally higher among young growing children compared with adults [63,64]. These nutritional aspects are more comprehensively described in other chapters in this book. Fish and marine invertebrate tissue contains from about 11%–24% (ww) crude protein depending on species, nutritional conditions, and the type of muscle. Although amino acid composition might vary among different types of tissue, there is a high similarity in the same tissue among species as pointed out by Mambrini and Kaushik [65]. The total body composition of amino acids shows high similarity among various cultured fish species [66].
270 ◾
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Handbook of Seafood and Seafood Products Analysis
Methods for Protein Determination
Several of the most important methods for protein determination in food date from the late 1800s (Dumas, Nessler’s reagent, Biuret, Kjeldahl, Folin-Ciocalteau, and Dye binding) [67]. Quantification of total protein in fish and fish products can be determined by total organic nitrogen followed by conversion into crude protein or by a set of direct methods.
16.4.3
Determination of Total Nitrogen
Determination of proteins by analysis of total nitrogen (N) multiplied by a specific factor is a common procedure in fish analysis [68]. The N content of food is commonly determined using the Kjeldahl [69] or the Dumas [70] methods. Kjeldahl includes digestion of material and quantifies only N that is transformable to NH4+ using titration, colorimetry, or an ion-specific electrode [71]. In the Dumas method, all N is converted to N2 through combustion using a nitrogen element analyzer. Generally, the Dumas method gives higher N values than the Kjeldahl method [72–74], and a Kjeldahl-N to Dumas-N ratio of 0.80 for fish has been calculated [71]. The conversion factor for N was originally 6.25, based on average nitrogen content in different proteins of 16%, which might not be suitable for all protein sources, as they vary in amino acid composition. Generally, studies on fish have shown lower values with a more specific conversion factor of 5.8 presented for fish filet [75,76], and a factor of 4.94 (nitrogen to net protein) for protein estimates for fish and fish products are suggested by Salo-Väänänen and Koivistoinen [77]. More specific conversion factors based on the N content in isolated proteins are frequently applied for different categories of food [78]. Salo-Väänänen and Koivistoinen [77] showed that the true conversion factor was 5%–20% lower than the general 6.25 in a line of food products. Moreover, up to 40% variations were found in a comparison study of the 6.25 factor against foodspecific factors or sum of amino acids [79]. These differences indicate a significant contribution of nitrogen from other than amino acids or protein structures. Large amounts of those compounds are found in fish and fish products, probably due to both natural composition and degradation products [77]. These other N contributions might originate from nucleic acids, nucleotides, trimethylamine n-oxide (TMAO), free amino acids, or others. Contributions of N from products such as urea might appear in sharks, skates, and rays. There are, however, options to separate protein N from nonprotein N by precipitation and filtration after solvent extraction if required [80]. The nitrogenous compounds that do not originate from proteins can also be separated using methods such as ion-exchange chromatography (IEC), gas chromatography (GC), thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) [81,82].
16.4.4
Direct Methods for Soluble Protein Determination
Protein is amino acids linked together via peptide bonds, and quantification of these amino acids might give more accurate values for protein estimates [68,77,83]. The term “net protein” is often used for those values that are corrected for added water during analysis. There are options to exclude or include the free amino acids during sample preparations, or they have also been analyzed separately using HPLC methods [84–86]. A more extensive description of various methods and techniques used in protein analyses are covered by Owusu-Apenten [67]. Acid hydrolysis followed by amino acid quantification such as by HPLC [87–90] or the more traditional IEC [89,91–93] are direct and specific methods for protein determination. During IEC, the derivatization of amino acids takes place postcolumn in most methods using, for
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example, ninhydrin [94,89] or O-phthalaldehyde (OPA) [95]. Common derivatization reagents for quantification of amino acids in HPLC methods are OPA [90,96] and 9-fluorenylmethyl chloroformate (FMOC) [96], which are often used in combination with 2-mercaptoethanol, ethanethiol [90], or 3-mercaptopropionic acid [90,96]. An additional derivatization agent 2-(9-anthryl)ethyl chloroformate showed good correlation with the use of FMOC and lower detection limits for amino acids when analyzed in UV absorbance due to better spectral properties of the produced chromophore [97]. Other derivatization reagents are discussed in Sarwar and Botting [91] and in Fekkes [92]. In HPLC methods both pre- and postcolumn derivatizations are used with variable mobile phases based on methanol and acetonitrile. The reaction time, choice of solvents, and the concentration of 2-mercaptoethanol determine the efficiency of the reaction between OPA and amino acids with influence on quantification of the amino acids [90] (generally, 2-mercaptoethanol should be kept in the lower concentration range for optimization of the method [90]). OPA does not react with secondary amino acids, and FMOC is, among others, less soluble and might create interference reactions, but by combining those both, the primary and secondary amino acids can be detected [98]. Further optimization of this approach and adding an online dialysis step have improved the method with separation of 25 amino acids, and quantification of most of them [96]. Hyp (hydroxyproline), which is primarily found in connective collagenous tissue [99], might otherwise be quantified through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [100,101] or N2-(5-fluoro-2,4-dinitrophenyl)-l-valine amide [102]. Alternative methods are the spectrophotometric determination of Hyp as a measure of collagen [103] or collagen/gelatin in fish skin [104], the latter using a modified spectrophotometric method for Hyp determination by Bergman and Loxley [105]. The destruction of Trp (tryptophan) during hydrolysis in hydrochloric acid can be omitted by replacing with a line of others, including methane sulfonic acid containing 3-(2-aminoethyl) indole [106,107]. Enhanced signal of tyrosine, phenylalanine, and Trp has also been obtained using online photolysis with chemoluminescence methods in the HPLC system [108]. A more comprehensive overview of alternative methods for quantification of Trp is otherwise reviewed by Molnar-Pearl [109] and includes both alkali hydrolyses along with more complex derivatization and detection methods. During amino acid determination with the HPLC methods, detection of Cys (cysteine/ cysteine) might require special procedures during extract preparations such as iodoacetic acid [110] or 3,3′-dithiodipropionic acid as used in Glencross et al. [111]. Some nitrogenous compounds such as nucleic acids and amines, the latter originating mainly from microbial decarboxylation of amino acids in food such as putrescine, cadaverine, spermidine, spermine, tyramine, and histamine [112], can also be separated using methods such as HPLC [113,114] and reverse-phase HPLC [115,116]. Amino acid determination is often used in nutritional studies on fish, and requirements are frequently determined after analysis using IEC or HPLC methods [111,117–119], or alternatively 13C-NMR after extraction has been applied in such studies [120]. Quantification of the individual amino acids in HPLC methods is based on standards (amino acids) and use of an internal analytical standard such as a-butyric acid (ABA), responses to those, and molecular weight make the basis for calculating the amino acids. The protein values are calculated as the sum of all amino acids corrected for water added during hydrolyses, and the free amino acids might be removed through the extraction procedure or analyzed separately. Proteins can also be determined by a number of spectrophotometric methods. Some of these analyses are based on the ability of proteins to absorb (or scatter) light, whereas in other analyses, proteins are chemically or physically modified to absorb (or scatter) light. Due to variation of amino acid composition in proteins, most of these methods give results that can be different from absolute protein concentrations [83].
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Methods where proteins are chemically or physically modified for determination (colorimetric assays) can also be divided in to two groups: dye-binding reaction and redox reaction with proteins [121]. In the redox spectrophotometric methods, analyses are based on reaction with Folin reagent, and the following methods could be mentioned: Biuret reaction [122], Lowry protein method [123], and bicinchoninic acid (BCA) assay [124]. In the Biuret reaction Cu(II) with proteins in alkaline medium is reduced to Cu(I), which binds to protein forming a Cu(I)–peptide complex with purplish-violet color [121]. The same principle is used in BCA assay, where Cu(I) is detected by reaction with BCA, which gives an intense purple color [125]. One of the most popular methods in this group is the Lowry protein method [123], which is initially based on the Biuret reaction, where peptide bonds react with Cu(II) in alkaline medium to produce Cu(I). Later Cu(I) reacts with the Folin reagent. The reaction gives a strong blue color [83]. The intensity of color partly depends on the amount of Tyr and Trp in samples but can also be influenced by other components such as N-containing buffer or carbohydrates [121]. The amounts of proteins in sardine determined by the Lowry method were comparable to those determined by Kjeldahl method [121]. The Lowry method is suitable for protein extracts such as actomyosin, which is an important component in surimi-based products [126]. However, the BCA assay is shorter compared with the Lowry method (where two steps are needed), more flexible and stable in alkaline conditions, and has a broad linear range. The BSA assay can also be interpreted by the usual chemical components such as EDTA, thiols, reducing sugars, hydrogen peroxide, or phospholipids [121,125]. The dye-binding spectrophotometric assay is based on the reaction between acid dye and positively charged amino acid residues in proteins [121]. In acidic conditions, the created insoluble complexes are removed and the unbound dye is determined by measuring its absorbance. The amount of protein is proportional to the amount of bound dye. Coomassie dye in acidic conditions binds to proteins and creates complexes that influence a color shift from a maximum from 465 nm to 595 nm, using the Bradford method [127]. Absorbance of Coomassie dye-protein complex is measured at 595 (575–615) nm, because the difference between the two forms of the dye is greatest in this area. Within the linear range of the assay (∼5–25 mg/mL), the protein amount is proportional to bounded Coomassie [127]. This method is suitable for determination of extractability of proteins [128] or protein content in extracts [129–131]. Th is technique is simple, sensitive, and uses shorter analysis time compared with the Lowry method. Moreover, the dye-binding assay is less affected by reagents and nonprotein components from biological samples [132]. Proteins in solution can be quantified in a simple spectrophotometric analysis by near- or farUV absorbance [133,134]. Absorption in the near UV by proteins depends mostly on the content of Tyr and Trp and less on the amount of phenylalanine (Phe) and disulfide bonds. This absorbance measurement is simple, sensitive, needs no reagents, and the sample is recoverable [133,134] Crude protein extracts or individual fractions of proteins [135] can be measured at 280 nm. Disadvantages of the method include interference with other components such as nucleic acid, which absorbs in the same wavelength region [133]. Far-UV absorption can also be used for determination of protein content: peptide bonds absorb in the area with the maximum at about 190 nm. Different proteins give a small variation in absorbance, and the method can be considered as accurate for protein determination. However, oxygen also absorbs at these wavelengths, and to avoid interference, measurements at 205 nm is used. It should also be mentioned that components such as carbohydrates, salts, lipids, amides, phosphates, and detergents interfere [133,134].
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16.4.5 Nondestructive Analysis of Proteins Recently, other advanced and nondestructive methods have become more common for determining protein. NIR is one of these [4,53], and it was originally developed for protein analysis and has since that time been developed and calibrated for a range of fish species. Low-field NMR is generally not suitable for protein determination in a nondestructive manner. See earlier text for more information on the nondestructive techniques.
16.5 Determination of Carbohydrate Content Carbohydrates are often classified into three broad groups: sugars (mono- and disaccharides), oligosaccharides (three to nine monosaccharides) and, polysaccharides (more than nine). The content of carbohydrates in fish muscle is low [136,137] and is further influenced by conditions experienced before and during capture, which may lead to depletion of glycogen stores and thereby a decrease in the carbohydrate level. Under anoxic conditions postmortem, glycogen will continue to be metabolized, resulting in increased lactic acid along with reduced pH and eventually a gradual loss of the sweet, meaty character of fresh fish. Some marine invertebrates on the other hand are characterized by a high content of carbohydrates; up to 10.2% and 12.5% total sugars can be found in subcuticular tissue of spiny lobster and blue crab, respectively, with the highest amounts of glucose followed by galactose and mannose [138]. Glycogen stores of scallops are highly dependent on season (temperature, food availability, and lifecycle), and highest levels are usually reached after the summer period [139], showing levels up to 23%–25% glycogen of dry weight of adductor muscle [139,140]. Seasonal variations of glycogen content in mussels (Mytilus edulis) are also high, showing values in the range 4%–37% of tissue dry weight [141,142]. Among the line of methods suitable for seafood, the amount of total carbohydrates in shellfish can be determined by using the phenol-sulfuric acid procedures described by Dubois et al. [143] as used for scallop (Pecten maximus) in Maguire et al. [144] and silver carp in Gnaiger and Bitterlich [144]. This method is based on hydrolysis of polysaccharides and does not measure all sugar molecules in the materials equally accurately, because the carbohydrates are absorbed at different maximum wavelengths and in addition differ in the ability to form the chromogenes formed in the method. If measurements are performed at 488 nm and a standard curve is prepared using glucose, this will lead to a possible underestimation in the case of chemical characteristics of monosaccharides deviant from glucose. This relatively simple method is often used, because it gives a good estimate of total carbohydrates in tissue that contain 10% or more of hexose polymers [145]. Glycogen from seafood can also be determined after preparation of solution of glucose units using a range of assay kits for glucose followed by colorimetric determination (Boehringer Mannheim, Cayman chemicals, Biovision or others), as described for Abalone tissue using a combination lipid and glucose extraction method in studies of Allen et al. [146]. Glycogen levels in small amounts of tissue can additionally be analyzed using the anthrone methods with spectrophotometric determinations [147–149], which have been demonstrated as useful for scallop [150]. Carbohydrates are frequently calculated and expressed as total carbohydrates by difference, which is the remainder after subtraction of moisture, crude protein, total fat, and ash and includes fibers if present in the analyzed material. An excellent overview of definitions and internationally used carbohydrate tag names along with applicable analytical procedures for food in general is given by Munro and Burlingame [151].
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16.6 Determination of Water Content Water content in fish can be determined by simple drying methods. Using conventional air ovens, a common practice has been to dry the sample at 105°C for 12 h, which by experience has shown satisfactory drying of fish and fish products. To ensure complete drying, the sample can be dried to constant weight. Other methods [40] refer to 101°C for 24 h by conventional ovens and 70°C for 24 h using vacuum ovens. The sample is weighed in a container, and after heating the sample is cooled and weighed again. The water content is determined by the following formula: Water content (%) =
(Weight of wet material − weight of dried material) × 100 Weight of wet material
Infrared and some microwave ovens may allow an analysis time of 1–2 h [152]. Further, the new nondestructive methods such as NIR/NIT, NMR, or Fatmeter, which are described previously in this chapter, may be used for fast determination of water, and the low-field NMR technique can even distinguish between free and bounded water [15,153]. In a volumetric method (Dean & Stark), the samples are boiled in toluene before measuring the volume of water. This method is relatively fast but uses toluene, which is hazardous to health [152].
16.7
Calories
The energy content of food is generally given in kilocalories (kcal) and kilojoules (kJ), which have a conversion factor of 1 kcal = 4.184 kJ. Seafood show variable composition of proteins and fat, and energy content is dependent on this distribution, which often might also be highly influenced by seasonal variations. In a seasonal study of 35 fish and shellfish species, Soriguer et al. [154] found a substantial variation in biochemical composition, where even mackerel known as fatty type of fish, in parts of the year could be classified within the lean fish category. The lipid level in particular has high significance for the calorie content of fish, with implications for calculations in dietary studies and databases; this is important to bear in mind when these are used.
16.7.1 Direct Measurement of Energy The gross energy content of food (measured as heat of combustion, kcal/g) may be determined directly by using a bomb calorimeter (micro- or macromethods), which includes burning food with oxygen in an insulated container of constant volume [155,156]. The heat is adsorbed in water, and the energy is determined from the mass of water, its temperature rise, and its specific heat. Dichromate wet oxidation with potassium dichromate is also sometimes used as a direct method, giving rise to slightly lower energy values in fish samples than when measured by bomb calorimetric methods [157,158]. Food composition databases are not based on direct measurements of gross energy, because those are not equal to energy requirements [159]. Instead the metabolizable food energy is used, which accounts for the energy in food remaining after losses through the feces, gas, urea, and the body surface [160].
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16.7.2 Indirect Measurements of Energy The energy released by oxidation of protein, fat, and carbohydrate is the basis for sets of conversion factors. The Atwater general factor system is the foundation for the most frequently used systems for energy conversion [161], which originates from combustion with adjustments for losses in digestion, absorption, and excretion of urea. The Atwater general energy conversion values are 4.0 kcal/g for proteins, 9.0 kcal/g for lipids, and 4.0 kcal/g for carbohydrates (calculated by difference, i.e., subtracting water, ash, proteins, and lipids). Originally no differences were determined between the fiber and available digestive carbohydrates, but exploring more specific heat of combustion led to factors of 3.75 kcal/g when used for monosaccharides and 4.2 kcal/g for polysaccharides, with application in the Atwater system [162]. However, the specific conversion factor used for carbohydrates in shellfish is 4.11 kcal/g [163]. For other food material, energy factors for dietary fiber have been developed, taking into account availability, provided also by the microorganisms in the colon giving values recommended by FAO [164] of 8.0 kJ/g (2.0 kcal/g). A more specific set of factors for energy conversion were developed due to different combustion rates and digestibility of various sources of proteins and fats and additional impact caused by processing. The specific set of factors presented in Merrill and Watt [163,165] arrived at 4.27 kcal/g for protein and 9.02 kcal/g for fat in meat and fish. It is, however, important to consider the choice of analytical methods regarding conversion of proteins to calories. Both the variable nonprotein N and the variations in amino acid composition in different protein sources might have implications on the calculated energy levels if based on N analysis (see above). When energy contributions from proteins are set, the most accurate method will be as the sum of amino acids (free and protein bound). Alternatively, Kjeldahl or Dumas techniques are used with more source-specific conversion factors such as those used by Jones [166] or others, when these are known. In terms of conversion to energy, the more specific conversion factor of 5.65 kcal/g for protein was suggested [167] and tested in combination with direct energy measurements for use with fish tissue, resulting in slightly higher values compared with bomb calorimetric methods [157]. Calculation of energy contribution from fat might include analysis of fatty acids with total fat calculated as triacylglycerol equivalents [160]. For fatty fish muscle the factor 0.90 is used in conversion of total fat to total fatty acids, whereas 0.70 is used for white fish muscle [169]. Gravimetric methods are also used for energy calculations, which (depending on methods used; see above) would include weight of the additional lipid components that are not transformed to energy, per se. The calorie content of extracted lipids (methanol/chloroform extraction) from fish tissue as found by microcalorimetric methods suggests the use of a lower energy conversion factor such as 8.49 kcal/g [157]. Gross energy levels obtained from bomb calorimetry might deviate from energy when based on analysis and conversion factors due to the lipid calculations. A high level of lipids in tissue is usually accompanied with high energetic content by both methods. However, with high levels of sterols, the gross energy by bomb calorimeter can be higher than the metabolic energy level calculated from the analysis by use of conversion factors. Th is method deviation was pointed out for low-lipid squid samples by Krishnamoorthy et al. [169]. In the study of feed, fish, and feces by Henken et al. [158] three different methods for calculating energy content were compared (I, dichromate wet oxidation; II, bomb calorimeter; or III, chemical analyses followed by conversion factors 5.65, 9.45, 4.2 kcal/g [proteins:fat:carbohydrates]). Proteins were calculated with N*6.25, fat analyzed by Soxhlet with hexane extraction, and carbohydrates calculated by difference. Agreements were obtained in methods II and III and lower energy values were obtained with method I. Inadequate protein oxidation by dichromate method
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[170] was solved by correction factors but still resulted in lower values in fish, feed, and feces compared with bomb calorimetry or direct analyses followed by conversion factors. In recent years the field of nutrition has become highly complex due to developments in both analytical and physiological methods. A variety of different analytical methods are in use along with various sets of conversion factors, which again are based on their own specific analytical methods. In scientific work it is particularly important to specify methods and calculations made in the presented results. Standardization of analytical methods and energy conversion factors might improve the use of nutrient databases for energy calculation.
16.7.3 Food Composition Tables and Databases Food composition databases are practical tools providing a line of useful information on foodrelated subjects. For the users it is convenient to find further links, reports, published works, nutrient composition tables, and so forth, through a database. Researchers are requested to make relevant publications available through these pages, adding to the up-front knowledge in the area. When food databases contain original analytical results, the values can be trusted to represent more accurate levels and are more useful for governmental and research purposes. There are several general databases available to the public both on international, regional, and national levels such as those of The International Network of Food Data Systems (FAO/INFOODS), United States Department of Agriculture (USDA), Pacific Island Food Composition Tables (PIFCT), and German Nutrient Database (BSL). The user groups for food databases are among others found within the groups of food researchers and industry, dieticians, epidemiological and health researchers, and national and governmental authorities. National and regional food composition tables are important, because they may reveal specific dietary traits of subpopulations important for health and epidemiological research. Differing nutritional definitions are also common as with different sets of energy conversion factors, which is important to be aware of when food tables are used. Databases as such FishBase provide specific tables for seafood such as proximate data and energy levels of different organs and ecological data of harvested species in specific regions. However, the databases might have a potential for improvement with regard to expected variability in the composition of food items, which might be due to seasonal variations, variations experienced during the growth, production phase, or as influenced by storage or processing conditions. Additionally, processed food with many ingredients is complex, some nutrients are labile, and constituents such as fat and moisture might be added and/or removed during food preparations. As it might be practically impossible to obtain the full detailed composition, there is selection of constituents in food tables. Most databases contain 10–25 food groups [160], but some also contain more than 100 nutrients and food components such as the Nutrition Data System for Research (NDS-R) in the United States [171]. Skills and knowledge in the analytical methods on which the values are based on, advantages, and drawbacks in the table values are required.
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3. Martens, H. and Russwurm, H., Food Research and Data Analysis, Applied Science Publisher, London, U.K., 1982. 4. Solberg, C., NIR–A rapid method for quality control. In: Seafood from Producer to Consumer, Integrated Approach to Quality, Eds., Luten, J.B., Børresen, T., and Oehlenschläger, J., Elsevier Science, Amsterdam, the Netherlands, pp. 529–534, 1997. 5. Wold, J.P. and Isaksson, T., Non-destructive determination of fat and moisture in whole Atlantic Salmon by near infrared diff use reflectance spectroscopy, J. Food Sci., 62, 734–736, 1997. 6. Downey, G., Non-invasive and non-destructive percutaneous analysis of farmed salmon flesh by near infrared spectroscopy, Food Chem., 55, 305–311, 1999. 7. Shimamoto, J., Hiratsuka, S., Hasegawa, K., Sato, M., and Kawano, S., Rapid non-destructive determination of fat content in frozen skipjack using a portable near infrared spectrophotometer, Fish. Sci., 69(4), 856–860, 2003. 8. Solberg, C., Saugen, E., Swenson, L.-P., Bruun, L., and Isaksson, T., Determination of fat in alive farmed Atlantic salmon using non-invasive NIR techniques, J. Sci. Food Agr., 83, 692–696, 2003. 9. Solberg, C., Rapid on line non-destructive measurement of fat in live and filleted salmon. In Near Infrared Spectroscopy: Proceedings of the 11th International Conference, Ed., Davies, A.M.C. and GarridoVaro, A., NIR Publications, Sussex, U.K., pp. 471–474, 2004. 10. Uddin, M., Okazaki, E., Turza, S., Yumiko, Y., Tanaka, M., and Fukuda, Y., Non-destructive visible/NIR spectroscopy for differentiation of fresh and frozen thawed fish, Food Chem. Toxicol., 70(8), 506–510, 2005. 11. Uddin, M., Okazaki, E., Ahmad, M.U., Fukuda, Y., and Tanaka, M., NIR spectroscopy: A nondestructive fast technique to verify heat treatment of fish-meat gel, Food Control, 17, 660–664, 2006. 12. Osborn, B.G., Fearn, T., and Hindle, P.H., Practical NIR Spectroscopy with Application in Food and Beverage Analysis. Longman Scientific and Technical, Essex, U.K., 90–144, 1993. 13. Lundby, F., Sørland, G.H., and Eilertsen, S., Determination of fat, moisture and protein in fish powder within 30 min, by combining low resolution NMR techniques and microwave technology, Abstract from the 8th International Conference on the Applications of Magnetic Resonance in Food Science, 2006, Nottingham, U.K. 14. Veliuylin, E., Van der Zwaag, C., Burk, W., and Erikson, U., In vivo determination of fat content in Atlantic salmon (Salmo salar) with a mobile NMR spectrometer, J. Sci. Food Agr., 85, 1299–1304, 2005. 15. Aursand, I., Veliuylin, E., and Erikson, U., Low-field NMR studies of Atlantic salmon (Salmo salar), In: Modern Magnetic Resonance, Modern Magnetic Resonance. Part 1: Applications in Chemistry, Biological and Marine Sciences, Ed., Webb, G.A., 895–903, 2006, Springer, Dordrecht, the Netherlands. 16. Blumich, B., Blumer, P., Eidman, G., Guthausen, A., Haken, R., Schmitz, U., Saito, K., and Zimmer, G., The NMR MOUSE: Construction, excitation and applications, Magn. Reson. Imaging, 16, 479–484, 1998. 17. Ghaedian, R., Coupland, J.N., Decker, E.A., and McClements, D.J., Ultrasonic determination of fish composition, J. Food Eng., 35(3), 323–337, 1998. 18. Suvanich, V., Ghaedian, R., Chanami, R., Decker, E.A., and McClements, D.J., Prediction of proximate fish composition from ultrasonic properties: Catfish, Cod, Flounder, Mackerel, and Salmon, J. Food Sci., 63(6), 966–968, 1998. 19. Sigfusson, H., Decker, E.A., and McClements, D.J., Ultrasonic characterisation of Atlantic mackerel (Scomber scombrus), Food Res. Int., 43, 15–23, 2001. 20. Shannon, R.A., Probert-Smith, P.J., Lines, J., and Mayia, F., Ultrasound velocity measurement to determine lipid content in salmon muscle; the effect of myosepta, Food Res. Int., 37, 611–620, 2004. 21. Jaramillo, F., Bai, S.C., Murphy, B.R., and Gatlin, D.M., Application of electrical-conductivity for nondestructive measurement of channel Catfish, Ictalurus punctatus, body-composition, Aquat. Living Resour., 7(2), 87–91, 1994.
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22. Veliuylin, E., Borge, A., Singstad, T., Gribbestad, I., and Erikson, U., Post-mortem studies of fish using magnetic resonance imaging. In: Modern Magnetic Resonance, Modern Magnetic Resonance. Part 1: Applications in Chemistry, Biological and Marine Sciences, Ed., Webb, G.A., 949–956, Springer, Dordrecht, the Netherlands, 2006. 23. Dyerberg, J., Bang, H.O., Stofferson, E., Monkada, S., and Vane, J.R., Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? Lancet, 117, 1978. 24. Uauy, R. and Valenzuela, A., Marine oils: The health benefits of n-3 fatty acids, Nutrition, 16(7–8), 680–684, 2000. 25. Vanshoonbecek, K., de Maat, M.P., and Heemssterk, J.W., Fish oil consumption and reduction of arterial disease, J. Nutr., 133, 657–660, 2003. 26. Ewald, G., Bremle, G., and Karlsson, A., Difference between Bligh and Dyer and Soxhlet extractions of PCBs and lipids from fat and lean fish muscle: Implications for data evaluations, Mar. Pollut. Bull., 36(3), 222–230, 1998. 27. Folch, J., Lees, M., and Stanley, G.H.S. Preparation of lipid extracts from brain tissue, J. Biol. Chem., 226, 497–509, 1957. 28. Bligh, E.G. and Dyer, W.J., A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911–917, 1959. 29. Iverson, S., Lang, S.L.C., and Cooper, M.H., Comparison of the Bligh and Dyer and Folch method for total lipid determination of a broad range of marine tissue, Lipids, 36(11), 1283–1287, 2001. 30. Lee, M.C., Trevino, B., and Chaiyawat, M., A simple and rapid solvent extraction method for determination of total lipids in fish, J. AOAC Int., 79(2), 487–492, 1996. 31. Smedes, F. and Askland, T.K., Revisiting the Bligh and Dyer total lipid determination method, Mar. Pollut. Bull., 38(3), 193–201, 1999. 32. Toge, Y. and Miyashita, K., Lipid extraction with electrolyzed cathode water from marine products, J. Oleo Sci., 52(2), 1–6, 2003. 33. Hara, A. and Radin, N.S., Lipid extraction of tissue with a low toxicity solvent, Anal. Biochem., 90, 420–426, 1978. 34. Burton, G.W., Webb, A., and Ingold, K.U., A mild, rapid and efficient method of lipid extraction for use in determining vitamin E/lipid ratios, Lipids, 20(1), 29–39, 1985. 35. Undeland, I., Harrod, M., and Lignert, H., Comparison between methods using low-toxicity solvents for the extraction of lipids from herring (Clupea harengus), Food Chem., 61(3), 355–365, 1998. 36. Woitke, P., Haarich, M., and Harms, U., Co-factors in biota: Results of a German interlaboratory exercise on the determination of total lipids in fi sh tissue, Accredit. Qual. Assur., 5, 499–503, 2000. 37. Jensen, S., Heggberg, L., Jorundsdottir, H., and Odham, G., A quantitative lipid extraction method for residue analysis of fish involving nonhalogenated solvents, J. Agr. Food Chem., 51(19), 5607–5611, 2003. 38. Christie, W.W., Ed., in Advances in Lipid Methodology–Two, Oily Press, Dundee, U.K., pp. 195–213, 1993. 39. Norwegian Standard (NS 9402), Atlantic Salmon: Measurement of Colour and Fat, 1st edn., Oslo, Norway, 1994. 40. AOAC Official methods, 960.39. In: Official Methods of Analysis, 15th edn., Association of Official Analytical Chemists, Arlington, VA, 1990. 41. Bremle, G., Okla, L., and Larsson, P., Uptake of PCBs in a contaminated river system: Bio concentration factors measured in the field, Environ. Sci. Technol., 29(8), 2010–2015, 1995. 42. Virot, M., Tomao, V., Colnagui, G., Visinoni, F., and Chemat, F., New microwave-integrated Soxhlet extraction. An advantageous tool for the extraction of lipids from food products, J. Chrom., 1174, 138–144, 2007. 43. Vogt, A., Gormley, T.R., Downey, G., and Somers, J., A comparison of selected rapid methods for fat measurement in fresh herring (Clupea harengus), J. Food Compos. Anal., 15, 205–215, 2002.
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171. Schakel, S.F., Maintaining a nutrient database in a changing marketplace: Keeping pace with changing food products–A research perspective, J. Food Comp. Anal., 14, 315–322, 2001. 172. Freese, M. and Hamid, M.A.K., Lipid content determination in whole fish using ultrasonic pulse backscatter. In: Ultrasonic Symposium Conference, 1974, Ed., De Klerk, J., Publisher IEEE, 69–76. 173. Lantry, B.F., Stewart, D.J., Rand, P.S., and Mills, E.L., Evaluation of total body electrical conductivity to estimate whole body water content of yellow perch (Perca flavescens) and alewife (Alosa pseudoharengu), Fish. Bull., 97, 71–79, 1999. 174. Novinger, D. C. and Martinez Del Rio, C., Failure of total body electrical conductivity to predict lipid content of brook trout, North Am. J. Fish. Manag., 19(4), 3, 1999. 175. Hancz, C., Milisits, G., and Horn, P., In vivo measurement of total body lipid content of common carp (Cyprinus carpio L.) by electrical conductivity, Arch. Tierzucht–Archives of Animal Breeding 46, 397–402, 2003. 176. Turgut, H., Drawbacks in the use of the Biuret method for determination of the same protein in differently treated fish samples, Food Chem., 4, 161–165, 1979. 177. Shono, N.I. and Baskaeva, E.M., Bradford’s method of determining protein: Application, advantages and disadvantages, Lab Delo., 4, 4–7, 1989. 178. Southgate, D.A.T., Availability of and needs for reliable analytical methods for the assay of foods, Food and Nutr. Bull., 5(2), 30–39, 1983. 179. Molnar-Perl, I. Derivatisation and chromatographic behaviour of the o-phthalaldehyde amino acid derivatives obtained with various SH-group containing additives, J. Chromatogr. A, 913, 283–302, 2001. 180. Jørgensen, B.M. and Jensen, K.N., Water distribution and mobility in fish products in relation to quality. In: Modern Magnetic Resonance, Part 1: Applications in Chemistry, Biological and Marine Sciences, Webb, Ed., G.A., 905–908, 2006, Springer, Dordercht, the Netherlands.
Chapter 17
Essential Amino Acids M. Concepción Aristoy and Fidel Toldrá Contents 17.1 Introduction ................................................................................................................. 287 17.2 Sample Preparation for the Analysis of Seafood Essential Amino Acids ....................... 288 17.2.1 Sample Preparation for Free Essential Amino Acid Analysis ........................... 288 17.2.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis................................................................................................... 289 17.3 Seafood Essential Amino Acid Analysis........................................................................ 290 17.3.1 High-Performance Liquid Chromatographic Methods.....................................291 17.3.1.1 Cation Exchange Chromatography ..................................................291 17.3.1.2 Reversed-Phase High-Performance Liquid Chromatography .......... 292 17.3.2 Gas Liquid Chromatographic Methods ........................................................... 298 17.3.3 Capillary Zone Electrophoretic Methods ........................................................ 298 17.3.4 Mass Spectrometry .......................................................................................... 299 17.4 Conclusions .................................................................................................................. 300 References ............................................................................................................................... 300
17.1 Introduction Amino acids are the basic components of the muscle protein structure of seafood. However, not all proteins have the same nutritional value, because protein quality strongly depends on its amino acid composition and digestibility.1 Fish and, in general, seafood proteins are considered as highquality proteins because of their balanced content in amino acids, especially in all the essential amino acids necessary for physical and mental well-being. Amino acids may also be found in free form, which contribute to fish taste and indirectly to aroma 2,3 by generation of volatile 287
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compounds through Maillard reactions and Strecker degradations.4 Branched-chain essential amino acids (valine, isoleucine, and leucine), sulfur-containing amino acids (methionine and cystine/cysteine), and aromatic amino acids (phenylalanine and tyrosine) are the most important from this point of view. Free amino acids initiate important changes at early postmortem and during storage and can be very useful as quality indices of processing and storage.5–10 Thus, the analysis of essential amino acids in seafood is important for the evaluation of both the nutritive value and the sensory quality of seafood. In this chapter, methods for the analysis of amino acids in seafood, especially of those considered essentials, are described. Special attention is also devoted to the analysis of the sulfur amino acid cysteine for several reasons: (1) the high reactivity of its thiol group, which confers numerous biological functions to this amino acid (precursor to the antioxidant glutathione), (2) its ability to cross-link proteins, which increases the protein stability in the harsh extracellular environment by conferring proteolytic resistance, and so forth, or (3) its Maillard reaction with sugars yielding characteristic flavors. Although classified as nonessential, in rare cases, cysteine may be essential for infants, the elderly, and individuals with certain metabolic disease or who suffer from malabsorption syndromes. A more detailed description of amino acid methods of analysis may be found in the work of Aristoy and Toldrá.11
17.2
Sample Preparation for the Analysis of Seafood Essential Amino Acids
Free or total essential amino acids are analyzed from the whole amino acid profile. Sample preparation will depend on whether free or total essential amino acids have to be analyzed.
17.2.1 Sample Preparation for Free Essential Amino Acid Analysis Sample preparation for free essential amino acids includes their extraction and the cleanup or deproteinization of the extract. The extraction consists in the separation of the free amino acid fraction from the insoluble portion of the matrix (fish muscle). It is usually achieved by homogenization of the ground sample in an appropriate solvent by using a Stomacher, Polytron, or by means of a simple stirring in warm solvent. The extraction solvent can be hot water, 0.01–0.1 N hydrochloric acid solution, or diluted phosphate buffers. In some cases, concentrated strong acid solutions such as 4% of 5-sulfosalicylic acid,12,13 5% of trichloroacetic acid,14 6% of perchloric acid,15 or a rich alcohol-containing solution (>75%) such as ethanol16–18 or methanol19,20 have been successfully used as extraction solvents, with the additional advantage that proteins are not extracted and, then, there is no need for further cleaning up of the sample. Once homogenized, the sample is centrifuged at more than 10,000 g under refrigeration (4°C) to separate the supernatant from the nonextracted materials (pellet) and filtered through glass wool to retain any fat material remaining on the surface of the supernatant. Sample cleanup is necessary to eliminate proteins and polypeptides by means of the deproteinization process, which can be achieved through different chemical or physical procedures. Several chemical methods include the use of concentrated strong acids such as phosphotungstic (PTA), sulfosalicylic (SSA),13,18,21 perchloric (PCA),16,22 trichloroacetic (TCA),23–25 and picric
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(PA)26–28 acids or organic solvents such as methanol, ethanol, or acetonitrile.22 Under these conditions, proteins precipitate by denaturation, whereas free amino acids remain in solution. Some physical methods consist in centrifugation through cutoff membrane filters (1,000, 5,000, 10,000, 30,000 Da) that allow free amino acids through while retaining large compounds.18,29,30 All these methods give a sample solution rich in free amino acids but free of proteins. Differences among all these chemical and physical methods are caused by several aspects such as differences in the cutoff molecular weight, recovery of amino acids, compatibility with derivatization (pH, presence of salts, etc.), or separation method (interferences in the chromatogram, etc.), and so forth. A good choice may be the use of 0.6 N PCA, which is easily neutralized by the addition of KOH or potassium bicarbonate, to rend insoluble potassium perchlorate, which is easily separated by centrifugation, resulting in a very simple deproteinization procedure with no interferences. The use of organic solvents, by mixing two or three volumes of organic solvent with one volume of extract, has also given very good results,31–33 with amino acid recoveries around 100% for all them. An additional advantage is the easy evaporation to concentrate the sample. Some comparative studies have been published on these deproteinization techniques.22,34
17.2.2 Sample Preparation for Total or Hydrolyzed Essential Amino Acid Analysis The total essential amino acid profile is usually requested, because it gives information on the nutritional value of fish meat. Proteins must be hydrolyzed into their constituent amino acids before the analysis. The most common method used for complete hydrolysis of proteins is acid digestion. Typically, samples are treated with constant boiling 6 N hydrochloric acid in an oven at around 110°C for 20–96 h. Digestion at 145°C for 4 h has also been proposed.35–38 These temperatures in such acidic and oxidative medium may degrade some amino acids. Nitrogen atmosphere and sealed vials are required during the hydrolysis to minimize the degradation. The hydrolysis may be accomplished using either liquid-phase or vapor-phase methods. Liquid-phase, where the hydrochloric acid contacts the sample directly, is well suited to hydrolyze large amounts or complex samples. When limited amounts of sample are available, the vapor-phase hydrolysis method is preferred to minimize contaminants coming from aqueous 6 N hydrochloric acid. In the vapor-phase hydrolysis method, the tubes containing the samples are located inside large vessels containing the acid. Upon heating, only the acid vapor comes into contact with the sample, thus excluding nonvolatile contaminants. In both cases, liquid phase or vapor phase, oxygen is removed and substituted by nitrogen or other inert gas, creating an appropriate atmosphere inside the vessels to ensure low amino acid degradation. Therefore, a system capable of alternative air evacuating/inert gas purging to get a correct deaeration inside is valuable.39 Some commercial systems are available. One of them is the Pico-Tag Workstation that includes special vessels (flat-bottom glass tubes) fitted with a heat-resistant plastic screw cap equipped with a Teflon valve, which permits the alternative air evacuating/inert gas purging, also disposes of an oven to accomplish the hydrolysis.40,41 The use of microwave technology for the hydrolysis has been assayed by some authors.39,42 Sample manipulation (sample evaporation to dryness, addition of constant boiling hydrochloric acid and additives, and performance under vacuum) is similar to that of a conventional oven, but the duration of the treatment is shorter (less than 20 min). Hydrolysis may be improved by optimizing the temperature and time of incubation41 or with the addition of amino acid oxidation protective compounds. The presence of appropriate antioxidants/scavengers during hydrolysis can prevent losses of the most labile amino acids, all of them
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essential amino acids, such as tyrosine, serine, threonine, methionine, and tryptophan. Thus, protective agents currently used, up to 1% phenol or 0.1% sodium sulfite, improve the recovery of nearly all of these amino acids except tryptophan and cysteine. Tryptophan is often completely destroyed by hydrochloric acid hydrolysis, although considerable recoveries have been found if no oxygen is present. Some additives have been proposed to protect tryptophan against oxidation as is the case of thioglycolic acid.38 Alkaline hydrolysis instead of acid hydrolysis is also proposed (see below). Cyst(e)ine is partially oxidized during acid hydrolysis yielding several adducts: cystine, cysteine, cysteine sulfinic acid, and cysteic acid making its analysis rather difficult. The previous performic acid oxidation of cysteine to cysteic acid, in which methionine is also oxidized to methionine sulfone,36,43–50 improves cysteine (and methionine) recoveries, making the posterior analysis easier. The use of alkylating agents to stabilize the previous hydrolysis of cysteine constitutes a valid alternative. Good recoveries have been achieved by using 3-bromopropionic acid,51 3-bromopropylamine,52 4-vinyl pyridine53,54 or 3,3¢-dithiodipropionic acid.30,55,56 As can be observed in this section, no single set of conditions will yield the accurate determination of all essential amino acids. In fact, a compromise of conditions offers the best overall estimation for the largest number of amino acids. In general, the 22–24 h acid hydrolysis at 110°C (vapor-phase or liquid-phase hydrolysis) with the addition of a protective agent like 1% phenol, yields acceptable results for the majority of amino acids, being enough for the requirements of any food industry. Additionally, when the analysis of cyst(e)ine would be necessary, adequate hydrolysis procedure as the performic acid oxidation before the hydrolysis is a good alternative. When high sensitivity is required, the pyrolysis from 500°C for 3 h57 to 600°C overnight58 of all glass material in contact with the sample is advisable as well as the analysis of some blank samples to control the level of background present. The optimization of conditions for hydrolysis based on the study of hydrolysis time and temperature, acid-to-protein ratio, presence and concentration of oxidation protective agents, importance of a correct deaeration, and so on, has been extensively reported in papers35,36, 41,59,60 and books.61,62 An alternative to acid hydrolysis is the alkaline hydrolysis with 4.2 M of either NaOH, KOH, LiOH, or BaOH, with or without the addition of 1% (w/v) thiodiglycol for 18 h at 110°C, which is recommended by many authors47,58,63–66 for a better tryptophan determination. A third way to hydrolyze proteins is enzymatic hydrolysis by proteolytic enzymes such as trypsin, chymotrypsin, carboxypeptidase, papain, thermolysin, or pronase. This option is chosen to analyze specific amino acid sequences or single amino acids because of their specific and well-defined activity.67–69
17.3
Seafood Essential Amino Acid Analysis
The analysis of individual amino acids needs a previous separation of all others, unless a very selective way of detection is used. The separation of the individual amino acids in a mixture requires very efficient separation, such as chromatographic (liquid or gas chromatography (GC)) or capillary electrophoresis (CE) techniques. The choice mainly depends on the equipment available or personal preferences, because each possible methodology has advantages and drawbacks. Before or after this separation, amino acids used to be derivatized to allow their separation or to enhance their detection. Derivatization is a usual practice in amino acid analysis. The effect of a derivatizing agent is evaluated based on the following aspects: (1) It must be able to react with both primary and secondary amino acids, (2) give a quantitative and reproducible reaction,
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(3) yield a single derivative of each amino acid, (4) have mild and simple reaction conditions, (5) have the possibility of automation, (6) have good stability of the derivatization products, and (7) have no interferences due to by-products or excess of reagent. It must be remarked that the use of sufficient amount of reagent is of special importance when dealing with biological samples, because reagent-consuming amines, although unidentified, are always present.12 Two types of derivatives are obtained depending on the chosen separation and/or detection technique. The first type are derivatives that enhance amino acid detection in liquid media, and they include derivatives for spectroscopic or for electrochemical detection. The formed derivatives will be separated by high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) as it is important to choose the most adequate derivative, because their spectral (high-ultraviolet (UV) absorbing or fluorescence properties) or electrochemical characteristics will affect the sensitivity and selectivity of detection. The second type are derivatives that allow gas chromatographic amino acid separation by increasing their volatility and temperature stability.
17.3.1
High-Performance Liquid Chromatographic Methods
HPLC is the preferred technique to analyze amino acids. Amino acids, in their native form, absorb at 210 nm and thus cannot be used for spectroscopic detection, as it is a very unspecific detection wavelength. Only three amino acids (phenylalanine, tyrosine, and tryptophan) have a chromophore moiety that confers a suitable maximum absorbance for more specific UV detection (280 nm for tyrosine and tryptophan and 254 nm for phenylalanine). Tryptophan also possesses native fluorescence (l ex = 295 nm, l em = 345 nm), which facilitates a more selective detection.68 Thus, the spectroscopic detection of amino acids requires their previous derivatization to obtain an UV absorbing or fluorescent molecule. The derivatization reaction can be performed after separation of the amino acids (postcolumn derivatization) or before separating them (precolumn derivatization). Although postcolumn techniques should be run online for maximum accuracy, precolumn techniques can be run either offline or online. The HPLC techniques to analyze amino acids are cation exchange and reversed-phase (RP) chromatography and are described in Sections 17.3.1.1 and 17.3.1.2.
17.3.1.1 Cation Exchange Chromatography This methodology is based on the amino acid charge, and thus the underivatized amino acids are separated using sulfonated polystyrene beads as the stationary phase and aqueous sodium citrate buffers as the mobile phase. The elution involves a stepwise increase in both pH and sodium or lithium ion concentration. Under these conditions, the more acidic amino acids elute first, and those with more than one primary amino group or possessing a guanidyl residue elute at the end of the chromatogram. The original method required two separate columns and needed about 4 h to achieve a complete analysis. After separation, amino acids were converted into colored ninhydrin derivatives for spectrophotometric (colorimetric) detection. The classical procedure has been improved with a new polystyrene matrix that offers better resolution power due to smaller particle size, speed, pellicular packaging, and better detection systems. The latest generation of Moore and Stein amino acid analyzers also use o-phthaldialdehyde (OPA),15,38,70 fluorescamine, or 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole postcolumn derivatization to obtain highly fluorescent derivatives with enhanced sensitivity, permitting 5–10 pmol sensitivity as standard. Nevertheless, recent improvements of the ninhydrin derivatization method71–73
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together with the low sensitivity requirements of fish amino acid analysis still make this method the most used.42,74 Nowadays, the separation times for the 20 amino acids naturally occurring in fish proteins take around 1 h and somewhat longer (2 h) for physiological amino acids.75,76 There are other reports of applying this technique to the amino acid analysis in food and tissues. 66,70,77 After separation, the derivatizing reagent is pumped into the effluent from the column system, through a mixing manifold, followed by a reaction coil, and finally the derivatized amino acids reach an online detector system. This method has been employed in the classical Moore and Steintype commercial amino acid analyzers. Obviously, the main drawback of this type of derivatization method is the required additional equipment: another pump to introduce the reagent as well as mixing and sometimes heating devices. Another disadvantage is the peak broadening produced by the dead volume introduced behind the column. Although this broadening may not affect when using standard-bore columns with flow rates above 1 mL/min, postcolumn derivatization is not suitable for narrow-bore HPLC. There are many manufacturers (Beckman, Amersham Biosciences, Biotronik, Dionex, Hitachi, LKB, Pickering, Kontron, etc.) who offer integrated commercial systems including the column, buffer system, and an optimized methodology with the advantage of ease of use and reliability. The advantage of this method is the accurate results for all known sample types (food, tissues, biological fluids, feed, plants), which makes it a reference method for amino acid analysis. In this way, each new methodology must contrast its results with those obtained by cation exchange chromatography (CEC). The main drawbacks of this methodology are the high cost of the ion exchange amino acid analyzer and its maintenance, the highly complex mobile phase composition, and the long time of analysis.
17.3.1.2 Reversed-Phase High-Performance Liquid Chromatography RP-HPLC has been widely used, because it requires only a standard equipment that can be shared by different types of analysis. This fact and the proliferation of precolumn derivatizing agents have stimulated the development of RP-HPLC methods to analyze amino acids in all kind of matrices (food, plants, biological fluids, and tissues). Precolumn amino acid derivatization may be necessary to confer hydrophobicity to the amino acid molecule, making it adequate for partition based on chromatography, but, also, the formed molecule improves sensitivity and selectivity at the detection by allowing the spectroscopic (UV or fluorescent) detection of amino acids. The resulting system is simpler and cheaper compared with the combination of cation-exchange plus postcolumn derivatization and permits choosing among a great number of possible methodologies, with which many of them have been marketed. To choose the most appropriate method some aspects must be taken into account such as the following: the disposable detector (fluorescence or UV), possibility of automation of the derivatization reaction (in the autosampler), the analysis requirements for free or hydrolyzed amino acids or required sensibility, time for sample preparation and amino acids separation, or the stability of formed derivatives, (i.e., some difficulties to analyze some essential or sulfur-containing amino acid derivatives). The most usual derivatizing agents for tissue amino acids are described below. Phenylisothiocyanate (PITC): This methodology involves the conversion of primary and secondary amino acids to their phenylthiocarbamyl (PTC) derivatives, which are detectable at UV (254 nm) with detection limits around 5–50 pmol. All PTC-amino acids have similar response factors, which constitutes an advantage. The PTC-amino acids are moderately stable at room temperature for 1 day and much longer when kept under frozen storage, especially in a dry condition.
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The methodology is well described in the literature.29,78–80 Sample preparation is quite tedious: it requires a basic medium (pH = 10.5), which is achieved by the addition of triethylamine and includes several drying steps, the last one being the elimination of the excess of reagent that may cause some damage to the chromatographic column. It is important to ensure a basic pH to get adequate derivatization recoveries, which is more critical when amino acids from acid hydrolysis are analyzed, because no buffer is used during the reaction. The reaction time is less than 10 min even though 20 min are recommended for a complete reaction.29,78–80 The chromatographic separation takes around 20 min for hydrolyzed amino acids and 60 min for physiological. Both examples applied to the analysis of total amino acids from hake and free amino acids from salmon are shown in Figures 17.1 and 17.2, respectively. Sarwar et al.81 reported a modification of the method in which the analysis of 27 physiological amino acids could be performed in 22 min (30 min including equilibration). The only limitation is the determination of PTC cystine that gives a poor linearity, which makes the quantitation of free cystine nonfeasible with this method.82 The selection of the column is critical to get a good resolved separation especially when the analysis of physiological amino acids is involved. Moreover, the residual PITC reagent left after evaporation will cause damage to the column package, as some columns are more resistant than others. This method is available as a commercially prepackaged system named Pico-Tag (Waters Associates, Milford, Massachusetts), which includes the analytical column, standards, and solvents. 700 Ala 600 Gly Absorbance at 254 nm (mAU)
500 Glu 400
lS Lys
300 Asp
Arg
Leu
200 Ser
Thr
Pro
100 OHpro
Tyr
His
Val
Met lle
Phe
0 0
2
4
6
8
10
12
14
16
18
Retention time (min)
Figure 17.1 Reversed-phase HPLC chromatogram of PTC amino acids from hydrolyzed hake muscle. IS, internal standard nor-Leucine.
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1200
Glu
Absorbance at 254 nm (mAU)
Ans 1000
800
Gly
600
Tau βAla His
400 Asp 200
Ala
Lys
Pro
OHpro
Tyr Arg
0
lS
Val
Thr
Ser
Met
Leu lle
Trp Phe Orn
Gln 0
Asn
10
20
30
40
50
Retention time (min)
Figure 17.2 Reversed-phase HPLC chromatogram of PITC-free amino acids from salmon muscle extract. Tau, taurine; Ans, anserine; IS, internal standard nor-Leucine.
4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl): This reagent was first described in 1975 for use in amino acid analysis.83,84 Detection is by absorption in the visible range, presenting a maximum from 448 to 468 nm. The high wavelength of absorption makes the baseline chromatogram very stable with a large variety of solvents and gradient systems. Detection limits are in the low picomole range.58,84 Derivatives are very stable (weeks) and can be formed from both primary and secondary amino acids. The reaction time is around 15 min at 70°C and takes place in a basic medium with an excess of reagent. Reaction efficiency is highly matrix dependent and variable for different amino acids, because it is especially affected by the presence of high levels of some chloride salts.33 To overcome this problem and obtain an accurate calibration, standard amino acid solution should be derivatized under similar conditions. By-products originating from an excess of reagent absorb at the same wavelength and thus they appear in the chromatogram. Nevertheless, Stocchi et al.58 obtained a good separation of 35 dabsyl-amino acids and by-products in a 15 cm C18 column packed with 3 mm particle size. Commercial System Gold/Dabsylation Kit™ uses this technique (Beckman Instruments, Palo Alto California, United States). 1-Dimethylamino-naphthalene-5-sulfonyl chloride (Dansyl-Cl): Dansyl-Cl reacts with both primary and secondary amines to give a highly fluorescent derivative (l ex = 350, l em = 510 nm) although UV (l = 250 nm) detection may also be used. The dansylated amino acids are stable for 1 day85 or until 7 days when kept at −4°C86 and protected from light. The sample derivatization is rather simple, and only needs a basic pH, around 9.5, and a reaction time of 1 h at room temperature (in the dark), 15 min at 60°C,87 or even 2 min at 100°C. However, the reaction conditions
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(pH, temperature, and excess of reagent) must be carefully fi xed to optimize the product yield and to minimize secondary reactions.86,88 Even so, this will commonly form multiple derivatives with histidine, lysine, and tyrosine. Histidine gives a very poor fluorescence response (10% of the other amino acids), reinforcing the poor reproducibility of its results.82 Another problem is the large excess of reagent needed to assure a quantitative reaction. This excess is hydrolyzed to dansyl sulfonic acid, which is present in excess as it is highly fluorescent and probably interferes into the chromatogram as a huge peak. On the contrary, this methodology reveals excellent linearity for cystine and also cystine-containing short-chain peptides.82,89 9-Fluorenylmethyl chloroformate (FMOC): This reagent yields stable derivatives (days) with primary and secondary amines. The derivative is fluorescent (l ex = 265 nm; l em = 315 nm) and is detected at the femtomole range. The major disadvantage is due to the reagent, itself or hydrolyzed, as it is highly fluorescent and then, the excess may interfere in the chromatogram and for this reason it must be extracted (with pentane or diethyl ether) or converted into noninterfering adduct before injection. The first option was included in the automated AminoTag method90 developed by Varian (Varian Associates Limited). In the second option, the reaction of the excess of reagent with a very hydrophobic amine as 1-adamantylamine (ADAM) gives a late-eluting noninterfering peak.91 This method is preferred because the addition of ADAM is more easily automatized. The reaction time is fast (45–90 s) and does not require any heating. In order to obtain reliable and precise results, reaction conditions, such as FMOC/amino acid ratio, as well as reaction time, have to be optimized very carefully. An automated precolumn derivatization routine, which includes the addition of ADAM, is of great advantage, because it guarantees the repeatability of parameters. Tryptophan adducts do not fluoresce and histidine and cyst(e)ine adducts fluoresce weakly. o-Phthaldialdehyde (OPA): This reagent reacts with primary amino acids in the presence of a mercaptan cofactor to give highly fluorescent 1-alkylthio-2-alkyl-substituted isoindols.92,93 The fluorescence is recorded at 455 or 470 nm after excitation at 230 or 330 nm, and the reagent itself is not fluorescent. OPA derivatives can be detected by UV absorption (338 nm) as well. The choice of the mercaptan can affect derivative stability, chromatographic selectivity, and fluorescent intensity;94–96 2-mercaptoethanol, ethanethiol, and 3-mercaptopropionic acid are the most frequently used. The derivatization is fast (1–3 min) and is performed at room temperature in alkaline (pH 9.5) medium. OPA amino acids are not stable; this problem is overcome by standardizing the time between sample derivatization and column injection by automation. This is relatively easy because the reaction is fast and no heating is necessary. Nowadays, many automatic injectors are programmable and able to achieve automatic derivatizations. Some reports have been published proposing several ways of automation,97,98 and some of them have been patented and commercially marketed (AutoTag OPA from Waters Associates). One of the main disadvantages of this procedure is the inability of OPA to react with secondary amines, which is not the case with any essential amino acid. The yield with lysine and cysteine is low and variable. The addition of detergents like Brij 35 to the derivatization reagent seems to increase the fluorescence response of lysine.99 In the case of cysteine, several methods have been proposed before derivatization. These methods include the conversion of cysteine and cystine to cysteic acid by oxidation with performic acid or carboxymethylation of the sulfhydryl residues with iodoacetic100,101 or the formation of the mixed disulfide S-2-carboxyethylthiocysteine (Cys-MPA) from cysteine and cystine, using 3,3′-dithiodipropionic acid55 and incorporated by Godel et al.94 into the automatic sample preparation protocol described by Schuster.32 In these methods, cysteine and cystine are quantified together. Another proposal102 consists of a slight modification in the OPA derivatization method by using 2-aminoethanol as a nucleophilic agent and altering the order of the addition of reagents in the automated derivatization procedure.32
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6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC): It reacts with primary and secondary amines from amino acids, peptides, and proteins, yielding very stable derivatives (1 week at room temperature) with fluorescent properties (l ex = 250 nm; l em = 395 nm), which are separated by RP-HPLC. UV detection (254 nm) may also be used. Sensitivity is in the femtomole range, making them very adequate for biochemical research.103 The main advantage of this reagent is that the yield and reproducibility of the derivatization reaction are scarcely interrupted by the presence of salts, detergents, lipids, and other compounds naturally occurring in biological samples and foods. Furthermore, the optimum pH for the reaction is in a broad range, from 8.2 to 10. Both facts facilitate sample preparation. The excess of reagent is consumed during the reaction to form aminoquinoline (AMQ), which is only weakly fluorescent at the amino acid derivatives detection conditions and does not interfere in the chromatogram. Reaction time is short, 1 min, but 10 min at 55°C would be necessary if a tyrosine monoderivative is required, because both mono- and di-derivatives are the initial adducts from tyrosine. The fluorescence of tryptophan derivative is very poor, and UV detection at 254 nm may be used for its analysis. In this case, the AMQ peak is very large at the beginning of the chromatogram and may interfere with the first eluting peaks (see Bosch et al.50). The chromatographic separation of these derivatives has been optimized for the amino acids from hydrolyzed proteins, and the separation of physiological amino acids is improved. Cystine and cysteine may be analyzed after their conversion to cysteic acid (CisH) by performic acid oxidation, because the resulting CisH is well separated inside the chromatogram. Figure 17.3A shows the separation of hydrolyzed amino acids from salmon, whereas Figure 17.3B shows the same sample but submitted to a performic acid oxidation before the hydrolysis in which the CisH peak appears by 7.5 min. The methodology has been marketed as a prepackaged AccQ Tag kit (Waters Corporation, Milford, Massachusetts, United States). Some of these derivatives are also susceptible to electrochemical detection, because they are molecules with electroactive functional groups. Indeed, OPA/mercaptoethanol or OPA/sulfite104,105 in addition to fluorescent properties possesses electroactivity (750 mV) and PITC106 has again the advantage of reacting with secondary amines. Electrochemical detection consists in one electrode or an array of electrodes mounted in a cell with an applied potential difference. Any electrical measure, such as current, potential, conductance, or charge, is related to the analyte concentration. Only amino acids with aromatic rings or sulfur-containing side chains are sufficiently electrochemically active to be detected by this method.107,108 If the choice of the derivative reaction is a challenge, the choice of the RP column is not an easy subject because of the great variability of commercially available RP columns. The most used column packaging consists of alkyl-bonded silica particles, mainly octadecylsilane. However, the selectivity obtained with each trademark column is different due to the particular chemistry employed in their manufacture rendering different density of bonded-phase coverage on the silica particle and hydrophobic behavior and, as a consequence, different selectivity. The presence of residual uncapped silanol groups on the silica surface, accessible to sample molecules, can cause unwanted tailing of peaks (especially for the basic amino acids). In these cases, the addition of a strong cation (i.e., triethylamine) to the mobile phase can overcome the problem. Nowadays, columns are more carefully manufactured with these silanol groups blocked or inaccessible by steric impediment avoiding the tailing. Due to these variables, different selectivity may be found among same columns, even those made by the same manufacturer. Only columns manufactured in the same batch are guaranteed to give the same selectivity if the rest of parameters are fi xed. It means that when transferring a published method to a particular set of samples, it will be necessary to readjust the chromatographic conditions to get a good separation of all amino acids.
Essential Amino Acids 200
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297
Leu
(A)
150 Val
lle Phe
αAba
100
Lys
Ala Glu
NH3 Arg Asp Thr Gly Ser His
Fluorescence (% FS)
50
Met Tyr Pro
0 1MeHis
βAla
200 (B)
150
100
MeS 50
CysH 0
0
5
10
15
20
25
30
35
Retention time (min)
Figure 17.3 Reversed-phase HPLC chromatogram of AQC amino acids from hydrolyzed salmon muscle (A) without and (B) after performic acid oxidation. 1MeHis, 1-methylhistidine; aAba, a aminobutyric acid used as internal standard; CysH, cysteic acid; MeS, methiomine sulfone.
Typical analytical column dimensions are 15 cm (for hydrolyzed amino acids) or 25–30 cm (for physiological amino acids), packed with 5 mm particle size or shorter columns (10 or 15 cm length) when packed with less than 3 mm particle size. Mobile-phase requirements consist in the ability to dissolve the sample while keeping it transparent to the detection system. Mobile-phase composition combines an aqueous buffered phase
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with an organic phase constituted by acetonitrile and/or methanol and/or tetrahydrofuran. The buffer may be constituted by less than 100 mM concentration of acetate or phosphate. A finely adjusted binary (most used) or ternary gradient elution is often necessary when the overall amino acid profile from hydrolyzed and, especially, physiological amino acids has to be analyzed.
17.3.2
Gas Liquid Chromatographic Methods
The extremely high-resolution capacity is the main advantage of GC, in comparison with liquid chromatographic techniques, especially since the capillary columns appeared. Gas liquid chromatography (GLC) is not often used for the determination of amino acids from tissues or foods. Some applications67,109,110 comparing GLC with cation exchange chromatography reported different conclusions when analyzing some hydrolyzed food samples. Nevertheless, the technique is very efficient and it is worth mentioning the separation of 32 nonprotein amino acids from edible seeds, nuts, and beans111 or other results obtained in honey,112 milk,113 and cheese.114 In their analysis by GLC, the amino acids must be converted to volatile and thermostable molecules. Reactions consist of two stages: an esterification with an acidified alcohol followed by N-acylation with an acid anhydride in an anhydrous medium. The detector used is the flame ionization detector (FID), which is universal and the most widely used, whereas thermionic-N-P (NPD) or flame photometric detector (FPD) are selective toward organic compounds containing phosphorous and nitrogen, which are much more sensitive than FID for such compounds. The NPD was used by Buser and Erbersdobler115 and FPD by Kataoka et al.116 to analyze phosphoserine, phosphothreonine, and phosphotyrosine. The main advantages of these detectors are their high sensitivity and wide linear range. In many cases, GLC has been combined with mass spectrometry (MS) for detection and identification, especially in the analysis of D isomers,113,117–119 where the separation was achieved by using chiral-GC stationary phases. Recently, a very fast GC analysis of physiological amino acids, capable of separating 50 compounds, including amino acids, dipeptides, and amines, has been developed. This methodology has been patented as EZ:faast and commercialized by Phenomenex (Torrance, California, United States). The method yields a full amino acid profile (33 amino acids) in 15 min including a 7 min extraction-derivatization step plus 8 min for the gas chromatographic separation. Protein removal is not required, and the derivatives are stable and ready for GC/FID, GC/NPD, or GC and LC with MS detection. Described applications are available for the analysis of physiological amino acids in blood, plasma, and urine matrices but not in tissues in which the presence of natural dipeptides, anserine, carnosine, and balenine may complicate the amino acid analysis. GLC is, in summary, a very highly efficient technique adequate for the amino acid analysis, although applications on meat samples are scarcely described. GLC is not very expensive because no solvent is used, and the equipment is very versatile and usually available in any analytical laboratory.
17.3.3
Capillary Zone Electrophoretic Methods
Capillary zone electrophoretic technique is extremely efficient for the separation of charged solutes.120,121 The high efficiency, speed, and low amount of sample make this technique very interesting when compared with classical electrophoresis and chromatographic techniques. The difficulty of separating amino acids by this technique relies on their structure. Amino acids constitute a mix of basic, neutral, and acidic constituents, and even though a particular pH can significantly
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improve the resolution of one kind, it is likely to cause overlap with the others. Under the conditions of electro-osmotic flow in CE, the species with different charge can be simultaneously analyzed but with serious doubts in their adequate resolution. CZE shows poor ability for the separation of neutral compounds, which constitutes an important limitation of this technique. Terabe et al.122,123 introduced a modified version of CZE in which surfactant-formed micelles were included in the running buffer to provide a two-phase chromatographic system for separating neutral compounds together with charged ones in a CE system. This technique has also been termed micellar electrokinetic capillary chromatography (MECC or MEKC).124 Basic theoretical considerations on this technique125 and its food applications126 are described elsewhere. With few exceptions,127–131 derivatization is used to improve separation, to enhance UV detection, or to allow fluorescence or electrochemical132 detection of amino acids. Good separations have been reported for precapillary derivatized amino acids with dansyl-Cl,125,133–135 PITC,136 phenylthiohydantoin,137,138 and OPA139 compared with the separation of OPA-amino acid derivatives by CZE with normal and micellar solutions, showing that higher efficiency is obtained by the MECC methods with sodium dodecyl sulfate (SDS) as micelle-forming substance. SDS is indeed the most used additive to form micelles in this kind of analysis, although other additives such as dodecyltrimethylammonium bromide,138 Tween 20,140 or even urea141 have been assayed. Other additives commonly used in this analysis are organic modifiers (acetonitrile, isobutanol, methanol, tetrahydrofurane, etc.). The effect of these additives on the electro-osmotic mobility and electrophoretic mobility of the micelle has been studied.141–143 The CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS) allows direct amino acid analysis without derivatization,131 and thus, 19 amino acids were analyzed by CE-ESI-MS in only 17 min with a minimal sample preparation and no matrix interference. This report includes the optimization of important parameters like the choice of a volatile electrolyte (1 M formic acid) for the electrophoresis, compatible with MS, and the composition and flow rate of the sheath liquid to obtain the best sensitivity. When sensitivity is the target, it is relatively easy to analyze low picomol levels of OPA derivatives in micellar solutions by using a conventional fluorometric detector,139 which is usually enough for food analysis or an LIF (laser-induced fluorescence) detector,136,144,145 when looking for more selective and sensitive detectors with a wide linear dynamic range (3 orders of magnitude) to cover new high-sensitivity applications (chiral analysis, o-tyrosine analysis, biomedical or pharmaceutical research, etc.) and instrumentation (CE, microcolumn liquid chromatography, etc.). Some reviews covering high-sensitivity detection following CE have been published.146,147
17.3.4
Mass Spectrometry
MS is based on the conversion of components of a sample into rapidly moving gaseous ions, which can be resolved on the basis on their mass-to-charge ratios that are characteristic of each ion and allow its identification. The identification of the 22 protein amino acids may not be a problem, although this detector may be used for more complex identifications as in d- and l-isomer mixtures, nonprotein amino acids, and so forth. Unfortunately, the high cost of purchase and maintenance of mass spectrometers has inhibited their more widespread use in the food industry and/or food control. Nevertheless, reports in the literature of its applications are increasing rapidly. Mass spectrometer detectors were first connected to GC equipments. A good compatibility between both techniques, in particular when capillary columns were available, allowed a rapid development and the onset of these complementary techniques. Application in foods such as in the identification of nonprotein amino acids,21 chiral amino acids,113 o-tyrosine in chicken148 or pork149 tissues, and others,118,119 have been reported.
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MS has also been used as a spectroscopic detector after HPLC or CZE, offering the additional advantage of analyzing the amino acids without derivatization, which means a minor sample manipulation, and, due to its high specificity, reduced problems related with matrix interferences or poor resolution between peaks. The connection of HPLC and MS detector is much more problematic than with GLC because of the incompatibility between both techniques (solvents from chromatography, high mobile-phase flow rate vs. vacuum). However, nowadays these difficulties have been overcome with the development of new interfaces, and the technique is widespread although it is still expensive. One of the main requirements for samples to be analyzed by MS is that analytes, amino acids in this case, must be ionized. Three types of ionization modes, atmospheric pressure microwave-induced plasma ionization (AP-MIPI), atmospheric pressure chemical ionization (APCI), and ESI, were compared by Kwon and Moini150 in relation to sensitivity. The best results were obtained by using AP-MIPI in conjunction with a dual oscillating capillary nebulizer.
17.4 Conclusions To obtain the total essential amino acids profile of a given seafood, the most important factors to take into account are the resolution power and selectivity. The highest resolution is obtained by GC with the capillary column technique, but tedious and time-consuming sample derivatization is required. In general, cation exchange and postcolumn derivatization or RP-HPLC precolumn derivatization techniques are the preferred methods. The majority of published reports in which seafood amino acids are analyzed have used the cation exchange method. Nevertheless, RP-HPLC methods with precolumn OPA or PITC derivatization are very convenient methods to use, and many applications can be found in other matrices like cheese or meat, which may be consulted.151,152 A very careful control of the derivatization reactions and chromatographic conditions are necessary for a consistent and reproducible analysis. Since many peaks corresponding to protein and nonprotein amino acids, nucleosides, small peptides, and so on, may appear in the chromatogram, a complete resolution of the whole peaks is really difficult. Therefore, the analytical technique for a determined sample must be carefully chosen based on the literature. The convenience of purchasing commercially available kits must be evaluated. When amino acids from seafood proteins have to be analyzed, the first decision is the choice of the hydrolysis method. In general, acid hydrolysis with HCl 6 N (110°C for 22 h or 145°C for 4 h) with an oxidation protective agent, such as phenol, and taking care of avoiding the presence of oxygen with vacuum and nitrogen purging, is enough for the majority of purposes. Particular hydrolysis problems related with certain amino acids are described in Section 17.2.2. The requirements in resolution are not so exigent as those for physiological amino acids, because fewer peaks appear in the chromatogram. Any separation strategy may give good results, and, once again, the convenience of purchasing commercially available prepackaged kits should be considered.
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4. Toldrá, F. Meat: Chemistry and biochemistry. In: Handbook of Food Science, Technology and Engineering. Hui, Y.H., Culbertson, J.D., Duncan, S., Guerrero-Legarreta, I., Li-Chan, E.C.Y., Ma, C.Y., Manley, C.H., McMeekin, T.A., Nip, W.K., Nollet, L.M.L., Rahman, M.S., Toldrá, F., Xiong, Y.L. (Eds.), Vol. 1, CRC Press, Boca Raton, FL, 2006, 28–1 to 28–18. 5. Sakaguchi, M., Murata, M., Kawai, A. Changes in free amino acids and creatine contents in yellowtail (Seriola quinqueradiata) muscle during ice storage. J. Food Sci. 47, 1662–1666, 1982. 6. Mendes, R., Goncalves, A., Nunes, M.L. Changes in free amino acids and biogenic amines during ripening of fresh and frozen sardine. J. Food Biochem. 23, 295–306, 1999. 7. Ruiz-Capillas, C., Moral, A. Changes in free amino acids during chilled storage of hake (Merluccius merluccius L.) in controlled atmospheres and their use as a quality control index. Eur. Food Res. Technol. A 212, 302–307, 2001. 8. Ruiz-Capillas, C., Moral, A. Effect of controlled and modified atmospheres on the production of biogenic amines and free amino acids during storage of hake. Eur. Food Res. Technol. A 214, 476–481, 2002. 9. Otsuka Y., Tanaka S., Nishigaki K. et al. Change in the contents of arginine, ornithine, and urea in the muscle of marine invertebrates stored in ice. Biosci. Biotech. Biochem. 56, 863–866, 1992. 10. Chang, C.H.M., Ohshima. T., Koizumi, C. Changes in composition of lipids, free amino acids and organic acids in rice bran fermented sardine (Etrumeus teres) during processing and subsequent storage. J. Sci. Food Agric. 59, 521–528, 1992. 11. Aristoy, M.C., Toldrá, F. Amino acids. In: Handbook of Food Analysis. Nollet, L.M.L. (Ed.), Vol. 1, Marcel Dekker, Inc., New York, 2004, 5–83 to 5–123. 12. Godel, H., Graser, T., Foldi, P. et al. Measurement of free amino acids in human biological fluids by high-performance liquid chromatography. J. Chromatogr. 297, 49–61, 1984. 13. Arnold, U., Ludiwg, E., Kuhn, R. et al. Analysis of free amino acids in green coffee beans. I. Determination of amino acids after precolumn derivatisation using 9-fluorenylmethylchloroformate. Z. Lebensm. Unters. Forsch. 199, 22–25, 1994. 14. Shibata, K., Onodera, M., Aihara, S. High-performance liquid chromatographic measurement of tryptophan in blood, tissues, urine and foodstuffs with electrochemical and fluorometric detections. Agric. Biol. Chem. 55, 1475–1481, 1991. 15. Ruiz-Capillas, C., Moral, A. Free amino acids in muscle of Norway lobster (Nephrops novergicus L.) in controlled and modified atmospheres during chilled storage. Food Chem. 86, 85–91, 2004. 16. Ali Qureschi, G., Fohlin, L., Bergström. Application of high-performance liquid chromatography to the determination of free amino acids in physiological fluids. J Chromatogr. 297, 91–100, 1984. 17. Nguyen, Q., Zarkadas, C.G. Comparison of the amino acid composition and connective tissue protein contents of selected bovine skeletal muscles. J. Agric. Food Chem. 37, 1279–1286, 1989. 18. Hagen, S.R., Augustin, J., Grings, E. et al. Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples. Food Chem. 16, 319–323, 1993. 19. Antoine, F.R., Wei, C.I., Littell, R.C. et al. HPLC method for analysis of free amino acids in fish using o-phthaldialdehyde precolumn derivatization. J. Agric. Food Chem. 47, 5100–5107, 1999. 20. Antoine, F.R., Wei, C.I., Littell, R.C. et al. Free amino acids in dark- and white-muscle fish as determined by o-phthaldialdehyde precolumn derivatization. J. Food Sci. 66, 72–77, 2001. 21. Izco, J.M., Torre, P., Barcina, Y. Ripening of Ossau-Iratzy cheese: Determination of free amino acids by RP-HPLC and of total free amino acids by the TNBS method. Food Control 11, 7–11, 2000. 22. Aristoy, M.C., Toldrá, F. Deproteinization techniques for HPLC amino acid analysis in fresh pork muscle and dry-cured ham. J. Agric. Food Chem. 39, 1792–1795, 1991. 23. Büetikofer, U., Ardö, Y. Quantitative determination of free amino acids in cheese. Bull. Int. Dairy Fed. 337, Part 2, 24–32, 1999. 24. Ordóñez, A.I., Ibañez, F.C., Torre, P. et al. Characterization of the casein hydrolysis of Idiazabal cheese manufactured from ovine milk. J. Dairy Sci. 81, 2089–2095, 1998. 25. Sanz, Y., Flores, J., Toldra, F. et al. Effect of pre-ripening on microbial and chemical changes in dry fermented sausages. Food Microbiol. 14, 575–582, 1997.
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26. Brüeckner, H., Hausch, M. d-amino acids in dairy products: Detection, origin and nutritional aspects. I. Milk, fermented milk, fresh cheese and acid curd cheese. Milchwissenschaft. 45, 357–360, 1990. 27. Oh, C.H., Kim, J.H., Kim, K.R. et al. Rapid gas chromatographic screening of edible seeds, nuts and beans for non-protein and protein amino acids. J. Chromatogr. A 708, 131–141, 1995. 28. Sugawara, H., Itoh, T., Adachi, S. Assignment of an unknown peak on the high performance liquid chromatogram of free amino acids isolated from fresh chicken egg albumen using picric acid as the deproteinizing agent. Jap. J. Zootech. Sci. 55, 892–893, 1984. 29. Cohen, S.A., Strydon, J. Amino acid analysis utilizing phenylisothiocyanate derivatives. Anal. Biochem. 174, 1–16, 1988. 30. Krause, I., Bockhardt, A., Neckermann, H. et al. Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives. J. Chromatogr. A 715, 67–79, 1995. 31. Sarwar, G., Botting, H.G. Rapid analysis of nutritionally important free amino acids in serum and organs (liver, brain, and heart) by liquid chromatography of precolumn phenylisothiocyanate. J. Assoc. Off. Anal. Chem. 73, 470–475, 1990. 32. Schuster, R. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. J. Chromatogr. 431, 271–284, 1988. 33. Jansen, E.H.J.M., Vandenberg, R.H., Bothmiedema, R. et al. Advantages and limitations of precolumn derivatization of amino acids with dabsyl chloride. J. Chromatogr. 553, 123–133, 1991. 34. Blanchard, J. Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis. J. Chromatogr. Biomed. Appl. 226, 455–460, 1981. 35. Lucas, B., Sotelo, A. Amino acid determination in pure proteins, foods, and feeds using two different acid hydrolysis methods. Anal. Biochem. 123, 349–356, 1982. 36. Gehrke, C.W., Wall, L.L., Absheer, J.S. et al. Sample preparation for chromatography of amino acids: Acid hydrolysis of proteins. J. Assoc. Off. Anal. Chem. 68, 811–821, 1985. 37. Gehrke, C.W., Rexroad, P.R., Schisla, R.M. et al. Quantitative analysis of cystine, methionine, lysine, and nine other amino acids by a single oxidation-4 h hydrolysis method. J. Assoc. Off. Anal. Chem. 70, 171–174, 1987. 38. Ashworth, R.B. Ion-exchange separation of amino acids with postcolumn orthophthalaldehyde detection. J. Assoc. Off. Anal. Chem. 70, 248–252, 1987a. 39. Woodward, C., Gilman, L.B., Engelhart W.G. An evaluation of microwave heating for the vapor phase hydrolysis of proteins. Int Lab. Sept. 40–45, 1990. 40. Molnár-Perl, I., Khalifa, M. Tryptophan analysis simultaneously with other amino acids in gas phase hydrochloric acid hydrolyzates using the Pico-Tag™ Work Station. Chromatographia 36, 43–46, 1993. 41. Molnár-Perl, I., Khalifa, M. Analysis of foodstuff amino acids using vapour-phase hydrolysis. LC-GC Int. 7, 395–398, 1994. 42. Rosa, R., Nunes, M.L. Nutritional quality of red shrimp, Aristeus antennatus (Risso), pink shrimp, Parapenaeus longirostris (Lucas), and Norway lobster, Nephrops norvegicus (Linnaeus). J. Sci. Food Agric. 84, 89–94, 2004. 43. Moore, S. On the determination of cystine and cysteic acid. J. Biol. Chem. 243, 235–237, 1963. 44. Hirs, C.H.W. Performic acid oxidation. Methods Enzymol. 11, 197–199, 1967. 45. MacDonald, J.L., Krueger, M.W., Keller, J.H. Oxidation and hydrolysis determination of sulfur amino acids in food and feed ingredients: Collaborative study. J. Assoc. Off. Anal. Chem. 68, 826–829, 1985. 46. Elkin, R.G., Griffith, J.E. Hydrolysate preparation for analysis of amino acids in sorghum grains: Effect of oxidative pre-treatment. J. Assoc. Off. Anal. Chem. 36, 1117–1121, 1985. 47. Meredith, F.I., McCarthy, M.A., Leffler, R. Amino acid concentrations and comparison of different hydrolysis procedures for American and foreign chestnuts. J. Agric. Food Chem. 36, 1172–1175, 1988. 48. Alegría, A., Barbera, R., Farre, R. et al. HPLC method for cyst(e)ine and methionine in infant formulas. J. Food Sci. 61, 1132–1135, 1170, 1996.
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49. Akinyele, A.F., Okogun, J.I., Faboya, O.P. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for determining cysteine and cystine in cereal and legume seeds. J. Agric. Food Chem. 47, 2303–2307, 1999. 50. Bosch, L., Alegría, A., Farré, R. Application of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant formula. J. Chromatogr. B 831, 176–183, 2006. 51. Bradbury, A.F., Smith, D.G. The use of 3-bromopropionic acid for the determination of protein thiol groups. J. Biochem. 131, 637–642, 1973. 52. Hale, J.E., Beidler, D.E., Jue, R.A. Quantitation of cysteine residues alkylated with 3-bromopropylamine by amino acid analysis. Anal. Biochem. 216, 61–66, 1994. 53. Fullmer, C.S. Identification of cysteine-containing peptides in protein digests by high-performance liquid chromatography. Anal. Biochem. 142, 336–339, 1984. 54. Morel, M.H., Bonicel, J. Determination of the number of cysteine residues in high molecular weight subunits of wheat glutenin. Electrophoresis 17, 493–496, 1996. 55. Barkholt, V., Jensen, A.L. Amino acid analysis: Determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal. Biochem. 177, 318–322, 1989. 56. Tuan, Y.H., Phillips, R.D. Optimized determination of cystine/cysteine and acid-stable amino acids from a single hydrolysate of casein- and sorghum-based diet and digesta samples. J. Agric. Food Chem. 45, 3535–3540, 1997. 57. Knecht, R., Chang, J.Y. Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)azobenzenesulfonyl chloride. Anal. Chem. 58, 2375–2379, 1986. 58. Stocchi, V., Piccoli, G., Magnani, M. et al. Reversed-phase high-performance liquid chromatography separation of dymethylaminoazobenzne sulfonyl- and dimethylaminoazobenzene thiohydantoinamino acid derivatives for amino acid analysis and microsequencing studies at the picomole level. Anal. Biochem. 178, 107–117, 1989. 59. Zumwalt, R.W., Absheer, J.S., Kaiser, F.E. et al. Acid hydrolysis of proteins for chromatographic analysis of amino acids. J. Assoc. Off. Anal. Chem. 70, 147–151, 1987. 60. Albin, D.M., Wubben, J.E., Gabert, V.M. Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate. J. Agric. Food Chem. 48, 1684–1691, 2000. 61. Ambler, R.P. Standards and accuracy in amino acid analysis. In: Amino Acid Analysis, Rattenbury, J.M. (Ed.), Chichester, U.K., Ellis Horwood, 1981, pp. 119–137. 62. Williams, A.P. Determination of amino acids and peptides. In HPLC in Food Analysis, McCrae, (Ed.), New York, Academic Press, 1982, pp. 285–311. 63. Hugli, T.E., Moore, S. Determination of the tryptophan content of proteins by ion-exchange chromatography of alkaline hydrolysates. J. Biol. Chem. 247, 2828–2834, 1972. 64. Zarkadas, C.G., Yu, Z.R., Zarkadas, G.C. et al. Assessment of the protein quality of beefstock bone isolates for use as an ingredient in meat and poultry products. J. Agric. Food Chem. 43, 77–83, 1995. 65. Viadel, B., Alegria, A., Farre, R. et al. Amino acid profile of milk-based infant formulas. Int. J. Food Sci. Nutr. 51, 367–372, 2000. 66. Jandasek, J., Kracmar, S., Milerski, M. et al. Comparison of the contents of intramuscular amino acids in different lamb hybrids. Cz. J. Animal Sci. 48, 301–306, 2003. 67. Ihekoronye A.I. Quantitative gas-liquid chromatography of amino acids in enzymic hydrolysates of food proteins. J. Sci. Food Agric., 36, 1004–1012, 1985. 68. García, S.E., Baxter, J.H. Determination of tryptophan content in infant formulas and medical nutritionals. J. Assoc. Off. Anal. Chem. Int. 75, 1112–1119, 1992. 69. Liaset, B., Espe, M. Nutritional composition of soluble and insoluble fractions obtained by enzymatic hydrolysis of fish-raw materials. Proc. Biochem. 43, 42, 2008. 70. Ashworth, R.B. Amino acids analysis for meat protein evaluation. J. Assoc. Off. Anal. Chem. 70, 80–85, 1987b.
304 ◾ Handbook of Seafood and Seafood Products Analysis 71. Sun, S.W., Lin, Y.C., Weng, Y.M. et al. Efficiency improvements on ninhydrin method for amino acid quantification. J. Food Comp. Anal. 19, 112–117, 2006. 72. Limin, L., Feng, X., Jing, H. Amino acids composition difference and nutritive evaluation of the muscle of five species of marine fish, Pseudosciaena crocea (large yellow croaker), Lateolabrax japonicus (common sea perch), Pagrosomus major (red seabream), Seriola dumerili (Dumeril’s amberjack) and Hapalogenys nitens (black grunt) from Xiamen Bay of China. Aq. Nutr. 12, 53–59, 2006. 73. Standara, S., Drdak, M., Vesela, M. Amino acid analysis: Reduction of ninhydrin by sodium borohydride. Nahrung 43, 410–413, 1999. 74. Rosa, R., Calado, R., Andrade, A.M. et al. Changes in amino acids and lipids during embryogenesis of European lobster, Homarus gammarus (Crustacea: Decapoda). Comp. Biochem. Physiol. B-Biochem. & Mol. 140, 241–249, 2005. 75. Buetikofer, U., Ardo, Y. Quantitative determination of free amino acids in cheese. Bull. Int. Dairy Fed., 337, Chemical methods for evaluating proteolysis in cheese maturation (part 2), 24–32, 1999. 76. Grunau, J.A., Swiader, J.M. Chromatography of 99 amino acids and other ninhydrin-reactive compounds in the Pickering lithium gradient system. J. Chromatogr. 594, 165–171, 1992. 77. Ardo, Y., Gripon, J.C. Comparative study of peptidolysis in some semi-hard round-eyed cheese varieties with different fat contents. J. Dairy Res. 62, 543–547, 1995. 78. Heinrikson, R.L., Meredith, S.C. Amino acid analysis by reverse-phase high-performance liquid chromatography, precolumn derivatization with phenylisothiocyanate. Anal. Biochem. 136, 65–74, 1984. 79. Bidlingmeyer, B.A., Cohen, S.A., Tarvin, T.L. Rapid analysis of amino acids using pre-column derivatization. J. Chromatogr. 336, 93–104, 1984. 80. Bidlingmeyer, B.A., Cohen S.A., Tarvin T.L. et al. New rapid high sensitivity analysis of amino acids in food type samples. J. Assoc. Off. Anal. Chem. 70, 241–247, 1987. 81. Sarwar, G., Botting, H.G., Peace, R.W. Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives. J. Assoc. Off. Anal. Chem. 71, 1172–1175, 1988. 82. Fürst, P., Pollack, L., Graser, T.A. et al. HPLC analysis of free amino acids in biological material—An appraisal of four pre-column derivatization methods. J. Liq. Chromatogr. 12, 2733–2760, 1989. 83. Liu, J.K., Chang, J.Y. Chromophore labelling of amino acids with 4-dimethyl-aminoazobenzene-4′sulfonyl chloride. Anal. Chem. 47, 1634–1638, 1975. 84. Chang, J.Y., Knecht, R., Braun, D.G. Amino acid analysis in the picomole range by precolumn derivatization and high-performance liquid chromatography. Meth. Enzymol. 91, 41–48, 1983. 85. Dejong, C., Hughes, G.J., Vanwieringen, E. et al. Amino acid analysis by high-performance liquid chromatography. An evaluation of the usefulness of pre-column Dns derivatization. J. Chromatogr. 241, 345–350, 1982. 86. Martín, P., Polo, C., Cabezudo, M.D. et al. Dansyl amino-acids behavior on a Radial Pak C-18 column. Derivatization of grape wine musts, wines and wine vinegar. J. Liq. Chromatogr. 7, 539–558, 1984. 87. Tapuhi, Y., Schmidt, D.E., Lindner, W. et al. Dansylation of amino acids for high-performance liquid chromatography analysis. Anal. Biochem. 115, 123–129, 1981. 88. Prieto, J.A., Collar, C., Benedito de Barber, C. Reversed-phase high-performance liquid chromatographic determination of biochemical changes in free amino acids during wheat flour mixing and bread baking. J. Chrom. Sci. 28, 572–577, 1990. 89. Stehle, P., Albers, S., Pollack, L. et al. In vivo utilization of cystine-containing synthetic short chain peptides after intravenous bolus injection in the rat. J. Nutr., 118, 1470–1474, 1988. 90. Burton, C. Fully automated amino acid analysis using precolumn derivatization. Int. Lab. 30, 32–34,36,38, 1986. 91. Bëtner, I., Foldi, P. The FMOC-ADAM approach to amino acid analysis. LC-GC Int. 6, 832–840, 1988. 92. Simons, S.S., Johnson, D.F. Reaction of o-phtalaldehyde and thiols with primary amines; formation of 1-alkyl(and aryl)thio-2-alkylisoindoles. J. Org. Chem. 43, 2886–2891, 1978.
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93. Alvarez-Coque, M.C.G., Hernandez, M.J.M., Camanas R.M.V. et al. Formation and instability of ortho-phtalaldehyde derivatives of amino acids. Anal. Biochem. 178, 1–7, 1989. 94. Godel, H., Seitz, P., Verhoef, M. Automated amino acid analysis using combined OPA and FMOC-Cl precolumn derivatization. LC-GC Int. 5, 44–49, 1992. 95. Lookhart, G.L., Jones, B.L. High-performance liquid chromatography analysis of amino acids at the picomole level. Cereal Chem. 62, 97–102, 1985. 96. Euerby, M.R. Effect of differing thiols on the reversed-phase high-performance liquid chromatographic behaviour of o-phthaldialdehyde-thiol-amino acids. J. Chromatogr. 454, 398–405, 1988. 97. Winspear, M.J., Oaks, A. Automated pre-column amino acid analyses by reversed-phase highperformance liquid chromatography. J. Chromatogr. 270, 378–382, 1983. 98. Willis, D.E. Automated pre-column derivatization of amino acids with o-phthalaldehyde by a reagent sandwich technique. J. Chromatogr. 408, 217–225, 1987. 99. Jarret, H.W., Cooksy K.D., Ellis, B. et al. The separation of ortho-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns. Anal. Biochem. 153, 189–198, 1986. 100. Gurd, F.R.N. Carboxymethylation. Meth. Enzymol. 25, 424–438, 1972. 101. Pripis-Nicolau, L., de Revel, G., Marchand, S. et al. Automated HPLC method for the measurement of free amino acids including cysteine in musts and wines; first applications. J. Sci. Food Agric. 81, 731–738, 2001. 102. Park, S.K., Boulton, R.B., Noble, A.C. Automated HPLC analysis of glutathion and thiol-containing compounds in grape juice and wine using pre-column derivatization with fluorescence detection. Food Chem. 68, 475–480, 2000. 103. Liu, H., Sanuda-Pena, M.C., Harvey-White, J.D. et al. Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis. J. Chromatogr A 828, 383–395, 1998. 104. Wang, J., Chatrathi, M.P., Tian, B. Micromachined separation chips with a precolumn reactor and end-column electrochemical detector. Anal. Chem. 72, 5774–5778, 2000. 105. Tcherkas, Y.V., Kartsova, L.A., Krasnova, I.M. Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection. J. Chromatogr. A 913, 303–308, 2001. 106. Sherwood, R.A., Titheradge, A.C., Richards, D.A. Measurement of plasma and urine amino acids by high-performance liquid chromatography with electrochemical detection using phenylisothiocyanate derivatization. J. Chromatogr. 528, 293–303, 1990. 107. Dou, L., Krull, I.S. Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection. Anal. Chem. 62, 2599–2606, 1990. 108. Mills, B.J., Stinson, C.T., Liu, M.C. et al. Glutathione and cyst(e)ine profiles of vegetables using high performance liquid chromatography with dual electrochemical detection. J. Food Comp. Anal. 10, 90–101, 1997. 109. Schrijver, R., de, Eenaeme, C., van, Fremaut, D. et al. Hydrolysate preparation and comparative amino acid determination by cation-exchange and gas-liquid chromatography in diet ingredients. Cerevisia Biotechnol. 16, 26–37, 1991. 110. Ogunsua, A.O. Amino acid determination in conophor nut by gas-liquid chromatography. Food Chem. 28, 287–298, 1988. 111. Oh, C.H., Kim, J.H., Kim, K.R. et al. Simultaneous gas chromatographic analysis of non-protein and protein amino acids as N(O,S)-isobutyloxycarbonyl tert-butyldimethylsilyl derivatives. J. Chromatogr. A 669, 125–137, 1994. 112. Paetzold, R., Brüeckner, H. Gas chromatographic detection of d-amino acids in natural and thermally treated bee honeys and studies on the mechanism of their formation as result of the Maillard reaction. Eur. Food Res. Technol. 223, 347–354, 2006.
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113. Brüeckner, H., Schieber, A. Determination of free D-amino acids in Mammalia by gas chromatography-mass spectrometry. HRC. 23, 576–582, 2000. 114. Bertacco, G., Boschelle, O., Lercker, G. Gas chromatographic determination of free amino acids in cheese. Milchwissenschaft. 47, 348–350, 1992. 115. Buser, W., Erbersdobler, H.F. Determination of amino acids by gas-liquid chromatography and nitrogen selective detection. Z. Lebensm. Unters. For. 186, 509–513, 1988. 116. Kataoka, H., Sakiyama, N., Makita, M. Distribution and contents of free o-phosphoamino acids in animal-tissues. J. Biochem. 109, 577–580, 1991. 117. Erbe, T., Brüeckner, H. Chiral amino acid analysis of vinegars using gas chromatography-selected ion monitoring mass spectrometry. Z. Lebensm. Unters. For. 207, 400–409, 1998. 118. Katona, Zs. F., Sass, P., Molnar-Perl, I. Simultaneous determination of sugars, sugar alcohols, acids and amino acids in apricots by gas chromatography-mass spectrometry. J Chromatogr. A 847, 91–102, 1999. 119. Starke, I., Kleinpeter, E., Kamm, B. Separation, identification, and quantification of amino acids in L-lysine fermentation potato juices by gas chromatography-mass spectrometry. Fresen. J. Anal. Chem. 371, 380–384, 2001. 120. Jorgenson, J.W., Lukacs, K.D. Free-zone electrophoresis in glass capillaries. Clin. Chim. 27, 1551–1553, 1981. 121. Jorgenson, J.W., Lukacs, K.D. Capillary zone electrophoresis. Science 222(4621), 266–272, 1983. 122. Terabe, S., Otsuka, K., Ichikawa, K. et al. Electrokinetic separations with micellar solutions and open-tubular capillaries. Anal. Chem. 56, 111–113, 1984. 123. Terabe, S., Otsuka, K., Ando, T. Electrokinetic chromatography with micellar solution and open-tubular capillary. Anal. Chem. 57, 834–841, 1985. 124. Burton, D.E., Sepaniak, M.J., Maskarinec, M.P. Analysis of B6 vitamers by micellar electrokinetic capillary chromatography with laser-excited fluorescence detection. J. Chromatogr. Sci. 24, 347–351, 1986. 125. Matsubara, N., Terabe, S. Separation of 24 dansylamino acids by capillary electrophoresis with a non-ionic surfactant. J. Chromatogr. A 680, 311–315, 1994. 126. Corradini, C., Cavazza, A. Application of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) in food analysis. Int. J. Food Sci. 10, 299–316, 1998. 127. Wu, J., Odake, T., Kitamori, T. et al. Ultrasensitive detection for capillary zone electrophoresis using laser-induced capillary vibration. Anal. Chem. 63, 2216–2218, 1991. 128. Ye, J., Baldwin, R.P. Determination of amino acids and peptides by capillary electrophoresis and electrochemical detection at a copper electrode. Anal. Chem. 66, 2669–2674, 1994. 129. Klampfl, C.W. Analysis of organic acids and inorganic anions in different types of beer using capillary electrophoresis. J. Agric. Food Chem. 47, 987–990, 1999. 130. Klampfl, C.W., Buchberger, W., Turner, M. et al. Determination of underivatized amino acids in beverage samples by capillary electrophoresis. J. Chromatogr. A 804, 347–355, 1998. 131. Soga, T., Heiger, D.N. Amino acid analysis by capillary electrophoresis electrospray ionisation mass spectrometry. Anal. Chem. 72, 1236–1241, 2000. 132. Weng, Q., Jin, W. Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene-2,3-dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection. Electrophoresis 22, 2797–2803, 2001. 133. Skocˆ ir, E., Prosek, M. Determination of amino acid ratios in natural products by micellar electrokinetic chromatography. Chromatographia 41, 638–644, 1995. 134. Skocˆ ir, E., Vindevogel, J., Sandra, P. Separation of 23 dansylated amino acids by micellar electrokinetic chromatography at low temperature. Chromatographia 39, 7–10, 1994. 135. Cavazza, A., Corradini, C., Lauria, A. et al. Rapid analysis of essential and branched-chain amino acids in nutraceutical products by micellar electrokinetic capillary chromatography. J. Agric. Food Chem. 48, 3324–3329, 2000.
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136. Arellano, M., Simeon, N., Puig, P. et al. Several applications of capillary electrophoresis for wines analysis. Quantitation of organic and inorganic acids, inorganic cations, amino acids and biogenic amines. J. Int. Sci. Vigne Vin. 31, 213–218, 1997. 137. Castagnola, D.V., Rossetti, D.V., Cassiano, L. et al. Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography. J. Chromatogr. 638, 327–333, 1993. 138. Otsuka, K., Terabe, S., Ando, T. Electrokinetic chromatography with micellar solutions separation of phenylthiohydantoin-amino acids. J. Chromatogr. 332, 219–226, 1985. 139. Liu, J., Cobb, K.A., Novotny, M. et al. Separation of pre-column ortho-phthalaldehyde-derivatized amino acids by capillary zone electrophoresis with normal and micellar solutions in the presence of organic modifiers. J. Chromatogr. 468, 55–65, 1989. 140. Matsubara, N., Terabe, S. Micellar electrokinetic chromatography. Meth Enzymol. 270, 319–341, Part A, 1996. 141. Otsuka, K., Terabe, S. Effect of methanol and urea on optical resolution of phenylthiohidantoinDL-amino acids by micellar electrokinetic chromatography with sodium N-dodecanoyl-l-valinate. Electrophoresis 11, 982–984, 1990. 142. Chen, N., Terabe, S. A quantitative study on the effect of organic modifiers in micellar electrokinetic chromatography. Electrophoresis 16, 2100–2103, 1995. 143. Chen, N., Terabe, S., Nakagawa, T. Effect of organic modifier concentrations in micellar electrokinetic chromatography. Electrophoresis 16, 1457–1462, 1995. 144. Jin, D.R., Miyahara, T., Oe, T. et al. Determination of d-amino acids labelled with fluorescent chiral reagents, R(−)and S(+)-a-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3benzoxadiazoles, in biological and food samples by liquid chromatography. Anal. Biochem. 269, 124– 132, 1999. 145. Novatchev, N., Ulrike, H. Evaluation of amino sugar, low molecular peptide and amino acid impurities of biotechnologically produced amino acids by means of CE. J. Pharm. Biomed. Anal. 28, 475–486, 2002. 146. Novotny, M.V., Cobb, K.A., Liu, J. Recent advances in capillary electrophoresis of proteins, peptides and amino acids. Electrophoresis 11, 735–749, 1990. 147. Issaq, H.J., Chan, K.C. Separation and detection of amino acids and their enantiomers by capillary electrophoresis: A review. Electrophoresis 16, 467–480, 1995. 148. Karam, L.R., Simic, M.G. Formation of o-tyrosine by radiation and organic solvents in chicken tissue. J. Biol. Chem. 265, 11581–11585, 1990. 149. Miyahara, M., Nagasawa, T., Izumi, K. et al. New LASER fluorometric detection for ortho-tyrosine in gamma-irradiated phenylalanine solution and pork. Food Irrad. Jap. 34, 3–8, 1999. 150. Kwon, J.Y., Moini, M. Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionisation-mass spectrometry. J. Am. Soc. Mass. Spectrom. 12, 117–122, 2001. 151. Aristoy, M.C., Toldra´, F. Essential amino acids. In: Handbook of Muscle Foods Analysis. Nollet, L.M.L., Toldra´, F. (Eds.), CRC Press, Boca Raton, FL, 2009, 385–397. 152. Aristoy, M.C., Toldra´, F. Essential amino acids. In: Handbook of Dairy Foods Analysis. Nollet, L.M.L., Toldra´, F. (Eds.), CRC Press, Boca Raton, FL, 2009, 9–32.
Chapter 18
Antioxidants Nick Kalogeropoulos and Antonia Chiou Contents 18.1 Introduction ..................................................................................................................310 18.1.1 Oxidation and Its Implications.........................................................................310 18.1.1.1 Oxidative Stress and Its Implications ...............................................310 18.1.1.2 Lipid Peroxidation............................................................................311 18.1.1.3 Marine Lipid Oxidation ...................................................................311 18.1.2 Antioxidants.....................................................................................................311 18.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems ..........................................................................................................313 18.3 Antioxidants in Seafood and Seafood Products .............................................................313 18.3.1 Antioxidant Enzymes .......................................................................................313 18.3.2 Ascorbic Acid ...................................................................................................314 18.3.2.1 Ascorbic Acid Functions ..................................................................314 18.3.2.2 Ascorbic Acid Analysis .....................................................................314 18.3.2.3 Occurrence of Ascorbic Acid in Marine Organisms ......................... 315 18.3.3 Vitamin E ........................................................................................................ 315 18.3.3.1 Vitamin E as an Antioxidant............................................................316 18.3.3.2 Vitamin E Determination ................................................................316 18.3.3.3 Occurrence of Vitamin E .................................................................317 18.3.4 Carotenoids ......................................................................................................317 18.3.4.1 Antioxidant and Other Functions of Carotenoids ............................317 18.3.4.2 Carotenoid Determination ...............................................................318 18.3.4.3 Occurrence of Carotenoids ..............................................................318
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18.3.5 Ubiquinone ......................................................................................................319 18.3.5.1 Function of Ubiquinone ..................................................................319 18.3.5.2 Determination of Ubiquinone .........................................................319 18.3.5.3 Occurrence of Ubiquinone...............................................................319 18.3.6 Other Endogenous Antioxidants ..................................................................... 320 18.4 Added Antioxidants ..................................................................................................... 320 18.4.1 Synthetic Antioxidants .................................................................................... 320 18.4.2 Natural Antioxidants .......................................................................................321 References ................................................................................................................................321
18.1
Introduction
Antioxidants evolved together with the emergence of photosynthesis by cyanobacteria, more than 2 billion years ago, as a defense against oxygen toxicity.1 Indeed cyanobacteria and the laterevolved green plants, being exposed to the oxygen they produce, are rich in antioxidants such as vitamins C and E, polyphenols, and carotenoids. Humans and most animals cannot synthesize the majority of these antioxidants and depend on the dietary intake from plant consumption. This is also followed in the marine environment, where antioxidants are mainly produced by photosynthetic organisms and are consequently transported through the trophic web.
18.1.1 Oxidation and Its Implications Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life, since oxygen is the final electron acceptor in the electron flow system that produces energy. When the electron flow becomes uncoupled (transfer of unpaired single electrons), generation of free radicals occurs, that is, electrically charged compounds that seek out and capture electrons from other compounds in order to neutralize themselves. Although the initial attack causes the neutralization of the free radical, another free radical is generated in the process, resulting in a chain reaction. Until subsequent free radicals are deactivated, thousands of free radical reactions may occur within a few seconds. Common free radicals in biological systems are the socalled reactive oxygen species (ROS), which include among others superoxide anion radical (O2•−), hydrogen peroxide (H2O2), nitric oxide (NO•), peroxyl (ROO•), alkoxyl (RO•), and hydroxyl (OH•) radicals, and the so-called reactive nitrogen species (RNS), such as peroxynitrite (ONOO−). ROS production in organisms is related to both the basal metabolism and the influence of environmental factors2; if ROS are not immediately intercepted by antioxidants, they may oxidize several cell components.
18.1.1.1
Oxidative Stress and Its Implications
Oxidative stress occurs when the prooxidant–antioxidant balance becomes too favorable to the prooxidants. Under conditions of oxidative stress, lipids, nucleic acids, and proteins may be damaged by reactive oxidants. Aging,3 pollution,4 and environmental stress5,6 have been reported to cause oxidative stress to fish or bivalves, resulting in an increased antioxidant activity and antioxidants
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loss or oxidation product development. There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases and conditions.3,7
18.1.1.2
Lipid Peroxidation
A very damaging effect of oxidant reactive intermediates is lipid peroxidation, in which polyunsaturated fatty acids on lipid molecules are attacked and oxidized. This can be especially damaging to lipid-rich cell membranes. The health implications of tissue lipid oxidation are numerous and well documented.8 Studies on the pathological significance of dietary lipid oxidation products have indicated that some lipid oxidation products have cytotoxic, mutagenic, carcinogenic, atherogenic, and angiotoxic effects. Lipid oxidation is a complex procedure induced by oxygen in the presence of initiators such as light, heat, free radicals, and metal ions. Three reaction pathways have been proposed: (1) nonenzymatic chain autoxidation, (2) nonenzymatic and nonradical photooxidation, and (3) enzymatic oxidation. In the first two cases a combination of reactions involving 3O2 and 1O2 occurs. Nonradical photooxidation seems to be a minor reaction compared with the 3O2-induced radical chain autoxidation. Autoxidation occurs through a three-phase process, that is, initiation, propagation, and termination.9
18.1.1.3
Marine Lipid Oxidation
Compared with other food lipids, marine lipids are relatively more susceptible to oxidation, because of their high degree of unsaturation.10 Lipids deteriorate in seafood products during processing, handling, and storage, being the major cause of the development of off-flavor compounds and rancidity as well as a number of other reactions that reduce the shelf life and nutritive value of food products. Lipid oxidation of omega-3 polyunsaturated fatty acids (PUFA)-rich food products results in the development of particularly unpleasant off flavors, for which the human sensory apparatus has a low threshold,11 and unhealthy compounds that reduce their shelf life and nutritive value.
18.1.2
Antioxidants
Antioxidants are defined as any substance that when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate.12 In biological systems various biochemical defense mechanisms, including enzymatic systems and nonenzymatic antioxidants, protect the cellular components from oxidative damage. Moreover, antioxidants supplied by foods, mainly of plant origin, are essential for counteracting oxidative stress. For food systems, antioxidants are molecules that protect macromolecules from being oxidized.13 Antioxidants counteract oxidation in two different ways: They protect lipids from oxidation initiators, preventive antioxidants, and they stall the propagation phase, chain-breaking antioxidants.9 Preventive antioxidants hinder ROS formation or scavenge species responsible for oxidation initiation (O2•−, 1O2, etc.). Chain-breaking antioxidants intercept radical oxidation propagators (LOO•) or participate in halting radical chain propagation. Nevertheless, antioxidants often act via more than one mechanism that combines different types of antioxidant activity. The major components of the antioxidant defense system together with their proposed mechanisms of action are presented in Table 18.1. In general, antioxidants are
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Table 18.1 Major Components of Antioxidant Defense System and Proposed Mechanism of Action Antioxidant Species
Mechanism of Action
Enzymes Catalase
ROS detoxification (reduction of H2O2 to water)
Glutathione peroxidase
ROS detoxification (reduction of H2O2 to water)
Superoxide dismutase (SOD)
ROS detoxification (removal of superoxide radical)
Thioredoxin
ROS detoxification (reduction of peroxides)
Metal Ion Sequestration Transferrin
Transient metal chelators (chelates Fe)
Albumin
Transient metal chelators (chelates Fe, Cu)
Ceruloplasmin
Transient metal chelators (chelates Cu)
Ferritin
Transient metal chelators (chelates Fe)
Lactalbumin
Transient metal chelators (chelates Fe)
Phytochelatins
Transient metal chelators (chelates Cd, Zn, Cu)
Low Molecular Mass Ascorbic acid (exogenous)
Chain-breaking antioxidant, regenerates oxidized vitamin E
Carotenoids (exogenous)
1
Coenzyme Q (endogenous)
Synergistic to vitamin E
Urate (endogenous)
Scavenges NO2
Phospholipids (endogenous)
Transient metal chelators, ROS detoxification (hydroperoxides), synergistic to vitamin E
Polyphosphates, EDTA, citric acid
Transient metal chelators
Polyphenols (exogenous)
Transient metal chelators (the ones with o-diphenolic structure), chain-breaking antioxidants, regenerate oxidized vitamin E
Vitamin E (exogenous)
Chain-breaking antioxidant, scavenges peroxyradicals, 1O2 quencher
Bilirubin, 2-oxo acids, sex hormones melatonin, lipoic acid, carnosine, anserine, melanins (endogenous)
Compounds with proven antioxidant activity in vitro, but uncertain in vivo
O2 quenchers, chain-breaking antioxidants
Source: Adapted from Willcox, J.K. et al., Crit. Rev. Food Sci., 44, 275, 2004.
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effective at very low concentration levels. At higher levels most of them behave as prooxidants possibly due to their involvement in the initiation reactions.
18.2 Determination of Antioxidants and Antioxidant Capacity in Biological and Food Systems Analytical techniques developed for some of the common endogenous or exogenous antioxidants range from qualitative detection by color-developing reactions to semiquantitative and quantitative determinations by means of spectroscopic, voltammetric, polarographic, and chromatographic methods, the latter including paper, thin-layer, column chromatography and the more sophisticated gas chromatography (GC), and high-performance liquid chromatography (HPLC) alone or combined with mass spectroscopy. The available methods have been reviewed by Rajalakshmi and Narasimhan,15 Griffiths et al.,16 and Laguerre et al.9 The determination of specific waterand fat-soluble antioxidants is discussed in Sections 18.3.2 and 18.3.3. The available methods for monitoring the antioxidant capacity in biological and food systems in vitro or in vivo were recently reviewed by MacDonald-Wicks et al.17 and Wood et al.18 In reviewing methods and tests for the assessment of lipid oxidation, Kolanowski et al.19 concluded that, for quality control of fish oil and fish oil-containing foods, the correlation of instrumental and sensory methods with multivariate data analysis should be followed.
18.3
Antioxidants in Seafood and Seafood Products
In living organisms, oxidative damage to macromolecules is controlled by two types of antioxidant systems, one represented by enzymes and the second represented by low molecular mass compounds, such as ascorbic acid, tocopherols, carotenoids, coenzyme Q, glutathione (GSH), bilirubin, thiols, and uric acid, together with traces of phenolic compounds. These antioxidants act in a concerted way to protect sensitive molecules such as the unsaturated fatty acids from oxidation.
18.3.1
Antioxidant Enzymes
Catalases are metal-containing enzymes, widely distributed in aerobic cells that help in preventing the accumulation of H2O2 within cells. Catalase activities ranged between 386 and 1523 mmol/ min/g tissue in several Atlantic fish and was higher in liver, kidneys, spleen, and heart, and in red muscle compared with that in white.20 Superoxide dismutases (SOD): SODs are metalloproteins, with Mn, Cu, Zn, or Fe in their active site, which act as primary preventive inhibitors and catalyze the dismutation of superoxide anion (O2•−) by reducing one O2•− to H2O2 and oxidizing another one to O2.1 Cu/Zn SOD were purified from marine fish tissues, whereas Fe SOD were purified from red algae, blue-green algae, and algae.21 In several species of teleosts, cephalopods, and crustaceans from the Mediterranean sea, Cu/Zn-SOD activities ranged between 1.9 and 9.7 U/mg of protein,22 whereas in nine Atlantic fish species total SOD values ranged between 157 and 796 U/g fish and Mn-SOD ranged between 45 and 751 U/g.20
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Glutathione peroxidase (GPx): GPx is a selenium-containing enzyme found in animals, plants, and some bacteria. It catalyses the reduction of hydrogen (H2O2) or lipid peroxides (LOOH) while oxidizing 2 mol of reduced GSH. In several Atlantic fish species, GPx ranged between less than 0.03 and 0.23 mmol/min per g fish, with higher activities observed in kidneys, spleen, and heart,20 whereas in Mediterranean fish, cephalopods, and crustaceans, GPx activities ranged between 0.16 and 0.40 U/mg of protein.22 Glutathione reductase (GR): The ratios of GSH/GSSG in cells are normally kept high by the reduction of GSSG back to GSH, which is catalyzed by GR. GR activities were around 84 mU/mg protein in the digestive gland and the gill of mussels Perna perna.5 Glutathione S-transferases (GST): GSTs are involved in the metabolism of xenobiotics and/or oxidized components in the liver.23 GST activities of 129.6 and 630.7 U/mg protein in the digestive gland and gill of the mussel P. perna were reported.5 Peroxiredoxins (Prx): Prxs are ubiquitous enzymes that can be simultaneously both antioxidants and modulators of signal transduction and may be the most important H2O2-removal systems in animals, bacteria, and possibly plants.24
18.3.2 Ascorbic Acid 18.3.2.1
Ascorbic Acid Functions
Ascorbic acid (AA), the most active water-soluble antioxidant, is ubiquitous in eukaryotic cells of both plants and animals. Crustaceans and most fish lack the ability to synthesize AA 25; therefore, they are dependent on dietary supply of vitamins. Most of the AA physiological functions are related to its ability to act as an electron donor,21 and it exerts its properties by neutralizing toxic peroxides and by stabilizing free radicals, thus protecting lipids, proteins, and other biocomponents from oxidative damage.26 Ascorbic acid is noted for its complex multifunctional effects; depending on conditions, AA can act as antioxidant, prooxidant, metal chelator, and reducing agent or as oxygen scavenger.27 In aquatic organisms AA is involved in several physiological functions including growth, development, reproduction, wound healing, collagen synthesis, response to stressors, tyrosine metabolism, metal ion metabolism, protection of cells from oxidative damage, and the regeneration of vitamin E in its metabolically active form. In fish, vitamin C is known to play important roles in improving immune response and resistance to infectious diseases,28 resisting stress,29 and oxidation, while additionally it has a positive effect on the wound healing process.30 Besides AA, its reversibly oxidized form, dehydroascorbic acid (DHAA), also exists in various dietary and biological samples. Additionally l-ascorbate 2-sulfate (AAS) has been isolated from undeveloped cysts of brine shrimp and in tissues of trout.31
18.3.2.2
Ascorbic Acid Analysis
Owing to the importance of AA, many analytical techniques have been proposed for its determination. However, due to AA and DHAA reactivity and instability, many assays suffer from inattention to stability, sensitivity, specificity, and interferences if derivatization products (colorimetric) or retention times (chromatographic) are not verified for the compounds under investigation.32 The main assay types for AA determination are spectroscopic, reviewed by Zaporozhets and Krushinskaya,33 chromatographic—mainly GC and HPLC—recently reviewed by Oliveira and
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Watson,34 and enzymatic. Flow injection techniques35 as well as capillary electrophoresis36 are also employed for AA determination. Methods for DHAA determination, which provides information about the oxidative stress within an organism, were reviewed by Deutsch.37 HPLC assays are generally preferred for AA and DHAA determination in biological samples, as they have certain advantages against spectroscopic or enzymatic methods. Sample preparation–extraction: Sample processing involves removal of protein usually with metaphosphoric acid, which seems to provide the most efficient extraction in food samples while simultaneously preventing oxidation of AA.38 AA is stabilized against oxidation with reagents such as dithioerythritol or monosodium glutamate39 and EDTA. Conditions that promote degradation should be minimized, for example, by purging of samples with an inert gas, storing, if necessary, at no more than −70°C and using amber glass. Determination: Spectroscopic methods have been applied for the determination of AA and DHAA in fish like Atlantic halibut40 and striped bass.41 HPLC methods with electrochemical,22,42,43 ultraviolet (UV),44 and fluorescence45 detection have been reported for the determination of AA and/or DHAA in marine organisms. Electrochemical detection allows the determination of the reduced form only, whereas lack of sensitivity and specificity are the main deficiencies for UV detection. Fluorimetric detection is considered more sensitive and selective, allowing the determination of both AA and DHAA after postcolumn derivatization.38 Total vitamin C (AA plus DHAA) can be measured after oxidation of AA to DHAA, followed by derivatization to give a fluorescent compound. AA is usually oxidized by chemical methods based on active charcoal46 or iodine.47 o-Phenylenediamine (OPDA) and several derivatives47 have been the most used fluorescent reagents for AA determination. If measurement of DHAA is required, the most frequently used approach is to carry out reduction, involving agents like dithioerythritol, 2,3-mercaptopropan-1-ol and mercaptoethanol, or tris-(2carboxyethyl) phosphine hydrochloride.48 HPLC assays have been applied in the analysis of AA and DHAA in fish and crustaceans,22,31,42,45,49 shrimps,50 microalgae, and live food organisms.43
18.3.2.3
Occurrence of Ascorbic Acid in Marine Organisms
Muscles of wild and cultivated marine and freshwater fish generally contain low amounts of vitamin C, usually not exceeding 1 mg/100 g.22,45,51 Vitamin C is known to be concentrated in vital organs with active metabolism such as brain, liver, kidneys, and the gonads,49,52 confirming the hypothesis of the importance of ascorbic acid in preserving vital tissues from oxidation processes. The AA content of Mediterranean cephalopods and crustaceans was low, not exceeding 0.5 mg/100 g.22 In cysts of various batches and strains of Artemia, the ascorbic acid-2-sulfate (AAS) content ranged from 296 to 517 mg AAS/g dw.43 The ovaries of Litopenaeus Yannamei shrimps contained 35.1–46.1 mg AA/100 g,50 whereas fresh and canned sea urchin gonads contained 26.6 and 14.2 mg AA/100 g, respectively.46 Several species of microalgae commonly used in mariculture contained 1.0–4 mg AA/g d.w.43
18.3.3 Vitamin E The term vitamin E refers to a group of eight chemically related compounds possessing a 6-chromanol ring structure attached to a carbon side chain. Based on the structure of the side chain, these compounds are further classified as tocopherols (tocols) and tocotrienols, further designated as a-, b-, g-, and d-vitamers.
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Vitamin E as an Antioxidant
Vitamin E is a natural, most effective lipid-soluble antioxidant and the principal membrane antioxidant in mammalian cells. Vitamin E antioxidant function, as a peroxyl radical scavenger that terminates chain reactions, is well known and well described.53,54 a-Tocopherol has been proposed to be a highly efficient antioxidant since (1) it reacts with peroxyl radicals faster than allowing the peroxyl radical to be involved in any other reactions, (2) it takes away the radical character from the oxidizing fatty acid and prevents it from further radical reactions, and (3) in the antioxidant reaction, a-tocopherol is turned into a fairly stable radical.54 Abstraction of the 6-OH hydrogen yields a tocopheroxyl radical. Tocopherol can be restored by reduction of the tocopheroxyl radical with redox-active reagents such as vitamin C, polyphenols, enzymes, or ubiquinol.54 In homogeneous solution phase autoxidation, the tocopheroxyl radical will react with a second peroxyl radical to give nonradical products. This second reaction leads to the destruction of tocopherol as an antioxidant. The relative antioxidant activity of different tocopherol homologs has been studied with much attention. According to Frankel,27 there are inconsistencies in these results, which can be attributed to the wide differences in substrates tested, the level of oxidation used in the tests, and the method used to analyze oxidation.
18.3.3.2 Vitamin E Determination Methods used for sample preparation and chromatographic analysis of tocopherols from various matrices have been reviewed.34,55,56 Vitamin E is not chemically bound to macromolecules, and using harsh reagents and conditions to liberate it (e.g., strong saponification) does not seem necessary, whereas on the other hand it can destroy the vitamins. However, efficient conditions are to be used for its release from lipophilic milieu, since vitamin E could be associated with other matrix components. Sample treatment for vitamin E analysis often includes saponification before extraction, frequently performed by heating with KOH in ethanol or methanol. Following saponification, the unsaponifiable components are extracted into an organic solvent. Several factors may interfere with the extraction of vitamin E from the saponification medium,55 among which are organic solvent, ethanol concentration, and the levels of lipids used in the digest. To overcome the oxidation of fat-soluble vitamins caused by saponification, the addition of antioxidants and internal standards is recommended. Vitamin E is commonly extracted using the Folch extraction with chloroform–methanol (2:1), acetone, diethyl ether, and Soxhlet extraction with a variety of solvents. Hexane, alone or with small amounts of more polar solvents such as ethanol and ethyl acetate, is the most frequently used extracting solvent. Alternative to liquid–liquid extraction the use of solid-phase extraction before HPLC analysis has been reported in the analysis of aquatic organisms.57 The use of supercritical fluid extraction for the determination of fat-soluble vitamins has also been reviewed.58 GC-flame ionization detection has been applied for the determination of vitamin since the early 1970s, but nowadays it is used at a lesser extent. HPLC separation of tocopherols provides a fast, simple, sensitive, selective, and more robust technique than GC. Separation of tocopherols is performed on both normal- and reversed-phase HPLC. Normal-phase HPLC via silica columns provides separation of all isomers, whereas b- and g-tocopherols are not easily separated in conventional reversed-phase columns; additionally, normal-phase HPLC operating with organic solvents allows high solubility of lipids. Reversed-phase C18 systems do not completely resolve b- and g-tocopherols, which may, however, be separated by a polymeric column. Nevertheless, when the separation of b- and g-tocopherols is not critical, C18 reversed-phase systems are preferred, since equilibration times are shorter, and better reproducibility is achieved. Electrochemical
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detection, fluorescence, UV absorbance detection, and light-scattering detection have been employed for determining tocopherols and related substances with sensitivities decreasing in the mentioned order.
18.3.3.3
Occurrence of Vitamin E
The vitamin E contents in the muscle tissue of 21 species of teleosts, 3 species of cephalopods, and 6 species of crustaceans caught from the central Mediterranean Sea has been found to range from 5.06 to 17.90 mg/kg, from 7.42 to 9.61 mg/kg, and from 5.77 to 11.70 mg/kg, respectively.59 In another study, the vitamin E level in 10 species of Mediterranean fish was reported to be 7.29 mg/kg.60 The total E vitamers (tocopherols and tocotrienols) of fish consumed in Hawaii61 has been found to range from 2.0 to 39.1 mg/kg wet weight. a-Tocopherol has been found to be the principal tocopherol in marine animals.61,62 Fish species have characteristic tocopherol levels in their tissue, which, since fish are unable to synthesize the vitamin, are related to diet. Considerable differences in a-tocopherol concentration have been reported between light and dark fish muscle, the latter presenting the higher values.51 Krill is a very good source of vitamin E (150 mg/kg tissue).63 Seasonal variations in tocopherol content of fish species62 and mussels64 have been reported. An age-dependent decline in lipophilic antioxidant levels has also been reported.59
18.3.4 Carotenoids Carotenoids are isoprenoid polyenes formed by joining eight isoprene units.65 A series of conjugated double bonds constitutes a chromatophore of variable length, resulting in characteristic yellow to red colors. Carotenoids may be divided into two major classes, based on the degree of substitution65: (1) highly unsaturated carotene hydrocarbons (C40H56) such as a-, b-carotene and lycopene, (2) xanthophylls, that is, oxygenated derivatives of carotene hydrocarbons. Carotenoids may occur in the free, esterified, or bound to macromolecules form. In invertebrates carotenoids may form stable complexes with proteins, that is, carotenoproteins, often with a different color than the original pigment.65 The red color of cooked crustaceans is produced by the release of the individual carotenoid (astaxanthin) from the carotenoproteins when denatured by the heat of cooking.
18.3.4.1
Antioxidant and Other Functions of Carotenoids
Among the proposed functions of carotenoids in aquaculture66 have been those of pigmentation, antioxidant functions, as a source of provitamin A, cellular protection from photodynamic damage, and enhancement of growth and reproductive potential. Some evidence suggests that these pigments may perform vital roles in growth and reproductive success in crustaceans. Dietary carotenoids are the sole biological precursors of retinoids in crustaceans. In terms of free-radical pathology, the most important biological functions of carotenoids appear to be their antioxidant nature, that is, their ability to quench free radical species such as 1O and 3O .65 Carotenoids may also act as chain-breaking antioxidants. There are at least three 2 2 possible mechanisms for the reaction of carotenoids with radical species: (1) radical addition, (2) electron transfer to the radical, or (3) allylic hydrogen abstraction.67 The antioxidant function of b-carotene complements the action of other antioxidants such as catalase, peroxidase, vitamin C, and vitamin E.21
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The relative antioxidant activity of carotenoids has been investigated in several studies. The presence of functional groups (hydroxyl, epoxide, or keto) and the type of ring located at the ends of the polyenoic chain can vary the rate of pigment oxidation. Carbonyl carotenoids such as canthaxanthin and astaxanthin are regarded as better free-radical scavengers than b-carotene. According to Naguib68 the higher antioxidant activity of astaxanthin compared with lutein, lycopene, and a- and b-carotenes could be attributed to an equilibrium with the enol form of the ketone astaxanthin in solution. In a complex system like seafood flesh, it was postulated that the higher hydrophilicity of astaxanthin compared with that of b-carotene provides a better contact to hydroperoxides and thus a more effective protection against lipid oxidation.69
18.3.4.2
Carotenoid Determination
Methods used for the analysis and chromatographic determination of carotenoids have been reviewed.70,71 The main problem associated with work on and manipulation of carotenoids is their instability, especially toward light, oxygen, and heat. During sample preparation and handling, precautions to minimize carotenoid oxidation should be taken (e.g., use of antioxidants, homogenization at low temperatures). The extraction procedures for seafood usually involve several solvents such as hexane isopropanol,72,73 methanol,74 and a mixture of methanol with other more apolar solvents e.g., chloroform; acetone alone or in combination with other solvents has also been used in crustaceans75,76 and salmonids77; supercritical fluid extraction has also been reported.58,78 In several cases alkaline saponification precedes carotenoid determination. Classical column chromatography, TLC, and spectroscopic methods were originally used for the determination of carotenoids. Nowadays, the most common method used in the analysis of carotenoids is HPLC employing various detection techniques. Both normal- and reversed-phase systems are used, either in isocratic or gradient elution modes, the latter generally preferred. Good separations of various carotenoids have been attained on a C-30 chemically bonded phase. UV/Vis detection is by far the most common used. Electrochemical, mass spectrometry, nuclear magnetic resonance detection, and Raman spectroscopy have also been used among others.70
18.3.4.3
Occurrence of Carotenoids
The occurrence of carotenoid species found in several seafoods and aquaculture has been reviewed by Shahidi et al.65 Although carotenoids are distributed in almost all living matter, their synthesis is restricted to plants and microorganisms; thus the presence of carotenoids in animal tissues is solely derived from their dietary intake. However, each species has its own carotenoid requirements, and each tissue even appears to have such specificity in the assimilation of carotenoids. Crustaceans, in general, contain the same major carotenoids among which are astaxanthin, canthaxanthin, and cryptoxanthin.65 The carapace and the ecdysial exoskeleton of crustaceans mainly contain astaxanthin and lutein; the recovery and quantification of carotenoids from several crustacean wastes have been the aim of several studies.72–74,76,79 The carotenoid content in the meat of the major marine crab from Indian waters was reported to be 3.4 mg/kg.75 The astaxanthin content of krill has been found in the range of 15–20 mg/kg tissue.63 In mollusks, various carotenoids have been identified.65 In fish, carotenoids are found in the skin, flesh, eggs, gonads, milt, liver, and eyes. Most of their skin and muscle pigments are xanthophylls.65 The color of flesh of salmon and trout is normally due to astaxanthin, while ovaries also contain high astaxanthin amounts; its concentration has been found to vary between 3 and 37 mg/kg.80,81 In the study of Czeczuga
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et al.82 the carotenoid content in the fillets of 22 species of fish from the fisheries of New Zealand ranged from 0.28 to 5.9 mg/kg. In the gonads of some of these fish species, the carotenoid content ranged from 0.38 to 1.03 mg/kg. The total carotenoid content of Sardina pilchardus from South Europe has been reported to be 10.5 mg/kg in the skin and muscle.83
18.3.5 Ubiquinone Coenzyme Q10 (also known as ubiquinone, coenzyme Q, and abbreviated at times to CoQ10, CoQ, Q10) is a benzoquinone, where the “Q” and the “10” in the name refer to the quinone chemical group and the 10 isoprenyl chemical subunits, respectively. The isoprenoid tail is found in various lengths (6–10 isoprene units) in plants and microorganisms.84 CoQ is found in two redox forms, and the redox cycle involves a reversible reduction to the respective ubiquinols (CoQH2).
18.3.5.1 Function of Ubiquinone In addition to its well-established function as a component of the mitochondrial respiratory chain, in recent years, ubiquinone has acquired increasing attention with regard to its function, in the reduced form (ubiquinol), as an antioxidant.85 Ubiquinone, partly in the reduced form, occurs in all cellular membranes as well as in blood serum and in serum lipoproteins. Ubiquinol efficiently protects membrane phospholipids and serum low-density lipoprotein (LDL) from lipid peroxidation, and, as recent data indicate, also mitochondrial membrane proteins, and DNA from free-radical–induced oxidative damage. Scientific evidence has indicated that ubiquinone may function together with tocopherols in protecting the function of biological membranes.86 Ubiquinol is the only known lipid-phase antioxidant that can be synthesized de novo in animal cells and for which an enzymatic mechanism in mitochondrial and microsomal electron-transport systems, which can regenerate the antioxidant ubiquinone form, exists.85
18.3.5.2
Determination of Ubiquinone
Extraction of ubiquinone is usually performed contemporaneously with other lipophilic compounds, using hexane after protein precipitation with methanol, ethanol,22,84,87,88 ethanol isopropanol,89 and tissue homogenization. Isopropanol has also been used as an extraction solvent.90 Several HPLC methods for the determination of total ubiquinone (the sum of ubiquinone and ubiquinol) have been described using electrochemical and/or UV detection.22,84,88,91
18.3.5.3 Occurrence of Ubiquinone Ubiquinone is widely distributed, as it is present in almost every cell of living organisms. The CoQ content varies in different organs, with the highest in energy-producing tissues, as the dark muscle of fish. The ubiquinone content of several food and marine organisms has been reported in several studies.22,51,84,87–89,91,92 The richest known food source of CoQ10 is the heart from mammal species.84,91 Concerning ubiquinone contents in fish, reported results show that fat fish flesh is the second most abundant source of CoQ10 after red meats.51,59,84,91 The ubiquinone content of pelagic fish flesh (mackerel and herring) was reported in the range of 12.3–67.7 mg/kg88; in the muscle of several Mediterranean species of teleosts, cephalopods, and crustaceans, the range of CoQ10
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content was 0.07–1.13, 0.10–1.06, and 0.21–1.15 mg/kg, respectively; the range of CoQ 10H2 content was 0.18–5.11, 0.27–7.18, and 2.11–8.37 mg/kg, respectively; CoQ 9 and CoQ 9H2 were also detected in some species of teleosts and cephalopods, at concentrations ranging from 0.10–2.5 to 0.18–6.35 mg/kg, respectively.22 The CoQ10H2 and CoQ10 levels in Mediterranean fish were reported to be 3.43 and 0.72 mg/kg, respectively.60 The total CoQ 10 (ubiquinone and ubiquinol) content in the muscle of fish and shellfish of Japanese diet has been found to be in the range of 1.8–130 and 1.7–4.9 mg/kg, respectively,90 in salmon, eel, and squid consumed in Japan, 3.8–7.6 mg/kg.92
18.3.6 Other Endogenous Antioxidants Glutathione: GSH is a tripeptide (Glu-Cys-Gly), existing in reduced GSH and oxidized (GSSG) forms. It is involved in free-radical scavenging, metabolism of toxic xenobiotic compounds, maintenance of thiol-disulfide status, and signal transduction.20 GSH provides a source of electrons that allow GPx to enzymically decompose hydrogen and lipid peroxides. Oxidized GSH, GSSG, is reduced back to GSH by GR, to maintain a high GSH/GSSG ratio, which could be considered as a quality parameter.22 GSH is usually determined enzymatically4,5 or by HPLC.22 Phospholipids: The antioxidant activity of phospholipids is mainly connected with their ability to function as synergists and metal chelators. Phospholipids exhibit synergism with tocopherols in fish oils.93,94 As phospholipids are involved in nonenzymic browning, they show higher antioxidant activity at elevated temperatures or where browning is considerable. Amino acids, amines, and peptides: Amines, peptides, and amino acids are known to exhibit antioxidant properties. Furthermore, amino acids are involved in nonenzymic browning reactions with carbonyls from oxidizing lipids, which lead to products with antioxidative properties.95 Protein hydrolysates from fish,96,97 and fish processing by-products,98 exert antioxidant and biological activities. Other antioxidants in fish and seafood: Aquatic plants like seagrass99 are known to possess antioxidant and anti-inflammatory activity. Phycocyanin, a blue-green algae pigment,100 the flesh and skin of hag fish101 and eel,102 and shrimp shell waste103 contain natural antioxidants capable of scavenging hydroxyl radicals. Phenolic antioxidants have been reported in green algae,104 microalgae and cyanobacteria,105 edible brown algae,106 and in mussels.6
18.4
Added Antioxidants
The naturally occurring antioxidants in seafood and seafood products impart a certain amount of protection by maintaining the balance between prooxidative and antioxidative factors. However, in postmortem conditions and during processing and prolonged storage, the endogenous antioxidants are consumed sequentially, and lipids are eventually oxidized,51,107 necessitating the addition of exogenous antioxidants to retard the onset of lipid oxidation and elongate the shelf life of seafood products.
18.4.1
Synthetic Antioxidants
Antioxidants allowed in food in the EU and the FDA are the calcium disodium salt of EDTA (E385), the phenolic antioxidants propyl gallate (E310), octyl gallate (E311), dodecyl gallate
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(E312), butylated hydroxyanisol (BHA, E320), butylated hydroxytoluene (BHT, E321), and tertbutylhydroquinone (TBHQ, E319), the more “natural” ascorbic acid (E300) and isoascorbic (erythorbic) acid (E315) and their salts, ascorbyl palmitate and stearate (E304), tocopherols (a-E307, d-E308, g-E309), and tocopherol concentrate (E306). However, there have been concerns about the possible negative health effects of synthetic antioxidants.108
18.4.2
Natural Antioxidants
Natural antioxidants isolated from aromatic herbs, tea, grapes, and seeds have recently gained interest as replacements for potentially toxic synthetic antioxidants.109,110 Natural antioxidants are readily accepted by the consumers, as they are considered to be safe, and no safety tests are required if they are components of foods that are consumed for centuries, whereas their main disadvantages are that (1) they are usually more expensive if purified and less efficient if not purified, (2) the properties of different preparations vary if not purified, (3) their safety is unknown, and (4) they may impart color, aftertaste or off flavor to the product.111 Several natural antioxidants have already been used in fish oils and fish products to retard oxidative deterioration. Dry oregano is effective in preventing oxidation in mackerel oil.112 Phenolic antioxidants from rosemary leaves have been successfully used in sardine oil, cod liver oil,113 horse mackerel, oil-in-water emulsions, and fish oils.114 Green tea polyphenols protect silver carp115 and fish oils113,116 from oxidation. Phenolic extracts from grape by-products were successfully applied in fish lipids and muscle.117–119 Polyphenols extracted from extra virgin olive oil retards oxidation of canned tuna,120 horse mackerel, and fish oils.114 Individual polyphenols, alone or in mixture with other antioxidants, are more effective than synthetic antioxidants in preventing oxidation of marine oils121,122 or frozen fish.123
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35. Yebra-Biurrun, M.C., Flow injection determination methods of ascorbic acid, Talanta, 52, 367, 2000. 36. Yao, X., Wang, Y., and Chen, G., Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection, Biomed. Chromatogr., 21, 520, 2007. 37. Deutsch, J.C., Dehydroascorbic acid, J. Chromatogr. A, 881, 299, 2000. 38. Kall, M.A. and Andersen, C., Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid and dehydroisoascorbic acid in food and biological samples, J. Chromatogr. B, 730, 101, 1999. 39. Iwase, H., Use of an amino acid in the mobile phase for the determination of ascorbic acid in food by high-performance liquid chromatography with electrochemical detection, J. Chromatogr. A, 881, 317, 2000. 40. Rønnestad, I. et al., Ascorbic acid and a-tocopherol levels in larvae of Atlantic halibut before and after exogenous feeding, J. Fish Biol., 55, 720, 1999. 41. Sealey, W.M. and Gatlin, D.M., Dietary vitamin C and vitamin E interact to influence growth and tissue composition of juvenile hybrid striped bass (Morone chrysops × M. saxatilis) but have limited effects on immune responses, J. Nutr., 132, 748, 2002. 42. Felton, S.P., Grace, R., and Halver, J.E., A non-ion-pairing HPLC method for measuring new forms of ascorbate and ascorbic acid, J. Liq. Chromatogr., 17, 123, 1994. 43. Merchie, G., Lavens, P., Dhert, Ph., Dehasque, M., Nelis, H., De Leenheer, A., and Sorgeloos, P., Variation of ascorbic acid content in different live food organisms, Aquaculture, 134, 325, 1995. 44. Wang, X., Kim, K.-W., and Bai, S.C., Comparison of L-ascorbyl-2-monophosphate-Ca with L-ascorbyl-2-monophosphate-Na/Ca on growth and tissue ascorbic acid concentrations in Korean rockfish (Sebastes schlegeli), Aquaculture, 225, 387, 2003. 45. Iglesias, J., González, M.J., and Medina, I., Determination of ascorbic and dehydroascorbic acid in lean and fatty fish species by high-performance liquid chromatography with fluorometric detection, Eur. Food Res. Technol., 223, 781, 2006. 46. Rodriguez-Bernaldo de Quirós, A.I., Lopez-Hernandez, J., and SirnaI-Lozano, J., Determination of vitamin C in sea urchin: Comparison of two HPLC methods, Chromatographia, 53 (Suppl. 1), S-246, 2001. 47. Mori, K. et al., A simple fluorometric determination of vitamin C, Chem. Pharm. Bull. (Tokyo), 46, 1474, 1998. 48. Lykkesfeldt, J., Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: Reliable reduction with tris[2carboxyethyl]phosphine hydrochloride, Anal. Biochem., 282, 89, 2000. 49. Alexis, M.N. et al., Tissue ascorbic acid levels in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata L.) fingerlings fed diets containing different forms of ascorbic acid, Aquaculture, 179, 447, 1999. 50. Wouters, R. et al., Lipid composition and vitamin content of wild female Litopenaeus Yannamei in different stages of sexual maturation, Aquaculture 198, 307, 2001. 51. Petillo, D. et al., Kinetics of antioxidant loss in mackerel light and dark muscle, J. Agric. Food Chem., 46, 4128, 1998. 52. Dabrowski, K. and Ciereszko, A., Ascorbic acid and reproduction in fish: Endocrine regulation and gamete quality, Aquaculture Res., 32, 623, 2001. 53. Traber, M.G. and Atkinson, J., Vitamin E, antioxidant and nothing more, Free Rad. Biol. Med., 43, 4, 2007. 54. Schneider, C., Chemistry and biology of vitamin E, Mol. Nutr. Food Res., 49, 7, 2005. 55. Luque-Garcia, J.L. and Luque de Castro, M.D., Extraction of fat-soluble vitamins, J. Chromatogr. A, 935 3, 2001. 56. Rupérez, F.J. et al., Chromatographic analysis of a-tocopherol and related compounds in various matrices, J. Chromatogr. A, 935, 45, 2001.
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57. Huo, J.Z. et al., Determination of vitamin E in aquatic organisms by high-performance liquid chromatography with fluorescence detection, Anal. Biochem., 242, 123, 1996. 58. Turner, C., King, J. W., and Mathiasson, L., Supercritical fluid extraction and chromatography for fat-soluble vitamin analysis, J. Chromatogr. A, 936, 215, 2001. 59. Passi, S. et al., Fatty acid pattern, oxidation product development, and antioxidant loss in muscle tissue of rainbow trout and Dicentrarchus labrax during growth, J. Agric. Food Chem., 52, 2587, 2004. 60. Passi, S. et al., Dynamics of lipid oxidation and antioxidant depletion in Mediterranean fish stored at different temperatures, BioFactors, 25, 241, 2005. 61. Franke, A.A. et al., Tocopherol and tocotrienol levels of foods consumed in Hawaii, J. Agric. Food Chem., 55, 769, 2007. 62. Syvaojaa, E.L. and Salminen, K., Tocopherols and tocotrienols in Finnish foods: Fish and fish products. J. Am. Oil Chem. Soc., 62, 1245, 1985. 63. Tou, J.C., Jaczynski, J., and Chen, Y.C., Krill for human consumption: Nutritional value and potential health benefits, Nutr. Rev., 65, 63, 2007. 64. Orban, E. et al., Seasonal changes in meat content, condition index and chemical composition of mussels (Mytilus galloprovincialis) cultured in two different Italian sites, Food Chem., 77, 57, 2002. 65. Shahidi, F., Metusalach, and Brown, J.A., Carotenoid pigments in seafoods and aquaculture, Crit. Rev. Food Sci., 38, 1, 1998. 66. Linan-Cabello, M.A., Paniagua-Michel, J., and Hopkins, P.M., Bioactive roles of carotenoids and retinoids in crustaceans, Aquaculture Nutr., 8, 299, 2002. 67. Krinsky, N.I. and Johnson, E.J., Carotenoid actions and their relation to health and disease, Mol. Aspects Med., 26, 459, 2005. 68. Naguib, Y.M.A., Antioxidant activities of astaxanthin and related carotenoids, J. Agric. Food Chem., 48, 1150, 2000. 69. Andersen, H.J., Development of rancidity in salmonoid steaks during retail display. A comparison of practical storage life of wild salmon and farmed rainbow trout, Z. Lebensm. Unters. Forsh, 191, 119, 1990. 70. Tee, E.S. and Lim C.L., The analysis of carotenoids and retinoids: A review, Food Chem., 41, 147, 1991. 71. Feltl, L. et al., Reliability of carotenoid analyses: A review, Curr. Anal. Chem., 1, 93, 2005. 72. Sachindra, N.M. et al., Recovery of carotenoids from ensilaged shrimp waste, Biores. Technol., 98, 1642, 2007. 73. Sachindra, N.M., Bhaskar, N., and Mahendrakar, N.S., Recovery of carotenoids from shrimp waste in organic solvents, Waste Manage., 26, 1092, 2006. 74. López-Cervantes, D.I. et al., Quantification of astaxanthin in shrimp waste hydrolysate by HPLC, J. Chromatogr. A, 20, 981, 2006. 75. Sachindra, N.M., Bhaskar, N., and Mahendrakar, N.S., Carotenoids in crabs from marine and fresh waters of India, LWT, 38, 221, 2005. 76. Lin, W.C., Chien, J.T., and Chen, B.H., Determination of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography, J. Agric. Food Chem., 53, 5144, 2005. 77. Tolasa, S., Cakli, S., and Ostermeyer, U., Determination of astaxanthin and canthaxanthin in salmonid, Eur. Food Res. Technol., 221, 787, 2005. 78. López, M. et al., Selective extraction of astaxanthin from crustaceans by use of supercritical carbon dioxide, Talanta, 64, 726, 2004. 79. Maoka, T. and Akimoto, N., Carotenoids and their fatty acid esters of spiny lobster Panulirus japonicus, J. Oleo Sci., 57, 145, 2008. 80. Torrissen, O.J., Hardy, R.W., and Shearer, K.D., Pigmentation of salmonids-carotenoid deposition and metabolism, Crit. Rev. Aquatic Sci., 1, 209, 1989. 81. Storebakken, T. and No, H.K., Pigmentation of rainbow trout, Aquaculture, 100, 209, 1992. 82. Czeczuga, B., Klyszejko, B., and Czeczuga-Semeniuk, E., The carotenoid content in certain fish species from the fisheries of New Zealand, Bull. Sea Fish. Inst., 1(149), 35, 2000. 83. Czeczuga, B., Carotenoids in fish. XXIV. Sardina pilchardus walb (clupeidae), Hydrobiologia, 69, 277, 1980.
Antioxidants
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84. Weber, C., Bysted, A., and Hølmer, G., The coenzyme Q10 content of the average Danish diet, Int. J. Vit. Nutr. Res., 67, 123, 1997. 85. Ernster, L. and Dallner, G., Biochemical, physiological and medical aspects of ubiquinone function, Biochim. Biophys. Acta, 1271, 195, 1995. 86. Kagan, V.E., Fabisiak, J.P., and Quinn, P.J., Coenzyme Q and vitamin E need each other as antioxidants, Protoplasma, 214, 11, 2000. 87. Mattila, P., Lehtonen, M., and Kumpulainen, J., Comparison of in-line connected diode array and electrochemical detectors in the high performance liquid chromatographic analysis of Co-enzymes Q9 and Q10 in food materials, J. Agric. Food Chem., 48, 1229, 2000. 88. Souchet, N. and Laplante, S., Seasonal variation of Co-enzyme Q10 content in pelagic fish tissues from Eastern Quebec, J. Food. Comp. Anal., 20, 403, 2007. 89. Giardina, B. et al., Coenzyme Q homologs and trace elements content of Antarctic fishes Chionodraco hamatus and Pugothenia bernucchii compared with the Mediterranean fish Mugil cephalus, Comp. Biochem. Physiol., 118A, 977, 1997. 90. Kubo, H. et al., Food content of ubiquinol-10 and ubiquinone-10 in the Japanese diet, J. Food Comp. Anal., 21, 199, 2008. 91. Mattila, P. and Kumpulainen, J., Co-enzyme Q9 and Q10: Contents in foods and dietary intake, J. Food. Comp. Anal., 14, 409, 2001. 92. Kettawan, A. et al., Food content of ubiquinol-10 and ubiquinone-10 in the Japanese diet, J. Clin. Biochem. Nutr., 41, 124, 2007. 93. Segawa, T., Hara, S., and Totanai, Y., Antioxidative behavior of pho⋅pholipids for polyunsaturated fatty acids of fish oil. II. Synergistic effect of phospholipids for tocopherol, J. Jpn. Oil Chem. Soc., 46, 515, 1994. 94. Bandarra, N.M. et al., Antioxidant synergy of a-tocopherol and phospholipids, J. Am. Oil Chem. Soc., 76, 905, 1999. 95. Alaiz, M., Zamora, R., and Hidalgo, F.J., Contribution of the formation of oxidized lipid/amino acid reaction products to the protective role of amino acids in oils and fats, J. Agric. Food Chem., 44, 1890, 1996. 96. Shahidi, F. and Amarowicz, R., Antioxidant activity of protein hydrolyzates from aquatic species, J. Am. Oil Chem. Soc., 73, 1197,1996. 97. Jun, S.H. et al., Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfin sole (Limanda aspera) frame protein, Eur. Food Res. Tech., 219, 20, 2004. 98. Kim, S.K. and Mendis, E., Bioactive compounds from marine processing byproducts—A review, Food Res. Intern., 39, 383, 2006. 99. Hua, K.F. et al., Study on the antiinflammatory activity of methanol extract from seagrass Zostera japonica, J. Agric. Food Chem., 54, 306, 2006. 100. Romay, C. et al., Antioxidant and anti-inflammatory properties of C-phycocyanin from blue-green algae, Inflamm. Res., 47, 36, 1998. 101. Ekanayake, P., Lee, Y.D., and Lee, J., Antioxidant activity of flesh and skin of Eptatretus burgeri (hag fish) and Enedrias nebulosus (white spotted eel), Food Sci. Technol. Int., 10, 171, 2004. 102. Ekanayake, P.M. et al., Antioxidant potential of eel (Anguilla japonica and Conger myriaster) flesh and skin, J. Food Lipids, 12, 34, 2005. 103. Seymour, T.A., Li, S.J., and Morrissey, M., Characterization of natural antioxidant from shrimp shell waste, J. Agric. Food Chem., 44, 682, 1996. 104. Markham, K.R. and Porter, L.J., Flavonoids in the green algae (chlorophyta), Phytochemistry, l8, 1777, 1969. 105. Scholz, B. and Liebezeit, G., Chemical screening for bioactive substances in culture media of microalgae and cyanobacteria from marine and brackish water habitats: First results, Pharm. Biol., 44, 544, 2006. 106. Kuda, T. et al., Antioxidant properties of dried “kayamo-nori”, a brown alga Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae), Food Chem., 89, 617, 2005. 107. Decker, E.A., Strategies for manipulating the prooxidative/antioxidative balance of foods to maximize oxidative stability, Trends Food Sci. Technol., 9, 241, 1998.
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108. Iqbal, S. and Bhanger, M.I., Stabilization of sunflower oil by garlic extract during accelerated storage, Food Chem., 100, 246, 2007. 109. Peschel, W. et al., An industrial approach in the search of natural antioxidants from vegetable and fruit wastes. Food Chem., 97, 227, 2006. 110. Yanishlieva, N.V., Marinova, E., and Pokorný, J., Natural antioxidants from herbs and spices, Eur. J. Lipid Sci. Technol., 108, 776, 2006. 111. Pokorný, J., Natural antioxidants for food use, Trends Food Sci. Technol., 2, 223, 1991. 112. Tsimidou, M., Papavergou, E., and Boskou, D., Evaluation of oregano antioxidant activity in mackerel oil, Food Res. Int., 28, 431, 1995. 113. O’Sullivan, A. et al., Use of natural antioxidants to stabilize fish oil systems, J. Aquat. Food Prod. Technol., 14, 75, 2005. 114. Medina, I. et al., Activity of plant extracts for preserving functional food containing n-3-PUFA, Eur. Food Res. Technol., 217, 301, 2003. 115. Fan, W., Chi, Y., and Zhang, S., The use of a tea polyphenol dip to extend the shelf life of silver carp (Hypophthalmicthys molitrix) during storage in ice, Food Chem., 108, 148, 2008. 116. Wanasundara, U.N. and Shahidi, F., Antioxidant and pro-oxidant activity of green tea extracts in marine oils, Food Chem., 63, 335, 1998. 117. Pazos, M. et al., Activity of grape polyphenols as inhibitors of the oxidation of fish lipids and frozen fish muscle, Food Chem., 92, 547, 2005. 118. Pazos, M. et al., Physicochemical properties of natural phenolics from grapes and olive oil byproducts and their antioxidant activity in frozen horse mackerel fillets, J. Agric. Food Chem., 54, 366, 2006. 119. Sánchez-Alonso, I. et al., Antioxidant protection of white grape pomace on restructured fish products during frozen storage, LWT, 41, 42, 2008. 120. Medina, I. et al., Comparison of natural polyphenol antioxidants from extra virgin olive oil with synthetic antioxidants in tuna lipids during thermal oxidation, J. Agric. Food Chem., 47, 4873, 1999. 121. Nieto, S. et al., Flavonoids as stabilizers in fish oil: An alternative to synthetic antioxidants, J. Am. Oil Chem. Soc., 70, 773, 1993. 122. Wanasundara, U.N. and Shahidi, F., Stabilization of marine oils with flavonoids, J. Food Lipids, 5, 183, 1998. 123. Pazos, M., Sánchez, L., and Medina, I., a-Tocopherol oxidation in fish muscle during chilling and frozen storage, J. Agric. Food Chem., 53, 4000, 2005.
Chapter 19
Vitamins Young-Nam Kim Contents 19.1 Fat-Soluble Vitamins .................................................................................................... 328 19.1.1 Vitamin A and Carotenoids ............................................................................ 328 19.1.2 Vitamin D........................................................................................................332 19.1.3 Vitamin E ........................................................................................................332 19.1.4 Vitamin K ........................................................................................................333 19.2 Water-Soluble Vitamins................................................................................................ 334 19.2.1 Thiamin (Vitamin B1) ..................................................................................... 334 19.2.2 Riboflavin (Vitamin B2) ...................................................................................335 19.2.3 Niacin (Vitamin B3) ........................................................................................ 336 19.2.4 Vitamin B6 .......................................................................................................337 19.2.5 Folate ...............................................................................................................338 19.2.6 Vitamin B12 ..................................................................................................... 340 19.2.7 Pantothenic Acid ............................................................................................. 341 19.2.8 Biotin .............................................................................................................. 341 19.2.9 Vitamin C ....................................................................................................... 342 19.3 Summary ..................................................................................................................... 343 References ............................................................................................................................... 344
Vitamins are a group of complex organic compounds that are essential to normal functioning and essential metabolic reactions in the body. Vitamins are not used as a source of energy or as a source of structural tissue components but rather as cofactors or coenzymes in biochemical reactions. Vitamins are divided into two categories based on their solubility—those soluble in fat organic solvents are known as fat-soluble vitamins and those soluble in water are known as 327
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◾ Handbook of Seafood and Seafood Products Analysis
water-soluble vitamins. Tables 19.1 and 19.2 list the concentrations of fat-soluble vitamins and water-soluble vitamins, respectively, in fish and seafood according to the U.S. Department of Agriculture National Nutrient Database [1]. The percentages of the daily value (DV) of vitamins estimated to be in 100 g of each seafood are included in Tables 19.1 and 19.2. The DV, established by the U.S. Food and Drug Administration, is a nutrient reference value intended to help consumers understand how foods fit into their overall diets [2]. Foods containing 20% or more of the DV of nutrients per reference amount are indicated to be “high,” “rich,” or “excellent” sources of the nutrients. Foods containing 10%–19% of the DV are categorized as “good” sources. Fish and shellfish are good to excellent sources of most of the B vitamins as defined by the U.S. Food and Drug Administration [3]. Fat-soluble vitamins A, D, and E are present in seafood in varying amounts, often in concentrations higher than those in other meats. Many species of fish and shellfish store high amounts of vitamins A, D, and E in their livers [4]. Little vitamin C is found in seafood, but it is considered a good to excellent source of B-complex vitamins. Generally, the methodologies used for determining the composition of various vitamins in seafood are the same as those used for other foods. Several high-performance liquid chromatography (HPLC) methods using various detections [5–9] have been proposed to determine several vitamins simultaneously. Internal standards are frequently used in the analytical methods for determination of vitamins. Several vitamins are lost or interconverted to their isomers in extraction and purification procedures during analyses. Various methods have been used for the determination of vitamins in foods. In-depth recent reviews of the methodologies for measuring vitamins are available [10,11].
19.1 Fat-Soluble Vitamins 19.1.1 Vitamin A and Carotenoids Vitamin A is a fat-soluble vitamin that is essential for humans and other vertebrates. Vitamin A as retinoids, primarily retinyl esters, is abundant in some animal-derived foods, whereas carotenoids are abundant in plant foods as pigments. Carotenoids are responsible for the color of fish and shellfish. Carotenoids cannot be synthesized by live animals but are obtained from their preys or aquafeeds, which have added carotenoids such as canthaxanthin and astaxanthin [12]. Because vitamin A and carotenoids are fat soluble, they are associated with the fat portion of foods. They are highly concentrated in fish liver oils, but small amounts are found in fish muscles or fillets. Fish are not a major source of this vitamin. In foods, retinyl esters and carotenoids are vulnerable to oxidation. Exposure to air, heat, and storage time also influence the destruction of vitamin A compounds. Thus, overcooking can cause loss of retinyl esters and provitamin A in foods. Reversed-phase HPLC followed by ultraviolet (UV) detection for retinoids and carotenoids is the most common method of analysis. HPLC methodologies are given in AOAC Official Methods 2001.13 and 2005.07 [13]. HPLC procedures for determination of retinoids and carotenoids in seafood [14–16] have also been published. During sample preparation and analysis, samples should be protected from heat, light, and oxidizing substances to avoid destructions and isomerizations of the retinoids and carotenoids. Antioxidants such as butylated hydroxytoluene (BHT), pyrogallol, or ascorbyl palmitate are used to prevent oxidation of retinoids and carotenoids. Alkali hydrolysis (saponification) is routinely used to extract retinoids and carotenoids from foods. Saponification removes chlorophylls, unwanted lipids, and other materials, which may interfere with chromatographic separation. Retinyl esters and carotenoid esters in foods are converted to retinol and carotenoids during saponification. However, the degradation and isomerization of
Vitamins ◾ TABLE 19.1
329
Concentrations of Fat-Soluble Vitamins in Selected Fish and Seafood Vitamin A
Food Product
IU/100 ga
Vitamin E
%DVb
Vitamin D
mg/100 gc
%DV
IU/100 gd
Vitamin K
%DV
μg/100 g
%DV
Catfish, raw
50
1
—e
—
500
125
—
—
Cod, raw
27
<1
0.64
3
—
—
0.1
<1
Flatfish, raw
33
<1
0.51
3
0.1
<1
Halibut, raw
157
3
0.85
4
—
—
0.1
<1
Herring, raw
93
2
1.07
5
1,628
407
0.1
<1
Mackerel, raw
167
3
1.52
8
360
90
5.0
6
Salmon, raw
117
2
0.64
3
—
—
0.4
1
Tuna, raw
60
1
0.50
3
—
—
0.1
<1
Clam, raw
300
6
0.31
2
0.2
<1
Lobster, raw
70
1
1.47
7
—
0.1
<1
Shrimp, raw
180
4
1.10
6
152
0.0
0
Mussel, raw
160
3
0.55
3
—
0.1
<1
Oyster, raw
100
2
0.85
4
320
0.1
<1
Scallop, raw
50
1
0.00
0
—
—
0.1
<1
Squid, raw
33
<1
1.20
6
—
—
0.0
0
Whelk, raw
87
2
0.13
<1
—
—
0.1
<1
Caviar
905
18
1.89
9
232
0.6
1
Mixed fish roe, raw
299
6
7.00
35
—
—
0.2
<1
0
0
—
—
—
—
—
—
100,000
2,000
—
—
2,500
—
—
Fish oil, herring Fish oil, cod liver
60
15
4
10,000
1 — 38 — 80
58
Source: Data obtained from U.S. Department of Agriculture, Agricultural Research Service, USDA National Nutrient Database for Standard Reference, Release 20, Nutrient Data Laboratory, 2007. a b c d e
1 IU vitamin A = 0.3 μg of all-trans-retinol or 0.6 μg of β-carotene. Percent daily value (DV), established by the U.S. Food and Drug Administration [2]. As mg α-tocopherol. 1 IU vitamin D = 0.025 μg cholecalciferol or ergocalciferol. No composition data provided by U.S. Department of Agriculture.
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Handbook of Seafood and Seafood Products Analysis
Table 19.2
Concentrations of Water-Soluble Vitamins in Selected Fish and Seafood Thiamin
Riboflavin
Niacin
mg/100 g
%DVa
mg/100 g
%DV
mg/100 g
%DV
Catfish, raw
0.210
14
0.072
4
1.907
10
Cod, raw
0.022
1
0.042
2
2.040
10
Flatfish, raw
0.089
6
0.076
4
2.899
14
Halibut, raw
0.060
4
0.075
4
5.848
29
Herring, raw
0.092
6
0.233
14
3.217
16
Mackerel, raw
0.176
12
0.312
18
9.080
45
Salmon, raw
0.170
11
0.060
4
7.000
35
Tuna, raw
0.434
29
0.047
3
9.800
49
Clam, raw
0.080
5
0.213
13
1.765
9
Lobster, raw
0.006
<1
0.048
3
1.455
7
Shrimp, raw
0.028
2
0.034
2
2.552
13
Mussel, raw
0.160
11
0.210
12
1.600
8
Oyster, raw
0.100
7
0.095
6
1.380
7
Scallop, raw
0.012
1
0.065
4
1.150
6
Squid, raw
0.020
1
0.412
24
2.175
11
Whelk, raw
0.026
2
0.107
6
1.050
5
Caviar
0.190
13
0.620
36
0.120
<1
Mixed fish roe, raw
0.240
16
0.740
44
1.800
9
Fish oil, herring
0.000
0
0.000
0
0.000
0
Fish oil, cod liver
0.000
0
0.000
0
0.000
0
Food Product
Source: Data from U.S. Department of Agriculture, Agricultural Research Service, USDA National a
Percent daily value (DV), established by the U.S. Food and Drug Administration [2].
retinol and carotenoids may occur during saponification. This is greater with higher concentrations of alkali and higher temperatures [17]. Hexane, petroleum ether, diethyl ether, dichloromethane, or mixtures of these solvents are common extracting solvents. The reversed-phase C18 column is commonly used to resolve retinoids and carotenoids in foods. The polymeric C30 column designed at the U.S. National Institute of Standards and Technology [18] provides high absolute retention and resolution of cis and trans isomers of carotenoids [19]. Acetonitrile- or methanol-based mobile phases are used by the addition of 1% or 0.1 M ammonium acetate or acetic acid [20]. Maximum absorbance is 320–380 nm for retinoids and 400–500 nm for carotenoids. Thus, to perform
Vitamins ◾
Vitamin B6
Folate
Vitamin B12
mg/100 g
%DV
μg/100 g
%DV
0.116
6
10
3
0.400
20
7
0.208
10
0.344
μg/100 g
Pantothenic Acid
331
Vitamin C
%DV
mg/100 g
%DV
mg/100 g
%DV
2.23
37
0.765
8
0.7
1
2
0.90
15
0.140
1
2.9
5
8
2
1.52
25
0.503
5
1.7
3
17
12
3
1.18
20
0.329
3
0.0
0
0.302
15
10
3
13.67
228
0.645
6
0.7
1
0.399
20
1
<1
8.71
145
0.856
9
0.4
<1
0.200
10
4
1
3.00
50
0.750
8
0.0
0
0.900
45
2
<1
0.52
9
0.750
8
1.0
2
0.060
3
16
4
49.44
824
0.362
4
13
22
0.063
3
9
2
0.93
16
1.630
16
0
0
0.104
5
3
<1
1.16
19
0.276
3
2
3
0.050
3
42
11
12.00
200
0.500
5
8
13
0.062
3
10
3
19.46
324
0.185
2
3.7
6
0.150
8
16
4
1.53
26
0.143
1
3.0
5
0.056
3
5
1
1.30
22
0.500
5
4.7
8
0.342
17
6
2
9.07
151
0.208
2
4
7
0.320
16
50
13
20.00
333
3.500
35
0
0
0.160
8
80
20
10.00
167
1.000
10
16.0
27
0.000
0
0
0
0.00
0
0.000
0
0.0
0
0.000
0
0
0
0.00
0
0.000
0
0.0
0
Nutrient Database for Standard Reference, Release 20, Nutrient Data Laboratory, 2007.
simultaneous HPLC analysis of retinoids and carotenoids, a photodiode array detector is essential to establish the identity of the compound in each peak and validate homogeneity. There are several units used for expressing vitamin A contents in foods. International units (IU) are defined by the relationship of 1 IU = 0.3 μg of all-trans-retinol or 0.6 μg of β-carotene. The biological activity of vitamin A is quantified by conversion of retinol and provitamin A carotenoids to retinol equivalents (RE). One RE is defined as 1 μg of retinol, 6 μg of β-carotene, or 12 μg of other provitamin A carotenoids. Therefore, 1 RE is equal to 3.33 IU based on retinol. In 2001, the U.S. Institute of Medicine [21] proposed the new vitamin A unit, retinol activity equivalents (RAE).
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One RAE is equivalent to 1 μg of retinol, which is nutritionally equivalent to 12 μg of β-carotene or 24 μg of other provitamin A carotenoids.
19.1.2
Vitamin D
Vitamin D is a fat-soluble vitamin found in foods and also synthesized in the body after exposure to UV rays from the sun. Several forms of vitamin D have been described, but the two major physiologically relevant ones are vitamin D2 and vitamin D3 [22]. Vitamin D2 (ergocalciferol) is a synthetic form of vitamin D that is produced by irradiation of plant and yeast steroid ergosterol. Vitamin D3 (cholecalciferol) is the naturally occurring form of vitamin D produced from 7-dehydrocholesterol when the skin of animals and humans is exposed to sunlight, specifically UV B radiation. Vitamin D is biologically inactive and is metabolized to 25-hydroxyvitamin D (25(OH)D) in the liver, which is the most abundant form of vitamin D in the circulatory system. This circulating metabolite is hydroxylated again to form its biologically active hormone, 1,25dihydroxyvitamin D (1,25(OH)2D), which acts as a hormone in controlling calcium homeostasis and regulating the growth of various cell types [23]. Most of the vitamin D in animal foods are vitamin D3 and 25(OH)D3. Fish liver oils are the richest sources of vitamin D, and finfish and shellfish are known to be natural vitamin D contributors in human diets [24]. Therefore, to estimate vitamin D values in seafood, analyses of 25(OH)D3 should be included. Food processing, cooking, and storage of foods do not generally affect the concentration of vitamin D [23]. HPLC methods using an UV absorbance detector are available for the quantitation of vitamin D from most food matrices. Several HPLC methodologies for vitamin D analyses are provided in published articles [25–29] including AOAC Official Method 995.05, which is the method for determination of vitamin D in infant formulas and enteral products [13]. Vitamin D oxidation can occur during the oxidation of fats. Thus, an antioxidant such as pyrogallol or ascorbic acid is added when analyzing food samples. Estimation of the low concentrations of vitamin D in seafood is often difficult due to interfering substances such as fats, cholesterol, vitamin A, and vitamin E. To remove fats, saponification and cleanup procedures should be applied. Hot saponification promotes thermal isomerization of vitamin D with the formation of previtamin D. Hence, several methods [27,30] have been used for saponification at ambient temperature overnight. After saponification, unsaponified lipids including vitamin D are extracted with diethyl ether:petroleum ether, 1:1 [27–29]. Vitamin A, vitamin E, sterols, and other interfering components in the unsaponified fraction are removed using a silica solid-phase extraction [26–32]. Both reversed-phase and normal-phase systems offer efficient resolution of vitamin D, 7-dehydrocholesterol, and hydroxylated metabolites. However, reversedphase chromatography can separate vitamin D2 from vitamin D3 [28,33]. Vitamin D shows identical UV absorption spectra with λmax at 265 nm, which is sensitive enough for the detection of vitamin D2, vitamin D3, and their metabolites. Vitamin D content in foods is expressed in either IU or μg of vitamin D. One IU of vitamin D is the activity obtained from 0.025 μg of cholecalciferol in bioassays. The activity of 25(OH)D is five times more potent than that of cholecalciferol. Therefore, the biological activity of 1 μg of vitamin D is 40 IU, and 1 IU is 0.005 μg of 25(OH)D or 0.025 μg D2 or D3 [22].
19.1.3
Vitamin E
Vitamin E is the most effective fat-soluble antioxidant known to occur in the human body. Natural vitamin E exists in eight different forms, four tocopherols (α-, β-, γ-, and δ-tocopherols)
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and four tocotrienols (α-, β-, γ-, and δ-tocotrienols). Vitamin E is found in plant and animal foods. Seafood provides small amounts of vitamin E. The fatty species of fish have greater quantities than lean varieties, and shellfish have very little vitamin E [34]. Tocopherol and/or antioxidants are usually added to fish oils to protect them from rancidity [35]. During processing, losses of vitamin E can occur quite rapidly. Losses are accelerated by oxygen, light, heat, and various metals, primarily iron and copper, and by the presence of free radicals in the fat, which can initiate autoxidation. For vitamin E assay in foods, HPLC methods using fluorescence or UV detection have largely replaced the colorimetric and polarimetric procedures of AOAC Official Methods 948.26, 971.30, and 975.45 [13]. Although gas chromatography (GC) methodologies (AOAC Official Methods 988.14 and 989.09) were developed to increase the precision of vitamin E quantification before the advent of HPLC procedures, HPLC is considered a suitable measurement of the individual tocopherols and tocotrienols as GC methodologies can be time consuming. HPLC methodologies are provided in AOAC Official Method 992.03 [13] and other published articles [5,36–38]. Vitamin E in oils can be measured after dilution with n-hexane or mobile phase with a normal-phase liquid chromatography. However, alkaline hydrolysis is usually required for seafood to release the α-tocopherol. Hydrolysis results in cleavage of the ester linkages of lipids in food samples, destroys pigments, and disrupts the sample matrix, which facilitates vitamin E extraction. Following saponification, the digest is extracted with ether, petroleum ether, hexane, ethyl acetate in hexane, or other organic solvent mixtures. During saponification, addition of antioxidants, pyrogallol, ascorbic acid, or BHT in extraction solvents, and protection from light are required to prevent losses of vitamin E. Both reversed-phase and normal-phase systems are useful for the resolution of vitamin E. The advantage of normal-phase HPLC systems is the ability to separate the eight tocopherols and tocotrienols that occur in nature. Reversedphase systems cannot resolve the β- and γ-isomers [40]. However, reversed-phase HPLC is the preferred system for the determination of α-tocopherol with retinol and carotenoids in foods. Fluorescence detection provides sensitivity, specificity, and cleaner chromatograms compared with UV detection [41]. Although the UV absorbance of tocopherols and tocotrienols is relatively weak, detection at the absorption maximum (292–298 nm) using a variable wavelength detector affords sufficient sensitivity for most applications [42]. For simultaneous detection of vitamin E with other fat-soluble vitamins and carotenoids, a multichannel UV detector or a photodiode array detector may be useful in a single sample assay. IUs of vitamin E activity (1 mg of α-tocopherol = 1.49 IU) may be used in food composition tables, but the IU is not commonly used. The vitamin E composition in foods is often expressed as mg of α-tocopherol equivalents based on the biological activity of the various forms of vitamin E [43]. However, the U.S. Institute of Medicine [44] has indicated that the only form of the tocopherols and tocotrienols that has vitamin E activity in humans is α-tocopherol.
19.1.4
Vitamin K
Vitamin K is a fat-soluble vitamin. Two forms of vitamin K exist in nature: phylloquinone and menaquinones. Phylloquinone, known as vitamin K1, is synthesized by plants. Menaquinones, known as vitamin K 2, are produced by bacteria and contain a polyisoprenyl side chain at the 3 position [45]. However, one of the menaquinones, menaquinone (MK)-4, is not a major bacterial product but is synthesized by animals from phylloquinone [46,47]. Menadione, vitamin K 3, is a synthetic form of vitamin K. Menadione and its derivatives are used as additives in the feed industry. Menadione can also be converted to MK-4 in animal tissues [46,48]. There are only minute
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amounts of vitamin K in most fish and shellfish. Vitamin K is quite stable to oxidation and most food processing and food preparation procedures, while it is unstable to light, alkali, strong acid, and reducing agents [49]. Current methods to determine vitamin K in foods are HPLC procedures using fluorescence or electrochemical detection systems. AOAC Official Methods 992.27 and 999.15 [13] for phylloquinone determination in infant formulas and several HPLC methodologies [50–53] simultaneously determining phylloquinone and menaquinones have been developed. GC procedures have been described but are not routinely used due to long retention times and the potential for on-column degradation from high-column temperatures. When analyzing vitamin K, saponification cannot be applied to remove fats and other components because of the instability of vitamin K under alkaline conditions. Vitamin K is extracted from foods with organic solvents such as ethanol, isopropanol, and acetonitrile and then purified by solid-phase extraction with silica cartridges before the resolution of vitamin K by reversedphase HPLC. Phylloquinone and menaquinones are detected by UV detection, but lipids and other interfering compounds remaining in extract solutions make UV detection unworkable for fish containing high fats. Although vitamin K does not fluoresce, the quinones are reduced to hydroquinones by the addition of zinc chloride using a postcolumn zinc metal reduction column, so that fluorescence detection provides a highly specific detection system for vitamin K determination in foods. Electrochemical detection is also used by adding an electrolyte such as sodium acetate or perchlorate in the mobile phase to support the conductivity. For the determination of phylloquinone and menaquinones in animal products, Koivu-Tikkanen et al. [50] extracted the samples with 2-propanol/hexane after adding internal standards. Sample extracts were purified by normal-phase HPLC, and the fraction containing vitamin K was analyzed by reversed-phase HPLC using postcolumn reduction and fluorescence detection. The vitamin K composition of foods is commonly expressed as microgram of vitamin K.
19.2 19.2.1
Water-Soluble Vitamins Thiamin (Vitamin B1 )
Thiamin, known as vitamin B1, is one of the B vitamins. Thiamin exists in interconvertible phosphorylated forms in nature: thiamin monophosphate, thiamin pyrophosphate, and thiamin triphosphate. Most fish and seafood have small amounts of thiamin. The amount of thiamin is less dependent on the species concerned [4]. Thiamin is most stable between pH 2 and 4 and unstable at alkaline pH [54]. In alkaline solution, thiamin is readily oxidized, even at room temperature. It is the most heat labile of the B vitamins, with its decomposition dependent on pH and exposure time to heat. In addition, thiamin is known as the most radiation-sensitive water-soluble vitamin [55]. Raw fish and shellfish contain thiaminase, an enzyme that destroys thiamin. Thiaminase reactions are initiated by brushing, blending, homogenization, or other processes that break tissue structure during sample preparation in raw seafood. Sulfhydryl groups and other reducing agents protect thiamin from thiaminase reaction [55]. Thiaminase is inactivated during the cooking process. Several different analytical methodologies have been used to determine the thiamin content of foods. These methodologies include the fluorometric methods of AOAC Official Methods 942.23, 953.17, 957.17, and 986.27 [13]; microbiological analyses [56–58]; HPLC methods [7,8,59–61]; and GC procedures [62,63].
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Extraction procedures for the microbiological, HPLC, and GC analyses generally follow the thiochrome analysis procedures of AOAC Official Method 942.23 [13]. Because thiamin is stable under acidic conditions, hot acid hydrolysis with HCl is used to release the thiamin and thiamin phosphate esters from their associations with proteins, followed by enzyme hydrolysis of the phosphorylated thiamin to free thiamin using takadiastase, Mylase 100 ® (U.S. Biochemical Corp.), or α-amylase. The use of the same extraction procedure allows both thiamin and riboflavin in foods to be separated and quantitated by HPLC simultaneously. Microbiological analyses depend on the extent of growth of a thiamin-dependent organism such as Lactobacillus fermentum and Lactobacillus viridescens. L. fermentum is susceptible to matrix effects such as carbohydrates, fats, and some minerals in a growth medium [64], but L. viridescens is more specific for thiamin and not susceptible to matrix effects. Microbiological assay is still used in food analysis, but AOAC International [13] does not provide an official method for thiamin determination using microorganisms. HPLC methods have recently been developed to allow the rapid, sensitive, and specific analysis of thiamin and its phosphorylated forms in drugs and biological materials. Th iamin is measured with absorbance detection, usually at 245–254 nm, or with fluorescence detection systems after conversion to thiochrome. Although absorbance detection has sensitivity for high-thiamin containing foods, it may not be appropriate for seafood containing small amount of thiamin. Fluorescence detection is much more sensitive than absorbance detection. Therefore, HPLC with fluorescence detection has been widely used for the determination of thiamin in foods including muscle foods. Thiamin itself does not fluoresce, so thiamin should be converted to thiochrome using reagents for alkaline oxidation either by postcolumn or precolumn derivatization. The maximum fluorescence of thiochrome excitation is λ 365–375 nm and emission, λ 425–435 nm. Recently, Lebiedziñska et al. [8] reported the simultaneous determination of thiamin, vitamin B6, and vitamin B12 in several types of seafood using the HPLC method with electrochemical and UV detection. The mobile-phase pH should be kept above 8, because the fluorescence intensity of thiochrome is pH dependent and reaches a steady state at a pH above 8 [65]. Milligrams of thiamin are frequently used for expressing the content of thiamin in foods.
19.2.2
Riboflavin (Vitamin B2 )
Riboflavin, known as vitamin B2, is a water-soluble vitamin naturally found in foods. Riboflavin acts as an integral component of two coenzymes: flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Riboflavin occurs naturally in foods as free riboflavin and as the proteinbound coenzymes, FAD and FMN. Seafood is generally a modest source of riboflavin. Some species like mackerel and squid are good to excellent sources. Fish consumed whole (e.g., smelt and sardines) are also good sources of riboflavin. Riboflavin is stable to heat, acidic conditions, and oxidation if light is excluded. Riboflavin is destroyed by exposure to UV and visible light within the range of 420–560 nm. The rate of destruction is accelerated by increasing temperature and pH. Thus, riboflavin is generally stable during heat processing and normal cooking of foods if protected from light. Several methods have been proposed for the determination of riboflavin in foods, usually involving the conversion of FAD and FMN to free riboflavin. The fluorometric methods of AOAC Official Methods 970.65 and 981.15 [13], microbiological assays [66], and HPLC methodologies using fluorescence detection [61,67–69] are used for measuring total riboflavin in foods. HPLC can separate individual free riboflavin, FAD, and FMN in foods.
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To measure total riboflavin contents as free riboflavin in foods using these methods, acid hydrolysis with autoclaving is used to release the riboflavin from association with proteins and to convert FAD and FMN to free riboflavin. However, to complete the conversion of FMN to free riboflavin, enzyme hydrolysis is required with diastatic enzymes after acid hydrolysis [70]. Combined extractions for thiamin and riboflavin assays are usually used for food analysis [7,58,61]. AOAC Official Method 940.33 [13] is an approved microbiological method only for riboflavin in vitamin preparations. However, a microbiological assay applying Lactobacillus rhamnosus (formerly L. casei) has been used to determine the total riboflavin content in foods. Because lactic acid bacteria use riboflavin and FMN, but not FAD, an acid hydrolysis step is necessary to convert FAD and FMN to free riboflavin, but enzyme hydrolysis for completing the conversion of FMN to riboflavin is not required. L. rhamnosus is affected by starch and fatty acids. The matrix effect by starch in the media is eliminated by acid hydrolysis. Fatty acids stimulate or inhibit the growth of L. rhamnosus. Hence, for high-fat fish such as mackerel, the fat extraction step with petroleum ether or hexane should be conducted before acid hydrolysis. Reversed-phase HPLC with fluorescence detection is frequently used for chromatography of riboflavin assays in foods. Extraction procedures for total riboflavin analyses include acid and enzyme hydrolysis. Chromatography is capable of separating FMN and free riboflavin, and the concentrations of FMN and free riboflavin by HPLC are summed to obtain total riboflavin concentration, so, in this case, enzyme hydrolysis can be skipped. To quantify individual free riboflavin, FAD, and FMN in foods, Viñas et al. [69] used an extraction method using acetonitrile without acid hydrolysis. Solid-phase cleanup procedures are often used before injection to remove some of the interfering materials. UV detection with reversed-phase HPLC has been used for riboflavin analyses in foods at 254 nm [60]; however, fluorescence detection (excitation λ: 440–500 nm, emission λ: 520–530 nm) is more sensitive and specific for riboflavin quantitation than UV detection [7,60,61]. Riboflavin content in foods is commonly expressed as milligram of riboflavin.
19.2.3
Niacin (Vitamin B3 )
Niacin is one of the water-soluble B-vitamins known as vitamin B3. The term “niacin” is the generic descriptor for nicotinic acid and nicotinamide, which are essential for formation of the coenzymes, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in the body. Niacin can be biosynthesized from the amino acid tryptophan. Niacin contents in fish and seafood considerably vary depending on the variety of fish or seafood. Lean white fish and shellfish tend to contain smaller amounts of niacin, whereas some varieties such as mackerel, salmon, and tuna are rich in the vitamin. Nicotinic acid is found mainly in plant foods, but animal foods contain nicotinamide, which is bioavailable. In uncooked foods, niacin is present as NAD and NADP, but these nucleotides may be hydrolyzed to nicotinamide by cooking [71]. Niacin is not affected by thermal processing, light, oxygen, and pH. It is stable during processing, storage, and cooking of foods. Thus, acid or alkali hydrolysis can be used for extraction of niacin from food samples. Niacin in foods can be determined by the colorimetric methods of AOAC Official Methods 961.14 and 975.41 [13] using the König reaction in which nicotinic acid and nicotinamide react with cyanogen bromide and the aromatic amine, sulfanilic acid. Microbiological assays [72], including AOAC Official Method 985.34 [13], and HPLC methodologies [73–76] are also currently used for determining niacin in foods. Because niacin in animal-based foods is present in free forms (nicotinic acid and nicotinamide) and bound forms (NAD and NADP), hydrolysis procedures are required. Acid hydrolysis
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with HCl or H2SO4 or alkaline hydrolysis with NaOH or Ca(OH)2 is used as the initial step in niacin extraction procedures. Acid hydrolysis liberates nicotinamide from bound forms and hydrolyzes it to nicotinic acid; however, nicotinic acid, mainly distributed in cereal products and biologically unavailable niacin, is not completely liberated from bound forms by acid hydrolysis. Alkaline hydrolysis liberates most bound forms and provides a measure of total niacin in cereal products [77]. Microbiological assay is used for the determination of total niacin using Lactobacillus plantarum, which responds to nicotinic acid, nicotinamide, nicotinuric acid, and NAD. However, this method does not account for tryptophan. To determine total niacin concentration, AOAC Official Methods 944.13 and 985.34 [13] using L. plantarum are provided for vitamin preparations and for ready-to-feed milk-based infant formulas, respectively. Solve et al. [78] developed an automated microplate method with L. plantarum, which reduced time expenditure and materials compared with conventional microbiological procedures. HPLC determination of niacin has generally been carried out with ion-pairing or reversedphase chromatography with UV detection. Either acid or alkali hydrolysis is used to liberate free niacin from bound forms. Water or methanol is used to measure only free forms of nicotinic acid and nicotinamide in foods by HPLC [76,79]. Cleanup procedures like cartridge extractions and column switching are usually performed to improve selectivity and sensitivity by eliminating interfering materials before HPLC. Most studies have used UV absorbance detection of nicotinic acid and nicotinamide at 254 or 264 nm. Fluorescence detection (excitation λ: 322 nm, emission λ: 380 nm) may be used to increase specificity and sensitivity of HPLC. Niacin is not naturally fluorescent, but fluorescent derivatives can be formed using cyanogen bromide and ρ-aminophenol [80]. A postcolumn UV irradiation in the presence of hydrogen peroxide and copper(II) ions also induces fluorescence [72,81]. Lombardi-Boccia et al. [7] determined niacin, thiamin, and riboflavin together by using reversed-phase HPLC with a photodiode array detector after acid and enzyme hydrolysis. Niacin content in foods is commonly expressed as mg of niacin equivalents (NE). Sixty milligrams of dietary tryptophan is considered equivalent to 1 mg of niacin [82]. Thus, 1 mg NE is equal to 1 mg of niacin or 60 mg of dietary tryptophan.
19.2.4
Vitamin B6
Vitamin B6 is a water-soluble vitamin. Vitamin B6 consists of derivatives of 3-hydroxy-2methylpyridine, that is, pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their respective 5′-phosphates (PLP, PNP, and PMP). PLP is a metabolically active B6 vitamer. Vitamin B6 in free and bound forms is found in a wide variety of foods including seafood. Fish are good-to-rich sources of vitamin B6. PLP, bound to the apoenzyme by a Schiff base in animal tissues, is the major form of vitamin B6 in fish tissue, which is bioavailable. PMP is also found in seafood. Hence, PL, PLP, PM, and PMP are determined in seafood as a result of interconversion of aldehyde and amine forms during processing and storage. However, PN and PNP, found in plants, are not detected in animal foods. Vitamin B6 is unstable to light, both visible and UV. The vitamin is stable in acidic conditions if protected from light. The stability of vitamin B6 toward heat treatment, processing, and storage depends on the pH of the media. Losses of the vitamin increase as pH increases. PN is more stable to heat than PL and PM. Several different methodologies have been developed to analyze vitamin B6 in foods. These include animal growth, microbiological, enzymatic, fluorometric, GC, and HPLC assays [83]. Currently, microbiological assays and HPLC methods are often used in the vitamin B6 determination
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in foods. AOAC International provides both microbiological (Official Methods 961.15 and 985.32) and HPLC (Official Method 2004.07) methods to measure total vitamin B6 in ready-to-feed milk-based infant formula and reconstituted infant formula, respectively [13]. In addition, several HPLC methodologies have been reported for determining total vitamin B6 as well as individual B6 derivatives in foods [8,84–86]. Before being analyzed by microbiological, chromatographic, and other methods, food samples are usually extracted by acid hydrolysis with autoclaving in HCl or H2SO4 to dissociate vitamin B6 from proteins. The phosphate esters of PNP, PLP, and PMP are also hydrolyzed by this procedure. In the AOAC microbiological methods [13] for measuring total vitamin B6, sample foods are autoclaved with 0.055 N HCl for 5 h at 121°C. However, the AOAC liquid chromatographic method for total vitamin B6 uses enzymatic hydrolysis using acid phosphatase followed by a reaction with glyoxylic acid in the presence of a Fe2+ catalyst to transform PM into PL [13]. For separation of individual B6 vitamers, metaphosphoric acid, perchloric acid, trichloroacetic acid, and/or sulfosalicylic acid are used as deproteinating agents. Phosphorylated forms in foods are preserved by using these agents. The total vitamin B6 composition in foods is usually estimated microbiologically using a turbidimetric assay. Saccharomyces uvarum (formerly S. carlsbergensis) is the commonly used microorganism, which is also used in the AOAC method. Acid hydrolysis is necessary for determining total vitamin B6 because the microorganism uses only nonphosphorylated B6 vitamers. S. uvarum responds unequally to PL, PM, and PN. The growth response of S. uvarum to PL relative to that to PN is practically equal, but the response of the microorganism to PM is frequently 60%–80% of that to PL and PN [87]. The problem of differential response of S. uvarum can be overcome by separating PL, PM, and PN chromatographically and analyzing each form of the vitamin. Cationexchange chromatography on Dowex AG 50 W-X8 resin resolves the vitamers in the acid extractant and allows their individual quantitation [88,89]. Kloekera brevis (formerly K. apiculata) may be used for vitamin B6 assay. However, the results in responses of the organism to PL, PM, and PN are not consistent [83], and K. brevis has not seen wide usage in food analysis. Ion-exchange and reversed-phase HPLC with fluorescence detection has been used for quantitative determination of vitamin B6 in foods. HPLC has the ability to separate and quantitate PL, PM, PN, and their 5′-phosphate esters and also vitamin B6 metabolites such as 4-pyridoxic acid. HPLC provides high resolution and high sensitivity to the B6 vitamers. To determine total vitamin B6 by quantitating PL, PM, and PN, hydrolysis of the phosphate esters is usually completed with a commercial phosphatase or treatment with H2SO4. For preservation of the phosphorylated vitamers and metabolites, deproteinizing agents are used. Because of the native fluorescence of PL, PM, PN, and their 5′-phosphorylated derivatives and a relatively low UV detection sensitivity, most of the HPLC methods have used fluorescence detection. The intensity of fluorescence among the B6 vitamers is pH dependent. The B6 vitamers are suited to ion exchange because of their pHdependent ionic nature. In reversed-phase HPLC, sample components are separated according to their relative affinity for a nonpolar bounded stationary phase and a polar mobile phase [90]. HPLC with fluorescence detection has been recommended for quantitative determination of vitamin B6 in foods because individual vitamers can be determined [91,92], but microbiological assays are still used in total vitamin B6 measurements in foods. Milligrams of vitamin B6 are generally used for expressing the content of vitamin B6 in foods.
19.2.5
Folate
Folate is a generic term for a water-soluble vitamin and includes naturally occurring food folates and folic acid found in dietary supplements and used in food fortification. Folate can vary in
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structure by reduction of the pteridine moiety to dihydrofolic acid and tetrahydrofolic acid (THF). Folate exists predominantly as polyglutamyl forms of THF, which are biologically active folate coenzymes in the body. Seafood provides small amounts of folate. Major folate vitamers in animal tissues are polyglutamyl forms of THF, 5-methyl-THF, and 10-formyl-THF [93]. The composition of 5-formyl-THF in animal tissues is low, but heating can increase it by isomerization of 10-formyl-THF [94]. Folate is sensitive to heat, acids, oxidation, and light. Folate losses by food processing and storage are variable by food matrices, oxygen availability, heating times, and forms of folate in foods. Folate is quite stable in dry products if protected from light and oxygen, but folate losses are large in water. Reducing agents such as ascorbic acid increases folate retention, whereas metals like Fe2+ increase folate losses. Folic acid is generally more stable than naturally occurring folates. Methodologies for the determination of folates in foods include microbiological assay [95] including AOAC Official Method 2004.05 for total folates in cereals and cereal foods [13], HPLC with UV, fluorescence, or electrochemical detection [93,96–98], and HPLC or GC with mass spectrometric (MS) methods [99–101] using stable isotopes. Microbiological assay for total folate determination is still the most widely used procedure. HPLC methods allow measurement of each form of folates. The traditional food folate extraction method includes heat treatment to release folate from its binding proteins and folate conjugase treatment to hydrolyze polyglutamyl folate to di- or monoglutamyl folate. Insufficient enzymatic deconjugation may result in underestimation of folate when measured by either microbiological or HPLC methods using single-enzyme digestion. Several studies have reported that treatment of food homogenates with α-amylase, protease, and folate conjugase (trienzyme extraction) enhances the yield of measurable folate in folate assays [102–104]. The use of α-amylase and protease allows for a more complete extraction of folate trapped in carbohydrates or proteins in foods. This trienzyme extraction method has become widely used in the extraction of folate from food samples. Antioxidants, such as ascorbic acid, 2-mercaptoethanol, and dithiothreitol, should be added to prevent the destruction of labile folates during heat treatment. For routine food analysis purposes, microbiological assay with L. rhamnosus (formerly L. casei) after extraction with folate conjugase is used for determination of folate. L. rhamnosus has greater capacity for response to the γ-glutamyl folate polymers compared with the other assay organisms. Although L. rhamnosus is the commonly used and accepted organism for folate analysis in foods, its ability to respond on a equimolar basis to metabolically active folates is controversial [105]. Chicken pancreas conjugase is used to hydrolyze polyglutamates to diglutamyl and monoglutamyl folates, which are used by L. rhamnosus. AOAC Official Method 2004.05 [13] is the microbiological method using L. rhamnosus after extracting samples by the trienzyme procedure. The method can determine turbidity semiautomatically by using 96-well microtiter plates and autoplate readers [106]. The major advantage of HPLC analysis is the ability to quantify the specific folate forms. Current HPLC systems for separating folates use either ion-pair or reversed-phase chromatography with UV, fluorometric, or electrochemical detection. Trienzyme extraction procedures are commonly used for HPLC analysis of food folates. Because HPLC systems for determining folate are able to detect only monoglutamates, human or rat plasma conjugase, not chicken pancreas conjugase, is used to deconjugate polyglutamyl folates to monoglutamyl folates. To remove interfering substances in extracts, purification is recommended with affinity chromatography, using immobilized folate-binding protein or solid-phase extraction using silica-based strong anion-exchange cartridges. Due to its sensitivity and selectivity, fluorescence detection is most commonly used,
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particularly for reduced folate forms. UV detection is useful in detecting the folic acid found in fortified foods but not for naturally occurring folates due to a lack of sensitivity. UV spectra and fluorescence excitation and emission spectra for the different forms of folates have been published by Ball [107]. Electrochemical detection has sensitivity for 5-methyl-THF; however, it has not been widely used in food analysis. A mass spectrometer with an HPLC system using stable isotope-labeled analyte standards has been used for folate detection to improve sensitivity and selectivity. The folate content of foods is expressed in either milligram or microgram of naturally occurring folate and fortified folic acid in the foods or dietary folate equivalents (DFE). Micrograms of DFE are calculated based on microgram of food folate plus fortified folic acid multiplied by the factor 1.7 [108].
19.2.6
Vitamin B12
Vitamin B12 is a water-soluble vitamin and a family of compounds called cobalamins, which contain cyanocobalamin, hydroxocobalamin, and the two coenzyme forms 5′-deoxyadenosylcobalamin (adenosylcobalamin) and methylcobalamin. Cyanocobalamin and hydroxocobalamin are the forms of vitamin B12 used in most dietary supplements and are converted to adenosylcobalamin and methylcobalamin in the body. Vitamin B12 found in nature appears to be from synthesis by bacteria and other microorganisms growing in soil and water. Fish and shellfish are rich sources of vitamin B12, with salmons, herrings, sardines, mackerels, oysters, and clams at the top of the list. The most prevalent forms of vitamin B12 in fish and seafood are adenosylcobalamin, hydroxocobalamin, and methylcobalamin. Sulfitocobalamin is found in canned fish. Vitamin B12 is generally stable if protected from light. Cobalamins are considered to be stable to thermal processing, but large losses of the vitamin occur by leaching into the cooking water. Cyanocobalamin is the most stable form of vitamin B12. Strong alkaline and acid conditions, intense visible light, and oxidizing agents inactivate the vitamin. The determination of total vitamin B12 may be performed by microbiological assays, including AOAC Official Methods 952.30 and 986.23 [13], radioisotope dilution methods [109,110], and HPLC methods [111,112]. Microbiological assay is most widely used for the determination of total vitamin B12 in foods. Radioassay kits for clinical samples are not useful for analysis of food samples. Radioisotope dilution methods lack selectivity as the intrinsic factor used for the assay could also bind other cobalamins or analogues [113]. Currently, these methods are not routinely used for vitamin B12 analysis of foods. The HPLC method lacks the sensitivity to measure vitamin B12 in nonfortified food products. The extraction procedures of the AOAC microbiological methods [13] are usually used for determining total vitamin B12 content in foods. Extraction procedures liberate cobalamins from protein and convert the labile naturally occurring forms to a single, stable form, which are cyanocobalamin or sulfitocobalamin [114]. The extraction is completed by homogenizing the sample in the extraction solution, autoclaving the sample at 121°C for 10 min. To protect cobalamins in samples, metabisulfite or ascorbic acid is added to the extracting solutions. Lactobacillus delbrueckii subsp. lactis (Lactobacillus leichmannii) is frequently used for determination of vitamin B12 in foods. L. delbrueckii has a similar response to nitritocobalamin, hydroxocobalamin, dicyanocobalamin, and sulfitocobalamin. However, adenosylcobalamin produces a greater response and methylcobalamin, a lesser growth response. If the sample extracts are exposed to light before analysis, adenosylcobalamin and methylcobalamin are completely converted to hydroxocobalamin, so that vitamin B12 activity can be measured accurately [115]. L. delbrueckii
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responds to deoxyribonucleosides. Treatment of the sample with alkali and heat destroys the vitamin cobalamins, leaving the deoxyribonucleosides intact; thus, the activity attributable to deoxyribonucleosides can be determined. Dilution of deoxyriboside concentrations to less than the 1 μg/mL of assay solution can also eliminate the effect [116]. The vitamin B12 concentrations in foods are expressed in milligram or microgram of vitamin B12.
19.2.7
Pantothenic Acid
Pantothenic acid, also known as vitamin B5, occurs primarily bound as part of coenzyme A and acyl-carrier proteins. Pantothenic acid is found in plant- and animal-derived foods because of its diverse metabolic functions as a structural component of coenzyme A. Fish and seafood contain a moderate amount of pantothenic acid. Pantothenic acid is stable to atmospheric oxygen and light, whereas large losses of the vitamin can occur in the blanching and boiling of foods. The stability of pantothenic acid is highly pH dependent. The vitamin is stable in slightly acidic solutions at pH 5–7. Microbiological assay [117] is most commonly used for determining pantothenic acid in foods. The microbiological assay has been accepted by AOAC (Official Methods 945.74 and 992.07) [13] for quantification of pantothenic acid in vitamin preparations and milk-based infant formula, respectively. Other methodologies for determination of the vitamin in foods include radiometric microbiological assay [118], radioimmunoassay [119,120], enzyme-linked immunosorbent assay [121], optical biosensor inhibition immunoassay [122], capillary electrophoresis [123], GC-MS using stable isotope dilution [124], and HPLC methods [125–127]. Methods for the pantothenic acid determination in foods vary widely in approach, because they remain limited by their low sensitivity and poor selectivity. In addition, applications of the analytic methods have several drawbacks including being time consuming, use of radioisotopes and scintillation counting, and sample derivatization procedures. The microbiological assay is time-consuming and lacks specificity [128], but determination of pantothenic acid in foods has most frequently been accomplished by this type of assay. The commonly used microorganism is L. plantarum. The organism does not respond to phosphopantetheine or intact forms of coenzyme A. Due to instability of the vitamin in acid and alkaline conditions, enzyme hydrolysis should be used to obtain free pantothenic acid and pantetheine. Intestinal phosphatase to cleave the phosphate linkage and avian liver peptidase to break the linkage between mercaptoethylamine and pantothenic acid are used in the enzyme hydrolysis, which is also used in the AOAC microbiological method for milk-based infant formulas [13]. Fatty acids in foods stimulate the growth of L. plantarum; therefore, a fat extraction step may be necessary before enzyme hydrolysis in fat-containing foods [117]. The pantothenic acid content of foods is commonly expressed as milligram of pantothenic acid.
19.2.8
Biotin
Biotin is a water-soluble B-vitamin, which contains sulfur. The biotin molecule contains three asymmetric carbon atoms, and therefore eight different isomers are possible. Of these isomers, only the dextrorotatory (+) d-biotin possesses biotin activity as a coenzyme. Biotin is widely distributed in many foods, but its concentrations are relatively low compared with that of other water-soluble vitamins. Liver contains considerable amounts of biotin. Most of biotin in animal products is in a protein-bound form. Biotin is generally stable to heat, but it is gradually destroyed
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by UV light. In strong acidic and alkaline solutions, biotin is unstable to heating. The sulfur atom in biotin is susceptible to oxidation with formation of biotin sulfoxide and biotin sulfone during food processing, which leads to loss of biotin activity. Microbiological assays [129], as well as protein-binding assays [130,131], and a biosensorbased immunoassay [132] have been developed for the determination of biotin content of foods. HPLC methods [133–135] have also been used for quantification of biotin and biocytin in foods. Currently an AOAC Official Method for biotin determination does not exist [13]. The most widely used method for determination of biotin in foods is a microbiological assay using L. plantarum. The microorganism, which requires biotin for growth and reproduction, is incubated with diluted sample extracts. The resulting increased turbidity of the extract is measured and correlated with the biotin content of the sample. The microorganism cannot use bound forms of biotin including biocytin, thus acid hydrolysis with autoclaving is required to liberate biotin completely from food samples. Although L. plantarum is more specific for biotin-active forms than other biotin-requiring organisms, L. plantarum responds to dethiobiotin, which spares biotin and can thus overestimate biotin content [136]. HPLC can separate free biotin, biotin sulfoxides, biotin sulfones, and other biotin analogs. Extractions with acid have commonly been used for foods to ensure liberation of bound biotin forms. The biotin molecule does not have enough UV absorbance and native fluorescence; thus UV and fluorescence detection are not useful. However, avidin can be labeled with a fluorescent marker and the complex used as a postcolumn derivatizing agent. The fluorescence of the labeled protein is enhanced on binding of its specific ligands, biotin, and biocytin. HPLC methods with avidin-binding detection, formation of fluorescent derivatives, or usage of other detection systems, including MS and electrochemical detection, are ways to increase the sensitivity of the detection after HPLC separation. Analytical results are expressed in microgram of biotin.
19.2.9
Vitamin C
Vitamin C is a water-soluble vitamin and occurs in two forms, the reduced ascorbic acid and the oxidized dehydroascorbic acid. Ascorbic acid is reversibly oxidized to dehydroascorbic acid. Further oxidations convert dehydroascorbic acid to the inactive and irreversible compound diketoglutamic acid. There are two enantiomeric pairs, l- and d-ascorbic acid and l- and d-isoascorbic acid. l-Ascorbic acid and d-isoascorbic acid (known as d-araboascorbic acid and erythorbic acid) have the biological activity of vitamin C. d-Isoascorbic acid has 1/20 the activity of l-ascorbic acid [137]. l-Ascorbic acid and dehydroascorbic acid are naturally occurring forms of vitamin C, but d-isoascorbic acid is not found in foods. d-Isoascorbic acid is synthesized commercially and used as an antioxidant in foods, usually processed and canned fish. Fish and seafood are not considered good sources of vitamin C. Vitamin C is very susceptible to oxidation during the processing, storage, and cooking of foods, especially under alkaline conditions. Maximal stability occurs between pH 4 and 6; however, degradation rates are influenced by oxygen availability, the presence of antioxidants, thermal processing conditions, transition metal catalysis, oxidizing lipid effects, and the presence of ascorbic acid oxidase [138]. Methodologies for determination of total vitamin C in foods include the titrimetric method using oxidation–reduction indicators [13], the fluorometric method including derivatization procedures [13], enzymatic methods [139], and electrochemical procedures [140]. Total vitamin C content is also usually determined by HPLC using UV, fluorescence, or electrochemical detection [141,142]. Only HPLC procedures simultaneously separate l-ascorbic acid, dehydroascorbic acid, and d-isoascorbic acid in foods [143–146]. The titrimetric method employed in AOAC Official
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Methods 967.21 and 985.33 [13] uses 2,6-dichloroindophenol in measuring ascorbic acid, not dehydroascorbic acid. This method cannot distinguish between l-ascorbic acid and d-isoascorbic acid; thus, the titrimetric method cannot be used for processed food products containing d-isoascorbic acid. The fluorometric method of AOAC Official Methods 967.22 and 984.26 [13] determines total vitamin C content in vitamin preparations and foods, respectively, by derivatization of dehydroascorbic acid using the o-phenylenediamine condensation reaction. Because vitamin C is destroyed easily, extraction procedures should be conducted to stabilize the vitamin. The choice of extracting solutions is dependent on the sample matrix and determination method, but the solutions should maintain an acidic environment, chelate metals, inactivate ascorbic acid oxidase, and precipitate proteins and starches [138]. The extracting solution is usually 3%–6% metaphosphoric acid dissolved in 8% glacial acetic acid or ethylenediaminetetraacetic acid (EDTA). Metaphosphoric acid prevents metal catalysis and activation of ascorbic acid oxidase and precipitates proteins. EDTA also chelates metals, and acetic acid precipitates starches in extractants. HPLC methodologies are widely used for determining ascorbic acid and its degradation products in foods. Reversed-phase chromatography with and without ion suppression, ion-pair reversed-phase chromatography with C18 columns, and ion-exchange chromatography are currently employed for analysis of vitamin C. UV, electrochemical, and fluorescence detection are commonly used for quantitation of l-ascorbic acid, dehydroascorbic acid, and d-isoascorbic acid in foods. UV detection is not suitable for low vitamin C content foods due to poor sensitivity of dehydroascorbic acid. Dehydroascorbic acid is electrochemically inactive; therefore, to measure total vitamin C contents, it is reduced to l-ascorbic acid before electrochemical detection. Fluorescence detection is accomplished after o-phenylenediamine derivatization using pre- or postcolumns. Both electrochemical and fluorescence detection have excellent selectivity and sensitivity to vitamin C analysis by HPLC. The vitamin C contents of foods are commonly expressed as milligram of vitamin C.
19.3
Summary
Vitamins are classified according to their solubility in fat organic solvents (fat-soluble vitamins) or water (water-soluble vitamins). The solubility properties are related to the distribution of vitamins in foods as well as the analytical methods employed. Fat-soluble vitamins A, D, and E are contained in fish and seafood in varying amounts. Fatty species of fish have high concentrations of fat-soluble vitamins. Fish and seafood are good to excellent dietary sources of most of the B vitamins. Vitamins are generally susceptible to oxidation, heat, pH, moisture, light, degradative enzymes, and metal trace elements. Thus, processing, storage, preparation, and cooking methods can affect the concentrations of vitamins in seafood. To liberate vitamins bound in lipid or protein fractions, food samples may need to be hydrolyzed using acids, alkalines, and/or enzymes or extracted directly with solvents without hydrolysis. The extract solutions may require some forms of cleanup before the vitamins are measured to remove interfering substances and to improve the sensitivity and selectivity of the analytical methods. Antioxidants such as BHT, pyrogallol, or ascorbic acid are frequently added in extraction solvents to prevent oxidation and conversion of vitamins. Most of the vitamins are liable to light, and, therefore, food samples must be protected from light during the entire analysis. Various methodologies, including colorimetric, fluorometric, titrimetric, and spectrophotometric methods, have been developed and used for determining the vitamins in foods. However,
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microbiological and HPLC methods are most frequently used in estimating the vitamins in foods, because these methods have sufficient sensitivity and selectivity to quantitate low concentrations of naturally occurring vitamins. Microbiological assay can be applied to all the B vitamins. Lactic acid bacteria are suitable for determining the B-vitamins turbidimetrically, except for vitamin B6, which may be determined using yeasts. Fat-soluble vitamins and vitamin C are most commonly determined by HPLC. HPLC methods can also be used for estimating the B vitamins. HPLC distinguishes between naturally occurring and added (fortified or enriched) vitamins and also separates the individual forms of vitamins. Some vitamins, with low UV absorbance or fluorescence responses, can be determined after conversion to fluorescent derivatives using pre- or postcolumn derivatization. Bio-specific methods for determining some of the water-soluble vitamins include immunoassays and protein-binding assays. Newer techniques continue to be developed for quantitating the concentrations of the various vitamins in all types of foods, including fish and seafood.
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15. Hwei, M.W. and Min, H.L., Identification of beta-carotene and astaxanthin in squid visceral oil, J. Clin. Agric. Chem. Soc., 31, 431, 1994. 16. López-Cervantes, J., Sánchez-Machado, D.I., and Ríos-Vázquiez, N.J., High-performance liquid chromatography method for the simultaneous quantification of retinol, α-tocopherol, and cholesterol in shrimp waste hydrolysate, J. Chromatogr. A, 1105, 135, 2006. 17. Kimura, M., Rodriguez-Amaya, D.B., and Godoy, H.T., Assessment of the saponification step in the quantitative determination of carotenoids and provitamin A, Food Chem., 35, 187, 1990. 18. Sander, L.C., Sharpless, K.E., Craft, N.E., and Wise, S.A., Development of engineered stationary phases for the separation of carotenoid isomers, Anal. Chem., 66, 1667, 1994. 19. Emenhiser, C., Simunovic, N., Sander, L.C., and Schwartz, S.J., Separation of geometrical carotenoid isomers in biological extracts using a polymeric C30 column in reversed-phase liquid chromatography. J. Agric. Food Chem., 44, 3887, 1996. 20. Eitenmiller, R., Ye, L., and Landen, Jr. W.O., Vitamin Analysis for the Health and Food Sciences, 2nd Edn., CRC Press, Boca Raton, FL, 2008, Chapter 1. 21. Institute of Medicine, Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc, National Academy Press, Washington, D.C., 2001, Chapter 4. 22. Institute of Medicine, Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride, National Academy Press, Washington, D.C., 1997, Chapter 7. 23. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 4. 24. Holden, J.M., Lemar, L.E., and Exler, J., Vitamin D in foods: Development of the U.S. Department of Agriculture database, Am. J. Clin. Nutr., 87(suppl), 1092S, 2008. 25. Kenny, D.E., O’Hara, T.M., Chen, T.C., Lu, Z., Tian, X., and Holick, M.F., Vitamin D content in Alaska Arctic zooplankton, fishes, and marine mammals, Zoo Biol., 23, 33, 2004. 26. Mattila, P., Piironen, V., Uusi-Rauva, E., and Koivistoinen, P., Cholecalciferol and 25-hydroxycholecalciferol content in fish and fish products, J. Food Compos. Anal., 8, 232, 1995. 27. Purchas, R., Zou, M., Pearce, P., and Jackson, F., Concentrations of vitamin D3 and 25-hydroxyvitamin D3 in raw and cooked New Zealand beef and lamb, J. Food Compos. Anal., 20, 90, 2007. 28. Jakobsen, J., Clausen, I., Leth, T., and Ovensen, L., A new method for the determination of vitamin D3 and 25-hydroxyvitamin D3 in meat, J. Food Compos. Anal., 17, 777, 2004. 29. Clausen, I., Jakobsen, J., Leth, T., and Ovesen, L., Vitamin D3 and 25-hydroxyvitamin D3 in raw and cooked pork cuts, J. Food Compos. Anal., 16, 575, 2003. 30. Mattila, P., Piironen, V., Bäckman, C., Asunmaa, A., Uusi-Rauva, E., and Koivistoinen, P., Determination of vitamin D3 in egg yolk by high-performance liquid chromatography with diode array detection, J. Food Compos. Anal., 5, 281, 1992. 31. Bui, M.H., Sample preparation and liquid chromatographic determination of vitamin D in food products, J. Assoc. Off. Anal. Chem., 70, 802, 1987. 32. Thompson, J.N. and Plouffe, L., Determination of cholecalciferol in meat and fat from livestock fed normal and excessive quantities of vitamin D, Food Chem., 46, 313, 1993. 33. Sliva, M.G., Green, A.E., Sanders, J.K., Euber, J.R., and Saucerman, J.R., Reversed-phase liquid chromatographic determination of vitamin D in infant formulas and enteral nutritionals, J. AOAC Int., 75, 566, 1992. 34. Nettleton, J., Seafood Nutrition: Facts, Issues and Marketing of Nutrition in Fish and Shellfish, Osprey Books, Huntington, NY, 1985, Chapter 2. 35. Pigott, G.M. and Tucker, B.W., Seafood: Eff ects of Technology on Nutrition, Marcel Dekker, New York, 1990, Chapter 2. 36. Driskell, J.A., Marchello, M.J., Giraud, D.W., and Sulaeman, A., Vitamin and selenium content of ribeye cuts from grass- and grain-finished bison of the same herd, J. Food Qual., 27, 3, 2004. 37. Bosco, A.D., Castellini, C., and Bernardini, M., Nutritional quality of rabbit meat as affected by cooking procedure and dietary vitamin E, J. Food Sci., 66, 1047, 2001.
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38. Chun, J., Lee, J., Ye, L., Exler, J., and Eitenmiller, R.R., Tocopherol and tocotrienol contents of raw and processed fruits and vegetables in the United States diet. J. Food Compos. Anal., 19, 196, 2006. 39. Eitenmiller, R., Ye, L., and Landen, Jr. W.O., Vitamin Analysis for the Health and Food Sciences, 2nd Edn., CRC Press, Boca Raton, FL, 2008, Chapter 3. 40. Bonvehi, J.S., Coll, F.V., and Rius, I.A., Liquid chromatographic determinations of tocopherols and tocotrienols in vegetable oils, formulated preparations, and biscuits, J. AOAC Int., 83, 627, 2000. 41. Thompson, J.N. and Hatina, G., Determination of tocopherols and tocotrienols in foods and tissues by high-performance liquid chromatography, J. Liq. Chromatogr., 2, 327, 1979. 42. Nelis, H.J., D’Haese, E., and Vermis, K., Vitamin E, in Modern Chromatographic Analysis of Vitamins, 3rd Edn., De Leenheer, A.P., Lambert, W.E., and van Bocxlaer, J.F., Eds., Marcel Dekker, New York, 2000, Chapter 2. 43. National Research Council, Recommended Dietary Allowance, 10th Edn., National Academy of Sciences, Washington, D.C., 1989, p. 78. 44. Institute of Medicine, Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, National Academy Press, Washington, D.C., 2000, Chapter 6. 45. Ferland, G., Vitamin K, in Present Knowledge in Nutrition, 9th Edn., Bowman, B.A., and Russell, R.M., Eds., ILSI Press, Washington, D.C., 2006, Chapter 16. 46. Thijssen, H. and Drittij-Reijnders, J., Vitamin K distribution in rat tissues; Dietary phylloquinone is a source of tissue menaquinone-4, Br. J. Nutr., 72, 415, 1994. 47. Davidson, R.T., Foley, A.L., Engelke, J.A., and Suttie, J.W., Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria, J. Nutr., 12, 220, 1998. 48. Suttie, J., The importance of menaquinones in human nutrition, Ann. Rev. Nutr., 15, 399, 1994. 49. Eitenmiller, R., Ye, L., and Landen, Jr. W.O., Vitamin Analysis for the Health and Food Sciences, 2nd Edn., CRC Press, Boca Raton, FL, 2008, Chapter 4. 50. Koivu-Tikkanen, T.J., Ollilainen, V., and Piironen, V., Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc, J. Agric. Food Chem., 48, 6325, 2000. 51. Elder, S.J., Haytowitz, D.B., Howe, J., Peterson, J.W., and Booth, S.L., Vitamin K contents of meat, dairy, and fast food in the U.S. diet, J. Agric. Food Chem., 54, 463, 2006. 52. Weizmann, N., Peterson, J.W., Haytowitz, D., Pehrsson, P.R., de Jesus, V.P., and Booth, S.L., Vitamin K content of fast foods and snack foods in the U.S. diet, J. Food Compos. Anal., 17, 379, 2004. 53. Booth, S.L., Sadowski, J.A., and Pennington, A.T., Phylloquinone (vitamin K1) content of foods in the U.S. Food and Drug Administration’s Total Diet Study, J. Agric. Food Chem., 43, 1574, 1995. 54. Bates, C.J., Thiamin, in Present Knowledge in Nutrition, 9th Edn., Bowman, B.A., and Russell, R.M., Eds., ILSI Press, Washington, D.C., 2006, Chapter 18. 55. Eitenmiller, R., Ye, L., and Landen, Jr. W.O., Vitamin Analysis for the Health and Food Sciences, 2nd Edn., CRC Press, Boca Raton, FL, 2008, Chapter 6. 56. Defibaugh, P.W., Smith, J.S., and Weeks, C.E., Assay of thiamin in foods using manual and semiautomated fluorometric and microbiological methods, J. Assoc. Off. Anal. Chem., 60, 552, 1977. 57. Bui, M.H., A microbiological assay on microtitre plates of thiamine in biological fluids and foods, Int. J. Vitam. Nutr. Res., 69, 362, 1999. 58. Ollilainen, V., Finglas, P.M., van den Berg, H., and de Froidmont-Görtz, I., Certification of B-group vitamins (B1, B2, B6, and B12) in four food reference materials, J. Agric. Food Chem., 49, 315, 2001. 59. Fellman, J.K., Artz, W.E., Tassinari, P.D., Cole, C.L., and Augustin, J., Simultaneous determination of thiamin and riboflavin in selected foods by high-performance liquid chromatography, J. Food Sci., 47, 2048, 1982. 60. Barna, É. and Dworschák, E., Determination of thiamine (vitamin B1) and riboflavin (vitamin B2) in meat and liver by high-performance liquid chromatography, J. Chromatogr., 668, 359, 1994. 61. Tang, X., Cronin, D.A., and Brunton, N.P., A simplified approach to the determination of thiamine and riboflavin in meats using reverse phase HPLC, J. Food Compos. Anal., 19, 831, 2006.
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62. Echols, R.E., Miler, R.H., Winzer, W., Carmen, D.J., and Ireland, Y.R., Gas chromatographic determination of thiamine in meats, vegetables and cereals with a nitrogen-phosphorus detector, J. Chromatogr., 262, 257, 1983. 63. Echols, R.E., Miller, R.H., and Foster, W., Analysis of thiamine in milk by gas chromatography and nitrogen-phosphorus detector, J. Dairy Sci., 69, 1246, 1986. 64. Voigt, M.N. and Eitenmiller, R.R., Comparative review of the thiochrome, microbial and protozoan analysis of B-vitamins, J. Food Prot., 41, 730, 1978. 65. Kawasaki, T. and Egi, Y., Thiamine, in Modern Chromatographic Analysis of Vitamins, 3rd Edn., De Leenheer, A.P., Lambert, W.E., and van Bocxlaer, J.F., Eds., Marcel Dekker, New York, 2000, Chapter 8. 66. Kornberg, H.A., Langdon, R.S., and Cheldelin, V.H., Microbiological assay for riboflavin, Anal. Chem., 20, 81, 1948. 67. Andrés-Lacueva, C., Mattivi, F., and Tonon, D., Determination of riboflavin, flavin mononucleotide and flavin-adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection, J. Chromatogr. A, 823, 355, 1998. 68. Russell, L.F., and Vanderslice, J.T., Non-degradative extraction and simultaneous quantitation of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in foods by HPLC, Food Chem., 43, 151, 1992. 69. Viñas, P., Balsalobre, N., López-Erroz, C., and Hernández-Córdoba, M., Liquid chromatographic analysis of riboflavin vitamers in food using fluorescence detection, J. Agric. Food Chem., 52, 1789, 2004. 70. Nielsen, P., Rauschenbach, P., and Bacher, A., Preparation, properties, and separation by highperformance liquid chromatography of riboflavin phosphates, Methods Enzymol., 122, 209, 1986. 71. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 9. 72. Rose-Sallin, C., Blake, C.J., Genoud, D., and Tagliaferri, E.G., Comparison of microbiological and HPLC-fluorescence detection methods for determination of niacin in fortified food products, Food Chem., 73, 473, 2001. 73. Tyler, T.A. and Genzale, J.A., Liquid chromatographic determination of total niacin in beef, semolina and cottage cheese, J. Assoc. Off. Anal. Chem., 73, 467, 1990. 74. Ward., C.M. and Trenerry, V.C., The determination of niacin in cereals, meat and selected foods by capillary electrophoresis and high performance liquid chromatography, Food Chem., 60, 667, 1997. 75. Vidal-Valverde, C. and Reche, A., Determination of available niacin in legumes and meat by high performance liquid chromatography, J. Agric. Food Chem., 39, 116, 1991. 76. Hamano, T., Mitsuhashi, Y., Aoki, N., Yamamoto, S., and Oji, Y., Simultaneous determination of niacin and niacinamide in meats by high performance liquid chromatography, J. Chromatogr., 457, 403, 1988. 77. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 16. 78. Solve, M., Eriksen, H., and Brogren, C.H., Automated microbiological assay for quantitation of niacin performed in culture microplates read by digital image processing, Food Chem., 49, 419, 1994. 79. Takatsuki, K., Suzuki, S., Sato, M., Sakai, K., and Ushizawa, I., Liquid chromatographic determination of free and added niacin and niacinamide in beef and pork, J. Assoc. Anal. Chem., 70, 69, 1987. 80. Krishnan, P.G., Mahmud, I., and Matthees, D.P., Postcolumn fluorimetric HPLC procedure for determination of niacin content of cereal, Cereal Chem., 76, 512, 1999. 81. Lahél, S., Bergaentzlé, M., and Hasselmann, C., Fluorimetric determination of niacin in foods by high-performance liquid chromatography with post-column derivatization, Food Chem., 65, 129, 1999. 82. Horwitt, M.K., Harper, A.E., and Henderson, L.M., Niacin-tryptophan relationships for evaluating niacin equivalents, Am. J. Clin. Nutr., 34, 423, 1981.
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83. Driskell, J.A., Vitamin B6, in Encyclopedia of Food Sciences and Nutrition, 2nd Edn., Caballero, B.L., Trugo, L., and Ginglas, P.M., Eds., Elsevier, Kent, U.K., 2003, p. 6012. 84. Valls, F., Sancho, M.T., Fernández-Muiño, M.A., and Checa, M.A., Determination of vitamin B6 in cooked sausages, J. Agric. Food Chem., 49, 38, 2001. 85. Esteve, M.J., Farré, R., Frígola, A., and Carcía-Cantabella, J.M., Determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in pork meat and pork meat products by liquid chromatography, J. Chromatogr. A, 795, 383, 1998. 86. van Schoonhoven, J., Schrijver, J., van den Berg, H., and Haenen, G.R.M.M., Reliable and sensitive high-performance liquid chromatographic method with fluorometric detection for the analysis of vitamin B6 in foods and feeds, J. Agric. Food Chem., 42, 1475, 1994. 87. Gregory, J.F., III, Relative activity of the nonphosphorylated B-6 vitamers for Saccharomyces uvarum and Kloeckera brevis in vitamin B-6 microbiological assay, J. Nutr., 112, 1643, 1982. 88. Toepfer, E.W. and Polansky, M.M., Microbiological assay of vitamin B-6 and its components, J. Assoc. Off. Anal. Chem., 53, 546, 1970. 89. Toepfer, E.W. and Lehmann, J., Procedure for chromatographic separation and microbiological assay of pyridoxine, pyridoxal, and pyridoxamine in food extracts, J. Assoc. Off. Anal. Chem., 44, 426, 1961. 90. Gregory, J.F. III, Methods for determination of vitamin B6 in foods and other biological materials: A critical review, J. Food Compos. Anal., 1, 105, 1988. 91. Gregory, J.F. III and Feldstein, D., Determination of vitamin B6 in foods and other biological materials by paired ion high performance liquid chromatography, J. Agric. Food Chem., 33, 359, 1985. 92. Morrison, L.A. and Driskell, J.A., Quantities of B6 vitamers in human milk by high-performance liquid chromatography: Influence of maternal vitamin B6 status. J. Chromatogr. Biomed. Appl., 337, 249, 1985. 93. Vahteristo, L.T., Ollilainen, V., and Varo, P., Liquid chromatographic determination of folate monoglutamates in fish, meat, egg, and dairy products, J. AOAC Int., 80, 373, 1997. 94. Gregory, J.F. III, Chemical and nutritional aspects of folate research: Analytical procedures, methods of folate synthesis, stability, and bioavailability of dietary folate, Adv. Food Nutr. Res., 33, 1, 1989. 95. Hyun, T.H. and Tamura, T., Trienzyme extraction in combination with microbiologic assay in food folate analysis: An updated review, Exp. Biol. Med., 230, 444, 2005. 96. Vahteristo, L.T., Ollilainen, V., and Varo, P., HPLC determination of folate in liver and liver products, J. Food Sci., 61, 524, 1996. 97. Póo-Prieto, R., Haytowitz, D.B., Holden, J.M., Rogers, G., Choumenkovitch, S.F., Jacques, P.F., and Selhub, J., Use of the affinity/HPLC method for quantitative estimation of folic acid in enriched cereal-grain products, J. Nutr., 136, 3079, 2006. 98. Konings, E.J.M., Roomans, H.H.S., Dorant, E., Goldbohm, R.A., Saris, W.H.M., and van den Brandt, P.A., Folate intake of the Dutch population according to newly established liquid chromatography data for foods, Am. J. Clin. Nutr., 73, 765, 2001. 99. Rychlik, M. and Freisleben, A., Quantification of pantothenic acid and folates by stable isotope dilution assays, J. Food Compos. Anal., 15, 399, 2002. 100. Stokes, P. and Webb, K., Analysis of some folate monoglutamates by high-perfomance liquid chromatography-mass spectrometry, J. Chromatogr., 864, 59, 1999. 101. Santhosh-Kumar, C.R. and Kolhouse, N.M., Molar quantitation of folates by gas chromatographymass spectrometry, Methods Enzymol., 281, 26, 1997. 102. Tamura, T., Mizuno, Y., Johnston, K.E., and Jacob, R.A., Food folate assay with protease, α-amylase, and folate conjugase treatments, J. Agric. Food Chem., 45, 135, 1997. 103. Johnston, K.E., DiRienzo, D.B., and Tamura, T., Folate concentrations of dairy products measured by microbiological assay with trienzyme treatment, J. Food Sci., 67, 17, 2001. 104. Yon, M. and Hyun, T.H., Folate content of foods commonly consumed in Korea measured after trienzyme extraction, Nutr. Res., 23, 735, 2003. 105. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 18.
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106. Tamura, T., Microbiological assay of folates, in Folic Acid Metabolism in Health and Diseases, Contemporary Issues in Clinical Nutrition, vol. 13, Picciano, M.F., Stockstad, E.L., and Gregory, J.F., III, Eds., Wiley-Liss, New York, 1990, p. 121. 107. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 21. 108. Institute of Medicine, Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6 , Folate, Vitamin B12, Pantothenic acid, Biotin, and Choline, National Academy Press, Washington, D.C., 2000, Chapter 8. 109. Casey, P.J., Speckman, K.R., Ebert, F.J., and Hobbs, W.E., Radioisotope dilution technique for determination of vitamin B12 in foods, J. Assoc. Off. Anal. Chem., 65, 85, 1982. 110. Osterdahl, B.G. and Johanosson, E., Radioisotope dilution determination of vitamin B12 in dietary supplements, Int. J. Vitam. Nutr. Res., 58, 300, 1988. 111. Heudi, O., Kilinç, T., Fontannaz, P., and Marley, E., Determination of vitamin B12 in food products and in premixes by reversed-phase high performance liquid chromatography and immunoaffinity extraction, J. Chromatogr. A, 1101, 63, 2006. 112. Choi, Y.J., Jang, J.H., Park, H.K., Koo, Y.E., Hwang, I.K., and Kim, D.B., Determination of vitamin B12 (cyanocobalamin) in fortified foods by HPLC, J. Food Sci. Nutr., 8, 301, 2003. 113. Indyk, H.E., Persson, B.S., Caselunghe, M.C., Moberg, A., Filonzi, E.L., and Woollard, D.C., Determination of vitamin B12 in milk products and selected foods by optical biosensor protein-binding assay: Method comparison, J. AOAC Int., 85, 72, 2002. 114. Ball, G.F.M., Vitamins in Foods: Analysis, Bioavailability, and Stability, CRC Press, Boca Raton, FL, 2006, Chapter 17. 115. Muhammad, K., Briggs, D., and Jones, G., The appropriateness of using cyanocobalamin as calibration standard in Lactobacillus leichmannii ATCC 7830 assay of vitamin B12, Food Chem., 4, 427, 1993. 116. Skeggs, H.R., Lactobacillus leichmannii assay for vitamin B12, in Analytical Microbiology, Kavanagh, F., Ed., Academic Press, New York, 1963, Chapter 7. 117. Skeggs, H.R. and Wright, L.D., The use of Lactobacillus arabinosus in the microbiological determination of pantothenic acid, J. Biol. Chem., 156, 21, 1944. 118. Guilarte, T.R., Radiometric microbiological assay of B vitamins. II. Extraction methods, J. Nutr. Biochem., 2, 399, 1991. 119. Walsh, J.H., Wyse, B.W., and Hansen, R.G., A comparison of microbiological and radioimmunoassay methods for the determination of pantothenic acid in foods, J. Food Biochem., 3, 175, 1979. 120. Walsh, J.H., Wyse, B.W., and Hansen, R.G., Pantothenic acid content of 75 processed and cooked foods, J. Am. Diet. Assoc., 78, 140, 1981. 121. Gonthier, A., Boullanger, P., Fayol, V., and Hartmann, D.J., Development of an ELISA for pantothenic acid (vitamin B5) for application in the nutrition and biological fields, J. Immunoassay, 19, 167, 1998. 122. Haughey, S.A., O’Kane, A.A., Baxter, G.A., Kalman, A., Trisconi, M.J., Indyk, H.E., and Watene, G.A., Determination of pantothenic acid in foods by optical biosensor immunoassay, J. AOAC Int., 88, 1008, 2005. 123. Kodama, S., Yamamoto, A., and Matsunaga, A., Direct chiral resolution of pantothenic acid using 2-hydroypropyl-β-cyclodextrin in capillary electrophoresis, J. Chromatogr. A, 811, 269, 1998. 124. Rychlik, M., Quantification of free and bound pantothenic acid in foods and blood plasma by a stable isotope dilution assay, J. Agric. Food Chem., 48, 1175, 2000. 125. Pakin, C., Bergaentzlé, M., Hubscher, V., Aoudé-Werner, D., and Hasselmann, C., Fluorimetric determination of pantothenic acid in foods by liquid chromatography with post-column derivatization, J. Chromatogr. A, 1035, 7, 2004. 126. Mittermayr, R., Kalman, A., Trisconi, M.-J., and Heudi, O., Determination of vitamin B5 in a range of fortified food products by reversed-phase liquid chromatography-mass spectrometry with electrospray ionization, J. Chromatogr. A, 1032, 1, 2004. 127. Woollard, D.C., Indyk, H.E., and Christiansen, S.K., The analysis of pantothenic acid in milk and infant formulas by HPLC, Food Chem., 69, 201, 2000.
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128. Tanner, J.T., Barnett, S.A., and Mountford, M.K., Analysis of milk-based infant formula. Phase V. Vitamins A and E, folic acid, and pantothenic acid: Food and Drug Administration-Infant Formula Council: Collaborative study, J. AOAC Int., 76, 399, 1993. 129. Livaniou, E., Costopoulou, D., Vassiliadou, I., Leondiadis, L., Nyalala, J.O., Ithakissios, D.S., and Evangelatos, G.P., Analytical techniques for determining biotin, J. Chromatogr. A, 881, 331, 2000. 130. Bitsch, R., Salz, I., and Hótzel, D., Biotin assessment in foods and body fluids by a protein binding assay (PBA), Int. J. Vitam. Nutr. Res., 59, 59, 1989. 131. Reyes, F.D., Romero, J.M.F., and de Castro, M.D.L., Determination of biotin in foodstuff s and pharmaceutical preparations using a biosensing system based on the streptavidin-biotin interaction, Anal. Chim. Acta, 436, 109, 2001. 132. Indyk, H.E., Evans, E.A., Caselunghe, M.C.B., Persson, B.S., Finglas, P.M., Woollard, D.C., and Filonzi, E.L., Determination of biotin and folate in infant formula and milk by optical biosensorbased immunoassay, J. AOAC Int., 83, 1141, 2000. 133. Staggs, C.G., Sealey, W.M., McCabe, B.J., Teague, A.M., and Mock, D.M., Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding, J. Food Compos. Anal., 17, 767, 2004. 134. Nelson, B.C., Sharpless, K.E., and Sander, L.C., Improved liquid chromatography methods for the separation and quantification of biotin in NIST Standard Reference Material 3280: Multivitamin/ multielement tablets, J. Agric. Food Chem., 54, 8710, 2006. 135. Höller, U., Wachter, F., Wehrli, C., and Fizet, C., Quantification of biotin in feed, food, tablets, and premises using HPLC-MS/MS, J. Chromatogr. B, 31, 8, 2006. 136. Eitenmiller, R.R. and Landen, W.O. Jr., Vitamin Analysis for the Health and Food Sciences, CRC Press, Boca Raton, FL, 1999, Chapter 12. 137. McDowell, L.R., Vitamins in Animal and Human Nutrition, 2nd Edn., Iowa State University Press, Ames, IA, 2000, Chapter 15. 138. Eitenmiller, R., Ye, L., and Landen, Jr. W.O., Vitamin Analysis for the Health and Food Sciences, 2nd Edn., CRC Press, Boca Raton, FL, 2008, Chapter 5. 139. Tsumura, F., Ohsako, Y., Haraguchi, Y., Kumagai, H., Sakurai, H., and Ishii, K., Rapid enzymatic assay for ascorbic acid in various foods using peroxidase, J. Food Sci., 58, 619, 1993. 140. Davey, M.W., Bauw, G., and van Montagu, M., Analysis of ascorbate in plant tissue by high performance capillary zone electrophoresis, Anal. Biochem., 239, 8, 1996. 141. Dodson, K.Y., Young, E.R., and Soliman, A.G.M., Determination of total vitamin C in various food matrixes by liquid chromatography and fluorescence detection, J. AOAC Int., 75, 87, 1992. 142. Brause, A.R., Woollard, D.C., and Indyk, H.E., Determination of total vitamin C in fruit juices and related products by liquid chromatography: Interlaboratory study, J. AOAC Int., 86, 367, 2003. 143. Hidiroglou, N., Madere, R., and Behrens, W., Electrochemical determination of ascorbic acid and isoascorbic acid in ground meat and in processed foods by high pressure liquid chromatography, J. Food Compos. Anal., 11, 89, 1998. 144. Nisperos-Carriedo, M.O., Buslig, B.S., and Shaw, P.E., Simultaneous detection of dehydroascorbic, ascorbic, and some organic acid in fruits and vegetables by HPLC, J. Agric. Food Chem., 40, 1127, 1992. 145. Kutnink, M.A. and Omaye, S.T., Determination of ascorbic acid, erythorbic acid, and uric acid in cured meats by high-performance liquid chromatography, J. Food Sci., 52, 53, 1987. 146. Doner, L.W. and Hicks, K.B., High-performance liquid chromatographic separation of ascorbic acid, erythobic acid, dehydroascorbic acid, dehydroerythobic acid, diketogulonic acid, and diketogluconic acid, Anal. Biochem., 115, 225, 1981.
Chapter 20
Minerals and Trace Elements Jörg Oehlenschläger Contents 20.1 Introduction ..................................................................................................................351 20.2 Elemental Composition .................................................................................................353 20.2.1 Seafood ............................................................................................................353 20.2.1.1 Aluminum .......................................................................................353 20.2.1.2 Arsenic .............................................................................................353 20.2.1.3 Chromium .......................................................................................354 20.2.1.4 Iodine ..............................................................................................358 20.2.1.5 Manganese ...................................................................................... 360 20.2.1.6 Molybdenum ...................................................................................361 20.2.1.7 Selenium ..........................................................................................361 20.2.1.8 Thallium ......................................................................................... 364 20.2.1.9 Vanadium ....................................................................................... 364 20.2.1.10 Zinc .................................................................................................365 20.2.1.11 Other Elements ................................................................................367 20.2.2 Crustacean and Molluscan Shellfish ................................................................ 368 20.3 Effects of Household Preparations and Processing........................................................ 368 20.4 Conclusions .................................................................................................................. 369 References ............................................................................................................................... 369
20.1
Introduction
Most publications and articles about the nutritional benefits of seafood, especially concerning marine fish, concentrate almost exclusively on the highly unsaturated long-chain fatty acids such as docosahexaenoic (22:6 n-3) and eicosapentaenoic acid (20:5 n-3). There are an abundant 351
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number of papers dealing with this issue, and the focus on these fatty acids sometimes gives the impression that they are the only constituents of importance in seafood. Other components that are also of great importance such as vitamins, amino acids, protein and peptides, and finally essential and other elements are much less in the focus of science and scientific literature. In this chapter, some trace elements that are present in seafood are described based on material that is published. This chapter does not focus on toxic heavy metals such as mercury, lead, and cadmium, on which extensive literature and reviews can be found elsewhere, and not on the nutritive and physiological properties of elements, on which specialized books and reviews are available. Emphasis is laid on information based on recent publications, which means that most of the material used was published in the last 10 years. When necessary and where appropriate, however, older literature is used too. Knowledge about minerals and elements in seafood, which is well documented in books, book chapters, and other literature is not repeated in this chapter. To get more basic information about this issue, readers are advised to consult the references section. A short look at the literature published until 1977 shows that there are only a few books existing, namely those of Anon [1], Eisler [5], Hall et al. [7], and Sidwell [14]. Reilly’s book [13] describes the nutritional properties of some elements. The chemical analysis of many elements is described in the work of Kiceniuk and Ray [8], and a few trace elements are described in a book chapter by Oehlenschläger [11]. The Joint Research Center of the European Commission has published the “Official Methods for the Determination of Heavy Metals in Feed and Food” including analytical methods for zinc, chromium, molybdenum, arsenic, selenium, nickel, and manganese [135]. For more general information about analytical methods, the work of Kellner et al. [136], is recommended. For the analytical determination of elements, mostly atomic absorption methods (flame, graphite furnace, or hydride generation), electro analytical techniques such as differential pulse scanning anodic voltammetry (DPSAV) or inductively coupled plasma (ICP) methods are used after appropriate sample preparation steps (commonly pressure digestion). Much emphasis must be laid on a critical analytical quality assurance during all analytical steps (e.g., use of certified standard reference materials, participation in proficiency tests) before results are published. Book chapters partly reviewing the issue have been written by Causeret [3], Gordon [6], Lall [9], Nunes et al. [10], and Oehlenschläger [12]. All these publications and books contain all basic facts in detail about the more common elements and minerals found in seafood as well as numerous references for further reading. Many useful data about concentrations of elements in seafood, which can be used by nutritionists, cooks, and people who are on a special diet, are present in food composition tables, e.g., Souci et al. [15], last edition in 2008. The book edited by Ebdon et al. [4] about trace element speciation for environment, food, and health gives a good overview about this aspect of elemental analysis. In this chapter therefore no, or only very limited, information is given about calcium, phosphorous, magnesium, iron, fluorine, sodium, and potassium. However, two older papers shall be mentioned in the introductory section: the one by Thurston [17] from 1958, which is about the sodium and potassium content of 34 species of fish and which is still a valuable source of information about these elements. Thurston found in the edible parts of 25 marine fish species an average sodium content of 68 mg/100 g and an average potassium content of 320 mg/100 g, whereas in 8 freshwater species 56 mg sodium/100 g and 282 mg potassium/100 g, respectively, were found. The ratio of sodium:potassium was between 1:5 and 1:6. The other work is that of Anthony et al. [102] providing considerable information about the mineral content in finfish and shellfish.
Minerals and Trace Elements
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353
The mean total mineral content (ash) of fish fillet is very close to 1% of its wet weight; some fatty fish species exceed this figure up to 1.6% (e.g., Sardina pilchardus) [12,111] and some mollusks even higher, more than 2%.
20.2 Elemental Composition 20.2.1 Seafood 20.2.1.1 Aluminum In the scientific literature almost nothing was found about the content of aluminum in edible parts (fillet, muscle tissue) of aquatic animals used for human nutrition almost until the end of the last century. Ranau et al. [18,19] developed an analytical method for the determination of aluminum in the edible parts of fish using graphite furnace atomic absorption spectrometry (GFAAS) after sample pretreatment with microwave-activated oxygen plasma. Applying this methodology, 25 fish species from North Sea, 25 species from North-Eastern Atlantic, 29 species from other areas, 17 species from coastal Norwegian waters, and 11 species of marine shellfish were investigated for their aluminum content. Values of North Sea fish species varied between 0.05 mg/kg wet weight and 0.28 mg/kg. Average overall value in species investigated was close to 0.1 mg/kg wet weight. The value in species from open Northeast Atlantic waters varied between 0.03 and 0.14 mg/kg (average overall species value was also close to 0.1 mg/kg). The species from other areas such as Barents Sea, Greenland waters, and Baltic Sea exhibit similar concentrations, never exceeding 0.2 mg/kg. Species from coastal Norwegian waters showed elevated aluminum concentrations up to 0.94 mg/kg. This can be explained by the presence of an aluminum smelter plant in that area. The conclusion based on all the results was that marine fish from waters not affected by aluminum-producing industry contains in its edible part approximately 0.1 mg aluminum/kg wet weight. Marine shellfish had generally higher aluminum levels compared with fish species: there was a variation from 0.2 mg/kg in squid (Loligo sp.) from North Sea to 4.95 mg/kg in blue mussels (Mytilus edulis) also from North Sea. An analysis of different organs of cod showed quite different aluminum concentrations in body compartments; all being higher than that in edible part: 0.06 mg/kg in female gonads, 0.22 in liver, 0.29 in spleen, 0.36 in kidney, 0.44 in brain, 0.45 in heart, and 0.63 in gills. In three fish species from Iskenderun Bay, North East Mediterranean Sea, Turkey, Türkmen et al. [130], determined the aluminum content and reported concentrations in edible parts of 0.83 mg/kg dry weight in Saurida undosquamis, 2.23 mg/kg dry weight in Sparus aurata, and 0.92 mg/kg dry weight in Mullus barbatus.
20.2.1.2
Arsenic
Arsenic concentration As (V) and As (III) in the surface water of the world’s oceans is close to 1 mg/L. Arsenic taken up by marine animals is concentrated in their bodies by metabolic processes and can be found in seafood in many different forms as inorganic arsenate or arsenite, as organic methyl arsonate and dimethyl arsonate, as arsenobetaine, arsenocholine, trimethyl arsine oxide, or as arseno sugars. Good introductions to the complex world of arsenic’s marine chemistry are given by Borak and Hosgood [21], Francesconi and Edmonds [26], Francesconi [27], and
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Francesconi [28]. Luten et al. [36] demonstrated with human volunteers who consumed 100 g plaice fillet containing 80 mg total arsenic/100 g that organic arsenic was almost quantitatively excreted as trimethyl arsine by man. Although the organic forms of arsenic are nontoxic to humans, the inorganic arsenic is the toxic form. Schoof et al. [40] made a market basket survey of inorganic arsenic in food and found in the seafood samples analyzed (saltwater finfish, canned tuna, shrimp, and freshwater finfish) no substantial amounts of inorganic arsenic. In two shrimp samples, inorganic arsenic was detected at levels of 2 and 3 ng/kg wet weight, whereas total arsenic concentration was 2790 and 2820 ng/kg wet weight, which is an inorganic arsenic proportion of 0.07% and 0.1% of total arsenic concentration. In 2002, De Dieter et al. [24] looked at the total and toxic arsenic levels in 25 North Sea fish species and four shellfish species. Although total arsenic concentration in species varied considerably between species (2–50 mg/kg wet weight), the concentration of toxic inorganic arsenic remained low (about 0.1 mg/kg). Larsen et al. [32] and Sloth et al. [41] made a survey of inorganic arsenic in marine animals using microwave-assisted alkaline alcoholic sample dissolution and high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLCICP-MS). In a variety of seafood samples including fish, crustaceans, bivalves, and marine mammals, the results for inorganic arsenic were in most cases below the limit of detection (0.0006 mg/ kg wet weight). In some samples inorganic arsenic concentrations up to 0.06 mg/kg were analyzed, and the inorganic arsenic content, however, constituted less than 1% of the total arsenic content. In addition, Li et al. [34] concluded that arsenic in seafood does not pose any risk to human health, because the major share of arsenic components in seafood is organic arsenic (>98%) with a low toxicity. In Table 20.1 an overview is given about the arsenic concentration reported in the literature. Arsenic seems to be higher in flatfish species such as plaice and lemon sole, whereas gadoid species are generally low in arsenic. There is also sufficient evidence that the geographic area where the species are caught or harvested has great influence on the arsenic content (e.g., species from South Adriatic Sea compared with species from Chile). Different arsenic species and especially arseno sugars were detected in marine algae. Hirata and Toshimitsu [30] found concentrations of four arseno sugars in four species of algae from Hiroshima Bay, Japan, in concentrations of 1.05 mg/kg dry weight (Myelophycus simplex) up to 10.04 mg/kg dry weight (Ecklonia cava). The total arsenic intake in Belgium based on elemental content of various foodstuffs including fish and other seafood was assessed recently [38]. Aqueous samples can be analyzed by IC-ICP-MS to determine arsenite, arsenate, and methylated and other organic forms of arsenic. Hydride generation atomic absorption spectrometry and atomic fluorescence spectrometry (HG-AAS and HG-AFS, respectively) are frequently used for total arsenic determination.
20.2.1.3 Chromium Vos and coworkers [47] analyzed chromium in Dutch fishery products and reported levels for different species: sole (Solea solea) from the coast of Holland, 0.02 mg/kg wet weight; cod (Gadus morhua) from the coast of Holland, 0.01 mg/kg wet weight; herring (Clupea harengus) from the coast of Holland, 0.02 mg/kg wet weight; eel (Anguilla anguilla) from lake Ijssel, 0.08 mg/kg wet weight; mussel (Mytilus edulis) from Eastern Scheldt, 0.43 mg/kg wet weight; and brown shrimp (Crangon crangon) from Western Wadden Sea, 0.26 mg/kg wet weight. Later Lendinez et al.
Minerals and Trace Elements Table 20.1
◾
355
Total Arsenic Concentrationsa in Marine Species based on Literature Values
Species
Geographic Area
Total Arsenic (mg/kg Wet Weight)
Ref.
Raja asterias
South Adriatic Sea
30.6
[42]
Raja clavata
South Adriatic Sea
43.7
[42]
Raja oxyrhynchus
South Adriatic Sea
49.4
[42]
Merluccius merluccius
South Adriatic Sea
9.7
[42]
Micromesistius poutassou
South Adriatic Sea
14.9
[42]
Trachurus murphyi
Chile
0.46
[39]
Merluccius gayi
Chile
0.32
[39]
Engraulis rigens
Chile
0.23
[39]
Clupea bentincki
Chile
0.11
[39]
Mytilus chilensis
Chile
1.17
[39]
Cancer edwardsii
Chile
2.46
[39]
Pleuroncodes monodon
Chile
6.57
[39]
Clupea harengus
Shetland Islands
4.29
[20]
Clupea harengus
Norway
1.5–1.7
[41]
Clupea harengus
Scottish waters
1–1.5
[25]
Clupea harengus
Baltic Sea
0.87
[20]
Gadus morhua
East Greenland
2.94
[20]
Gadus morhua
Belgian coastal waters
2.5–5.4
[23]
Gadus morhua
Scottish waters
0.7–4.6
[25]
Gadus morhua
Newfoundland and Labrador
0.8
[31]
Gadus morhua
Belgium
4.7
[22]
Gadus morhua
Norway
15–17
[41]
Gadus morhua
Baltic Sea
0.56
[20]
Scomber scombrus
Shetland Islands
2.08
[20]
Scomber scombrus
Scottish waters
0.4–1
[25]
Scomber scombrus
Norway
1.7–2.8
[41]
Scomber scombrus
German Bight
0.72
[20]
Pleuronectes platessa
The Netherlands
23
[36] (continued)
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Table 20.1 (continued) Literature Values
Total Arsenic Concentrationsa in Marine Species based on
Species
Geographic Area
Total Arsenic (mg/kg Wet Weight)
Ref.
Pleuronectes platessa
Irish Sea
4.9–21.3
[33]
Pleuronectes platessa
Belgium
19.8
[22]
Pleuronectes platessa
German Bight
9.33
[20]
Melanogrammus aeglefinus
Bear Island
10.48
[20]
Melanogrammus aeglefinus
Scottish waters
1.5–4.9
[25]
Melanogrammus aeglefinus
German Bight
9.73
[20]
Many unspecified species
North Adriatic Sea
5.36
[29]
Platichthys flesus
Netherlands
0.45–6.8
[35]
Platichthys flesus
Belgian coastal waters
2.4–4.1
[23]
Crangon crangon
Belgian coastal waters
4–10
[23]
Invertebrates
Alaska
5–120 (dry weight)
[37]
Invertebrates
California
3 to >1000 (dry weight)
[37]
Microstomus kitt
The Netherlands
24.9–31.4
[35]
Microstomus kitt
Scottish waters
11.5–15.3
[25]
Glyptocephalus cynoglossus
Scottish waters
12.7–15.6
[25]
Limanda limanda
The Netherlands
2.4–12.5
[35]
Limanda limanda
Scottish waters
4.5
[25]
Lepidorhombus whiffiagonis
Scottish waters
2.2
[25]
Lophius piscatorius
Norway
15–44
[41]
Lophius piscatorius
Scottish waters
7.9
[25]
Squalus acanthias
Scottish waters
2.7
[25]
Molva molva
Scottish waters
2.4
[25]
Merlangius merlangus
Irish Sea
2.4–6.2
[33]
Merlangius merlangus
Scottish waters
1–1.7
[25]
Pollachius virens
Scottish waters
0.6–1.4
[25]
Loligo forbesi
Scottish waters
3.8–8.0
[25]
Nephrops norvegicus
Scottish waters
2.7–5.8
[25]
Pecten maximus
Scottish waters
1.0–1.4
[25]
Minerals and Trace Elements Table 20.1 (continued) Literature Values
357
Total Arsenic Concentrationsa in Marine Species based on
Species
a
◾
Geographic Area
Total Arsenic (mg/kg Wet Weight)
Ref.
Littorina littorea
Scottish waters
2.2–16.1
[25]
Sebastes marinus
Newfoundland and Labrador
0.8
[31]
Hippoglossoides platessoides
Newfoundland and Labrador
4.4
[31]
Rheinhardtius hippoglossoides
Newfoundland and Labrador
0.8
[31]
Solea solea
Belgium
5.1
[22]
Solea solea
The Netherlands
6.2–10.2
[35]
Salmo salar
Norway
1.9
[41]
Anarhichas lupus
Norway
4.1
[41]
Pandalus borealis
Norway
3.8–67
[41]
Engraulis encrasicolus
Black Sea
11.1
[113]
Sprattus sprattus
Black Sea
1.7
[113]
Trachurus mediterraneus
Black Sea
7.9
[113]
Mullus barbatus
Black Sea
3.5
[113]
Belone belone
Black Sea
0.8
[113]
Sarda sarda
Black Sea
11.8
[113]
Merlangius merlangus
Black Sea
18.7
[113]
Venerupis rhomboides
Galicia, Spain
18.3 (dry weight)
[76]
Cardium edule
Galicia, Spain
13.4 (dry weight)
[76]
Cancer pagurus
Galicia, Spain
39.3 (dry weight)
[76]
Lepidorhombus whiffiagonis
Galicia, Spain
42.5 (dry weight)
[76]
Ensis ensis
Galicia, Spain
18.1 (dry weight)
[76]
Average values, when not otherwise indicated.
[46] investigated chromium in basic foods of the Spanish diet including seafood. They analyzed 33 species of marine animals and reported an average concentration of chromium in fish of 0.032 mg/kg wet weight, in mollusks of 0.015 mg/kg wet weight, and in crustaceans of 0.016 mg/ kg wet weight. Highest chromium levels were found in Trachurus trachurus (0.04 mg/kg), Scomber scombrus (0.042 mg/kg), Brama raii (0.045 mg/kg), Sardina pilchardus (0.047 mg/kg), Cepola rubescens (0.052 mg/kg), Helicolenus sp. (0.067 mg/kg), and Mullus surmuletus (0,079 mg/kg). In the Greek diet the chromium content was evaluated by Bratakos et al. [44]. In seafood samples, the following chromium contents were reported on a wet weight basis: fish, Boops boops
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(0.11 mg/kg), Chrysophrys auratus (0.18 mg/kg), Merluccius merluccius (0.13 mg/kg), Mullus surmuletus (0.16 mg/kg), Pagelus erythrinus (0.19 mg/kg), Sardina pilchardus (0.08 mg/kg); shellfish, Sepia officinalis (0.15 mg/kg), Homarus vulgaris (0.15 mg/kg), Octopus vulgaris (0.17 mg/kg), Ostrea edulis (0.28 mg/kg), and Crangon vulgaris (0.21 mg/kg). In six marine species from the Adriatic Sea (anchovy, angler, hake, mackerel, red mullet, and sole) chromium levels between <0.001 mg/kg and 0.61 mg/kg fresh weight were reported by Sepe et al. [79]. Chromium was analyzed in the squid Todarodes sagittatus from Japan in a concentration of 0.052 mg/kg wet weight [92]. In the clam Chamelea galena from the Adriatic Sea chromium was found at levels ranging from 0.78 mg/kg wet weight to 1.39 mg/kg wet weight depending on the season [95]. An extensive review about chromium presence in foods and beverages was recently published by Cabrera-Vique [45]. Chromium levels from different literature sources and species were in the range from 0.01 mg/kg (Boops salpa, Sepia officinalis, Merluccius merluccius, Octopus vulgaris, and Maena smaris) up to >0.5 mg/kg (Tilapia sp., Mugil sp., Crassostrea gigas). The chromium content in seafood seems to vary considerably according to species and geographic origin between 0.01 mg/kg wet weight and 0.5 mg/kg wet weight. Pacific oysters (Crassostrea gigas) purchased from markets in Hong Kong exhibited a very pronounced seasonal variation in chromium content ranging from 1 mg/kg dry weight in May, June to >35 mg/kg dry weight in September and December [86]. In a long-term study from 1985 to 1996 Vyncke et al. [99] reported average chromium content in the cut trough shell (Spisula subtruncata) from Belgian coastal waters of 0.47 mg/kg wet weight. Vernocchi et al. [98] found rather low chromium concentrations in Mediterranean mussel (Mytilus galloprovincialis) harvested in Adriatic Sea (Italy) ranging from 0.11 mg/kg wet weight in November to 0.51 mg/kg in December. For chromium content determination graphite furnace atomic absorption spectroscopy (GFAAS) is commonly used after previous mineralization with HNO3 and V2O5.
20.2.1.4 Iodine Iodine is an essential trace element for human beings required for normal activity of the thyroid hormones thyroxine and triiodothyronine. Seafood is the only natural source that contains a considerable amount of iodine. In 1998 and 1999 Karl and Münkner [52,53] could demonstrate that the iodine content in the skin of fish is much higher than that in the edible part (0.68 mg/kg wet weight in saithe fillet and 0.29 mg/kg wet weight in cod fillet versus 13, 26, and 5.95 mg/kg, respectively, in skin). The authors could also show that a considerable part of iodine is located in the lipids of fish (28% in mackerel, 45% in Greenland halibut, 68% in Atlantic salmon, and 62% in ocean perch). It was further revealed that the range of iodine concentration between specimens within a given species can be extremely big (e.g., variation in haddock from 0.14 up to 6 mg/kg). In a Norwegian project by Dahl et al. [49] about the iodine content in Norwegian foods, 393 lean fish and 313 fatty fish were analyzed. Lean fish from Norwegian waters had an average iodine content of 0.86 mg/kg (range, 0.03–12.7) and fatty fish 0.4 mg/kg (range, 0.05– 1.61 mg/kg). A similar investigation was carried out in Switzerland by Haldimann et al. [50], where the iodine content was measured in different food groups consumed in Switzerland. The authors found an average concentration of 2.1 mg/kg (range, 0.39–6.9) in 34 marine fish samples and of 0.38 mg/kg (range, 0.01–1.57) in 17 samples of freshwater fish.
Minerals and Trace Elements
◾
359
More concentrations of iodine in edible parts of aquatic species reported in the literature are listed in Table 20.2. In a preliminary study Julshamn et al. [51] increased the iodine concentration in edible parts of Atlantic salmon during a feeding experiment with iodine-enriched feed to 1.4 mg/kg wet weight. Iodine is nowadays preferably analyzed by inductively coupled plasma/mass spectrometry (ICP/MS). An alternative GC method is described in Ref. [132]. Table 20.2
Iodine Concentrations in Edible Part of Seafood
Species
Geographic Area
Iodine (mg/kg Wet Weight)
Ref.
Melanogrammus aeglefinus
Northeast Atlantic
1.86
[53]
Pollachius virens
Northeast Atlantic
1.21
[53]
Gadus morhua
Northeast Atlantic
1.87
[53]
Merlangius merlangus
Northeast Atlantic
1.38
[53]
Molva molva
Northeast Atlantic
1.75
[53]
Sebastes sp.
Northeast Atlantic
0.7
[53]
Clupea harengus
Northeast Atlantic
0.41
[53]
Scomber scombrus
Northeast Atlantic
1.09
[53]
Sprattur sprattus
Northeast Atlantic
0.24
[53]
Psetta maximus
Northeast Atlantic
1.8
[53]
Pleuronectes platessa
Northeast Atlantic
0.46
[53]
Limanda limanda
Northeast Atlantic
0.66
[53]
Platichthys flesus
Northeast Atlantic
0.65
[53]
Crangon crangon
Northeast Atlantic
0.74
[53]
Mytilus edulis
Northeast Atlantic
0.99
[53]
Salmo salar
Northeast Atlantic
0.45
[53]
Boops boops
Libyan waters
1.6
[48]
Mugil sp.
Libyan waters
1.7
[48]
Dentex dentex
Libyan waters
2.4
[48]
Scomber japonicus
Libyan waters
2.1
[48]
Euthynnus alletteratus
Libyan waters
4.3
[48]
Sardinella aurita
Libyan waters
3.9
[48]
Thunnus thynnus
Libyan waters
0.3
[48]
Thunnus albacores
Libyan waters
0.8
[48]
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20.2.1.5 Manganese Yebra and Moreno-Cid [55] determined manganese in solid seafood samples by flame atomic absorption spectrometry. By this method manganese contents of 2.4–15.9 mg/kg dry mass in mussel, of 4.9 mg/kg dry matter in clam, of 5.8 mg/kg dry matter in tuna, and of 4.8 mg/kg dry matter in sardine all from the Galician coast in Spain were analyzed. Da Silva et al. [54] have analyzed shellfish samples from Brazilian waters for their manganese concentration. Mussel (Mytella guiyanensis) exhibited 0.22 and 0.30 mg/kg wet weight and another mussel species (Perna perna), 0.18 mg/kg. The clam (Anomalocardia brasiliana) contained 0.20 mg/kg and the oyster (Crassostrea rhizophorae), 0.27 mg/kg. Ogunlade et al. [121] measured the manganese content in shellfish samples from Nigeria. For five crustacean species, a mean vanadium concentration of 10.2 mg/kg wet weight was reported ranging from 4 to 19 mg/kg. In three shrimp species from Turkey, Penaeus semisulcatus, Parapenaeus longirostris, and Paleomon serratus, manganese was found at a level of 0.60 mg/kg wet weight, 1.52 mg/kg wet weight, and 0.25 mg/kg wet weight, respectively [89]. Manganese concentration ranging from 11.2 mg/kg wet weight to 15.2 mg/kg wet weight has been reported for five tropical shrimp species from Indian waters (Chilika lagoon) [94]. The manganese concentration in two crustacean species from the Strait of Magellan, Chile, varied from 1.6 to 3.2 mg/kg in Mytilus chilensis and from 1.5 to 4.5 mg/kg in Perunytitus purpuratus depending on the season [83]. The edible part (arms) of Octopus vulgaris from the Portuguese coast analyzed for manganese showed concentrations ranging from 1.5 to 2.39 mg/kg wet weight depending on the year and catching location [96]. The manganese content in claw meat of Blue crab (Callinectes sapidus) and swim crab (Portunus pelagicus) was estimated to be 3.9 and 0.6 mg/kg wet weight, respectively, by Gökodlu and Yerlikaya [87]. The same authors also demonstrated the annual variation of manganese content in Pearl oyster (Pinctada radiata) from the Gulf of Antalya, Turkey, ranging from 1.9 mg/kg wet weight in November to about 6 mg/kg wet weight in July [88]. The manganese content in some fish species from Turkey was also assessed by Demirezen and Uruc [107] who reported 46 mg/kg for mackerel, 43 mg/kg for anchovy, 41 mg/kg for Blue whiting, and 45 mg/kg for bonito. In Baltic herring fillets (Clupea harengus) a manganese content of 0.47 mg/kg fresh weight (variation 0.30–0.63 mg/kg) was estimated [127]. The muscle concentration of manganese in nine species of fish caught in the coastal waters of Southwest Taiwan ranged from 0.20 to 0.83 mg/kg wet weight [106], and the muscle concentration in 18 marine species from the Arabian Gulf ranged from 0.05 to 0.52 mg/kg wet weight [108]. In fish and shellfish from internal markets of India, manganese levels were assessed to be between more than 0.08–9.2 mg/kg wet weight [126]. In edible parts of three fish species from Dhanmondi lake in Bangladesh, an average manganese level of 17.7 mg/kg dry weight was reported [103]. The manganese content of nine fish species from the Eastern Gulf of California was published by Ruelas-Inzunza and Paez-Osuna [122]. They reported manganese concentrations from 0.06 mg/kg dry weight in Mugil cephalus to a maximum of 6.6 mg/kg dry weight in Sphyrna lewini. Manganese was analyzed in freshwater and saltwater fish, some invertebrates, and in their products by Krelowska-Kulas [117], showing a very similar concentration of 0.13 mg/kg fresh weight with the exception of carp (0.25 mg/kg fresh weight). Manganese determination is commonly made by flame atomic absorption spectrometry or by Zeeman atomic absorption spectrometry.
Minerals and Trace Elements
◾
361
20.2.1.6 Molybdenum In Chinese shrimps a molybdenum amount of 3.74 mg/kg was published by Zaijun et al. [56] using a spectrophotometric method with p-carboxyphenylfluorone.
20.2.1.7 Selenium The trace element selenium is like iodine an essential nutrient of fundamental importance to human biology. This is well documented by many reviews [60,68,69]. Seafood is the major natural source for this element. It was also demonstrated that the bioavailability of selenium from fish was high for humans [61] and rats [65]. A proportion of 23%–34% of total selenium present in fish was found to be soluble selenium [64]. Characteristics of selenium in Australian marine biota have been provided by Maher et al. [119]. The literature about selenium in fish is huge and for Table 20.3 only newer data produced during the last 10 years were used. A good source for selected data about the selenium content in seafood and about dietary intake of selenium in France is the work of Bourre and Paquotte [57]. Table 20.3 Selenium Content in Edible Parts of Seafood from Different Origins Species
Geographic Area
Selenium (mg/kg Wet Weight)
Ref.
Pleuronectes platessa
Irish waters
0.28
[63]
Gadus morhua
Irish waters
0.27
[63]
Shrimp
Northern Mexico
0.53
[73]
Scallops
Northern Mexico
1.03
[73]
Oyster
Northern Mexico
0.48
[73]
Octopus
Northern Mexico
0.38
[73]
Fish
Northern Mexico
0.9
[73]
Mackerel
Australia
0.31
[62]
Morwong
Australia
0.63
[62]
Spotted cod
Australia
0.39
[62]
Trevally
Australia
0.48
[62]
Yellow tail kingfish
Australia
0.45
[62]
Oyster
Australia
0.77
[62]
Salmon
Australia
0.32
[62] (continued)
362
◾
Handbook of Seafood and Seafood Products Analysis Table 20.3 (continued) from Different Origins Species
Selenium Content in Edible Parts of Seafood
Geographic Area
Selenium (mg/kg Wet Weight)
Ref.
Sardine
Australia
0.57
[62]
Trachurus trachurus
Portugal
0.26
[58]
Octopus vulgaris
Portugal
0.13
[58]
Aphanopus carbo
Portugal
0.47
[58]
Sardina pilchardus
Portugal
0.43
[58]
Thunnus sp.
Portugal
0.92
[58]
Trachurus trachurus
Portugal
0.62
[72]
Aphanopus carbo
Portugal
0.54
[72]
Squid
Portugal
0.26
[72]
Octopus vulgaris
Portugal
0.23
[72]
Sardina pilchardus
Portugal
0.43
[72]
Lophius piscatorius
Italy
0.17
[67]
Gadus morhua
Iceland
0.31
[67]
Gadus morhua
Norway
0.19
[67]
Merluccius merluccius
Italy
0.47
[67]
Salmo salar
Norway
0.20
[67]
Xiphias gladius
Italy
0.28
[67]
Thunnus thynnus
Italy
0.73
[67]
Theragra chalcogramma
North Pacific
0.16
[67]
Trigla lucerna
Italy
0.37
[67]
Mullus surmuletus
Italy
0.43
[67]
Sardina pilchardus
Italy
0.68
[67]
Callinectes sapidus
Italy
0.49
[67]
Carcinus maenas
Italy
0.45
[67]
Sepia officinalis
Italy
0.20
[67]
Squilla mantis
Italy
0.40
[67]
Parapenaeus longirostris
Italy
0.51
[67]
Mytilus edulis
Italy
0.61
[67]
Minerals and Trace Elements Table 20.3 (continued) from Different Origins Species
Selenium Content in Edible Parts of Seafood
Geographic Area
Selenium (mg/kg Wet Weight)
Ref.
Octopus vulgaris
Italy
0.18
[67]
Palinurus borealis
Italy
0.07
[67]
Loligo vulgaris
Italy
0.44
[67]
Silver pomfret
Thailand
0.52
[71]
Splendid squid
Thailand
0.41
[71]
Short bodied mackerel
Thailand
0.88
[71]
Ark shell
Thailand
0.44
[71]
Green mussel
Thailand
0.43
[71]
Pacific oyster
Thailand
0.29
[71]
Green tiger prawn
Thailand
0.35
[71]
Sardina pilchardus
Mid Adriatic Sea
0.71
[70]
Sardina pilchardus
Northern Adriatic Sea
0.47
[70]
Crangon vulgaris
Southeastern Spain
0.53
[59]
Mullus surmuletus
Southeastern Spain
0.43
[59]
Sarda sarda
Southeastern Spain
0.65
[59]
Belone belone
Southeastern Spain
0.27
[59]
Boops boops
Southeastern Spain
0.26
[59]
Sardina pilchardus
Southeastern Spain
0.67
[59]
Engraulis encrasicolus
Southeastern Spain
0.31
[59]
Trachurus trachurus
Southeastern Spain
0.29
[59]
Merluccius merluccius
Southeastern Spain
0.32
[59]
Helicolenus dactylopterus
Southeastern Spain
0.23
[59]
Lophius piscatorius
Southeastern Spain
2.01
[59]
Bogue
Greece
0.51
[66]
Gilthead seabream
Greece
0.12
[66]
Sardina pilchardus
Greece
0.30
[66]
Trout
Greece
0.63
[66]
◾
363
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Handbook of Seafood and Seafood Products Analysis
The data in Table 20.3 demonstrate that there is no big variation in selenium content. The range of data in this table is from 0.1 to 1 mg/kg, with average selenium content over all species of about 0.4 mg/kg. In 2005, Burger and Gochfeld [104] reported the selenium content in edible parts of 12 different seafood species of different origin, ranging from 0.05 mg/kg wet weight in scallops to 1.02 mg/kg wet weight in Chilean sea bass. Selenium content in eight Indian fish species amounted to 4.4 mg/kg on a dry weight basis on an average [123]. Trends in selenium determination and speciation by hyphenated techniques based on AAS or AFS are reviewed by Capelo et al. [134]. Generally, selenium is analyzed by hydride generation atomic absorption spectrometry (HGAAS) after pressure digestion.
20.2.1.8 Thallium Information about the natural content of thallium in seafood is rare. In a short review about the determination of thallium in biological samples by Das et al. [74] a thallium content in fish muscle of 1 mg/kg, in dogfish of 3.2 mg/kg, and in trout of 110.5 mg/kg was cited. Thallium was also assessed in different sturgeon species from Caspian Sea [100]; 0.004 mg/kg dry weight was reported for Huso huso, less than 0.001 mg/kg dry weight in all other species, Acipenser persicus, Acipenser gueldenstaedtii, Acipenser nudiventris, and Acipenser stellatus. In a large number of marine species from Southeast Asia (Cambodia, Indonesia, Malaysia, and Thailand), thallium was found to be present in concentrations from less than 0.001 mg/kg dry weight to 0.038 mg/kg dry weight [101].
20.2.1.9 Vanadium The vanadium content in seafood was already reported in 1977 by Myron et al. [77]. The authors found levels of vanadium in the following edible parts of seafood (not further specified): codfish, 28 mg/kg; scallops, 22 mg/kg; lobster, 5 mg/kg; and tuna (canned), 11 mg/kg. In an exposure assessment for Kuwaiti consumers Bu-Olayan and Al-Yakoob [75] analyzed vanadium in nine fish and shellfish samples from the Arabian Gulf. Concentrations of vanadium ranging from 0.48 mg/kg dry weight in Pomadasys argenteus up to 1.48 mg/kg dry weight in Acanthopagrus latus were presented. Sepe et al. [79] published the content of vanadium in edible parts of six fish species from Adriatic Sea. Vanadium levels ranging from less than 4.0 mg/kg fresh weight (angler, hake) to 74.4 mg/kg fresh weight (sardine) were found. The majority of samples exhibited vanadium concentrations around 10 and 30 mg/kg. In mantle and arms of Octopus vulgaris, vanadium was not detectable by Seixas and Pierce [78], whereas vanadium could be detected in other body compartments as brachial hearts at a concentration of 25–53 mg/kg dry weight. In some marine fish and shellfish species from Galicia, Spain, vanadium levels were investigated by Lavilla et al. [76]. In fish samples vanadium was present at concentrations of 0.82 mg/kg dry weight (Merluccius merluccius), 1.25 mg/kg (Lepidorhombus whiffiagonis), and 1.17 mg/kg (Solea solea); in shellfish samples, 2.93 mg/kg (Venerupis rhomboides), 5.14 mg/kg (Cardium edule), 0.95 mg/kg (Sepia officinalis), 3.54 mg/kg (Cancer pagurus), 2.73 mg/kg (Mytilus galloprovincialis), 2.04 mg/kg (Penaeus kerathurus), 3.75 mg/kg (Ensis ensis), and 2.14 mg/kg (Palaemon elegans). In the Japanese common squid a vanadium content of 0.56 mg/kg wet weight was described [92]. Vanadium can be analyzed by liquid chromatography–inductively coupled plasma-mass spectrometry or by electrothermal atomic absorption spectrometry following ultrasound-assisted extraction [76].
Minerals and Trace Elements
◾
365
20.2.1.10 Zinc Zinc is an essential element and zinc-containing foods must be a part of the human diet. There is a lot of information about zinc given in the literature; in this chapter only some results from the recent years are mentioned. In a review of the zinc content of raw foods, where data originating from different geographic regions of the world were compared [82], data about the average zinc content of seafood and its variation were presented: flatfish species such as plaice and sole, 5.04 mg/ kg (variation 3.45–6.8); cod, 3.95 mg/kg (variation 3.25–4.5); herring, 7.18 mg/kg (3.2–9.9); mackerel, 4.66 mg/kg (variation 1.0–7.0); eel, 21.6 mg/kg (variation 16.2–27.0); pike perch, 4.28 (variation 3.1–5.7); trout, 5.24 mg/kg (variation 4.4–6.6); mussel, 20.7 mg/kg (variation 4.2– 34.0); shrimp, 16.8 mg/kg (variation 11.1–27.0); and oyster, 49.1 mg/kg (variation 23.0–120). This demonstrates that oysters are by far the best zinc suppliers. Celik and Oehlenschläger [80,81] assessed the zinc content in fish samples from Northeast Atlantic and from Eastern Mediterranean Sea, and Carvalho et al. [105] as well as Nunes et al. [10] reported the zinc concentration in seafood from the Portuguese coast. Some results of these publications are given in Table 20.4, showing that zinc content can vary largely depending on fishing area, probably season, and intrinsic factors such as state of maturity.
Table 20.4 Zinc Concentrations in Edible Parts of Seafood from Different Geographic Origins Species
Geographic Area
Zinc (mg/kg Wet Weight)
Ref.
Phycis phycis
Portuguese coast
16
[105]
Argyrosomus regius
Portuguese coast
15
[105]
Diplodus sargus
Eastern Mediterranean Sea
3.8
[81]
Diplodus sargus
Portuguese coast
16
[105]
Pagellus acarne
Portuguese coast
20
[105]
Pagellus bogaraveo
Portuguese coast
17
[105]
Pagrus pagrus
Portuguese coast
14
[105]
Helicolenus dactylopterus
Portuguese coast
18
[105]
Solea vulgaris
Eastern Mediterranean Sea
3.7
[81]
Solea vulgaris
Portuguese coast
22
[105]
Lophius piscatorius
Northeast Atlantic
3.0
[80]
Lophius piscatorius
Portuguese coast
21
[105]
Octopus vulgaris
Portuguese coast
109
[105]
Aphanopus carbo
Portuguese coast
5
[10]
Nephrops novegicus
Portuguese coast
45
[10] (continued)
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Table 20.4 (continued) Zinc Concentrations in Edible Parts of Seafood from Different Geographic Origins Species
Geographic Area
Zinc (mg/kg Wet Weight)
Ref.
Octopus vulgaris
Portuguese coast
13
[10]
Anguilla anguilla
Portuguese coast
25
[10]
Pleuronectes platessa
Portuguese coast
6
[10]
Sardina pilchardus
Eastern Mediterranean Sea
9.4
[81]
Sardina pilchardus
Portuguese coast
17
[10]
Melanogrammus aeglefinus
Northeast Atlantic
2.9
[80]
Gadus morhua
Northeast Atlantic
3.3
[80]
Pollachius virens
Northeast Atlantic
3.7
[80]
Molva molva
Northeast Atlantic
3.1
[80]
Merluccius merluccius
Eastern Mediterranean Sea
3.3
[81]
Merluccius merluccius
Northeast Atlantic
3.3
[80]
Zeus faber
Northeast Atlantic
3.1
[80]
Merlangius merlangus
Northeast Atlantic
3.0
[80]
Scomber scombrus
Northeast Atlantic
4.1
[80]
Belone belone
Eastern Mediterranean Sea
9.3
[81]
Trachurus trachurus
Portuguese coast
12
[10]
Trachurus trachurus
Eastern Mediterranean Sea
4.8
[81]
Engraulis encrasicolus
Eastern Mediterranean Sea
6.2
[81]
Dicentrarchus labrax
Eastern Mediterranean Sea
4.9
[81]
Mullus surmuletus
Eastern Mediterranean Sea
4.9
[81]
Scomber japonicus
Eastern Mediterranean Sea
5.6
[81]
Boops boops
Eastern Mediterranean Sea
6.4
[81]
Sparus aurata
Eastern Mediterranean Sea
5.6
[81]
Mullus barbatus
Eastern Mediterranean Sea
4.2
[81]
Sarda sarda
Eastern Mediterranean Sea
4.7
[81]
Pomatomus saltator
Eastern Mediterranean Sea
5.3
[81]
Pagellus erythrinus
Eastern Mediterranean Sea
5.7
[81]
Diplodus vulgaris
Eastern Mediterranean Sea
5.4
[81]
Minerals and Trace Elements
◾
367
Table 20.4 (continued) Zinc Concentrations in Edible Parts of Seafood from Different Geographic Origins Species
Geographic Area
Zinc (mg/kg Wet Weight)
Ref.
Sarpa salpa
Eastern Mediterranean Sea
7.8
[81]
Mugil cephalus
Western Africa
14.2 (dry weight)
[125]
Argyrosomus regius
Western Africa
8.63 (dry weight)
[125]
Epinephelus marginatus
Western Africa
15.85 (dry weight)
[125]
Mycteroperca rubra
Western Africa
11.22 (dry weight)
[125]
Pegusa lascaris
Western Africa
15.05 (dry weight)
[125]
Pagrus auriga
Western Africa
7.73 (dry weight)
[125]
Haliotis rubra
Australia
11.3
[110]
Pagrus auratus
Australia
5.2
[110]
Jausus edwardsii
Australia
19.8
[110]
Nezumia aequalis
West off Scotland
3.9
[120]
Lepidion eques
West off Scotland
2.6
[120]
Raja fyllae
West off Scotland
5.5
[120]
Zinc in edible parts of octopuses from Kerguelen Islands, Southern Indian Ocean, was analyzed by Bustamante et al. [85]. In the muscles of Graneledone sp. and Benthoctopus thielei 113 and 138 mg/kg dry weight were detected. An average zinc content of 3.79 ± 0.57 mg/kg wet weight in edible tissue of 20 fish species from coastal waters of Eastern Taiwan was reported [114]. Zinc is mostly determined by atomic absorption spectrometry (AAS) after microwave digestion.
20.2.1.11 Other Elements The lithium content in some marine smoked fish was described by Nabrrzyski and Gajewska [16] and they reported an average of 0.55 mg lithium/kg in 10 fish species. Lithium was also investigated earlier by Teeny et al. [129] in edible parts of fish species from Northeast Pacific, reporting concentrations in the range of 0.002–0.11 mg/kg wet weight. The concentration of rubidium was found to be only slightly varying around 2.2 mg/kg wet weight (Carvalho et al. [105]). Another source [92] reported 1.2 mg/kg wet weight in Todarodes sagittatus. In freshwater fish species from Bangladesh the average rubidium concentration was estimated at 20 mg/kg dry weight, ranging from 5.5–37.5 mg/kg [124]. For cesium in marine species there is only one reference: Izak-Biran and Guinn [43]. Cobalt was measured by Engman and Jorhem [109] in fish species from Nordic waters. Cobalt concentration was generally low with a minimum of 0.002 mg/kg wet weight and a maximum of 0.008 mg/kg wet weight. Most concentrations were close to 0.003 mg/kg. An exception was eel with 0.02 mg/kg wet weight.
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Bromine was found in the muscle tissue of Zosterisessor ophiocephalus at a concentration of 33.4 mg/kg on a dry weight basis by Tallandini et al. [128]. Earlier, bromine has been assessed in some Chilean seafood with low values in sardine and albacore tuna (8.6 and 8.7 mg/kg dry weight) and higher values in clam species (48.4 and 54.1 mg/kg dry weight) [112]. Nickel was assessed in three species from Northeast Mediterranean Sea by Kalay et al. [115]. Values reported were 2.9–6.1 mg/kg dry weight in Mullus barbatus, 1.9–4.8 mg/kg dry weight in Caranx crysos, and 2.3–4.3 mg/kg dry weight in Mugil cephalus.
20.2.2
Crustacean and Molluscan Shellfish
In this section only literature that could not be used in the other sections and that seems to be worth mentioning as source of valuable information is referenced. A multielemental analysis with 43 elements analyzed was performed with Purpleback Flying squid from Japanese waters. Emphasis was laid on elemental content of livers and stomachs [91]. Zinc concentration in the mussel Perna perna from Moroccan coast was determined at 38 sampling sites. Zinc content in this mussel varied considerably between sampling sites (90–450 mg/kg dry weight), with an average zinc content of 231 mg/kg dry weight [84]. In 2006 the mineral content Ca (3.98 g/kg), Mg (1.17 g/kg), P (1.76 g/kg), K (0.69 g/kg), and Na (6.63 g/kg) of cooked claw meat of Blue crab (Callinectes sapidus) from East Mediterranean Sea, Turkey, was measured [93]. Skonberg and Perkins [97] analyzed the same species in North America. The elemental composition (22 elements) of the fresh Cannonball jellyfish (Stomolophus meleagris) has been reported by Hsieh et al. [90].
20.3 Effects of Household Preparations and Processing An overview about 15 essential and toxic elements in processed shellfish products available in Poland (local markets in Gdansk), originating from different geographical regions of the world, was presented by Kwoczek et al. [118]. Karakotsidis provided information about some commercially important processed Mediterranean finfish, crustaceans, and mollusks [116]. The losses or gains of elements during household preparations or industrial processing are described in detail and quantified only in a few publications. In the following text four examples were chosen to illustrate what happens with elements when seafood undergoes preparation processes involving mostly drastic thermal treatment. Karl et al. [132] investigated the changes of iodine content in fish during household preparation and smoking. It was found that thawing of deep-frozen fillets reduced the iodine content of approximately 10%. The same losses were observed when fish balls were steamed. Frying did not influence the original iodine content significantly. The influence of hot smoking was studied on ocean perch and herring fillets. No changes in iodine content could be observed. Devesa et al. [131] studied the effect of cooking (grilling, roasting, baking, stewing, boiling, steaming, and microwaving) on total and inorganic arsenic content in cooked seafood products. It was revealed that after cooking there was a significant increase in the concentration of total arsenic for salted cod and bivalves and in the concentration of inorganic arsenic for bivalves and squid. The mean content of inorganic arsenic was significantly higher in bivalves than in any other type of seafood.
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Mierke-Klemeyer et al. [133] assessed the retention of selenium during household preparations (boiling in pouches, baking in aluminum foil and deep-frying) of selenium-enriched African catfish (Clarias gariepinus) fillets. The retention of selenium was 91%, 97%, and 104% for baking, boiling, and deep-frying, and no significant difference was found between any of the cooking procedures. Badiani et al. [2] analyzed the effect of sample preparation (cooking) with and without shell liquor on contents and retentions of 11 macro- and microelements in three species of bivalve mollusks (Tapes philippinarum, Callista chione, and Mytilus galloprovincialis). Mollusks prepared (cooked) without shell liquor were more significant sources of several minerals than their counterparts prepared with shell liquor.
20.4 Conclusions In 1997 the author of this chapter asked the question: “Marine fish—a source for essential elements?!” in a book chapter about elemental composition of seafood [12] and came to the conclusion: “for some elements yes, for others not.” As an example a typical fish portion of about 200 g can cover the recommended daily intake of iodine by 100% and of selenium by more than 100%. Ten years later this conclusion is still valid, although the knowledge and database about elemental composition of seafood have increased tremendously.
References Books and book chapters 1. Anon., Chemical Composition and Processing Properties of Marine and Ocean Fishes, VNIRO Publishing, Moscow, 2000, 376. 2. Badiani, A. et al., Effect of sample preparation with or without shell liquor on contents and retentions of macro and micro elements in three species of bivalve molluscs, in Seafood Research from Fish to Dish, Luten, J.B., Jacobsen, C., Bekaert, K., Sæbø, A., and Oehlenschläger, J., Eds., Wageningen Academic Publishers, Wageningen, the Netherlands, 2006, 459. 3. Causeret, J., Fish as a source of mineral nutrition, in Fish as Food, Vol II, Nutrition, Sanitation and Utilization, Borgstrom G., Ed., Academic Press, New York, 1962, 205. 4. Ebdon, L., Pitts, L., Cornelis, R., Crews, H., Donard, O.F.X., and Quevauviller, Ph., Eds., Trace Element Speciation for Environment, Food and Health, Royal Society of Chemistry, Cambridge, U.K., 2001, 391. 5. Eisler, R., Trace Metal Concentrations in Marine Organisms, Pergamon Press, New York, 1981, 687. 6. Gordon, D.T., Minerals in Seafoods: Availability and interactions, in Seafood Quality Determination, Kramer D.E. and Liston J.L., Eds., Elsevier Science Publishers B.V., Amsterdam, the Netherlands, 1987, 517. 7. Hall, R.A., Zook, E.G., and Meaburn, G.M., National Marine Fisheries Service Survey of Trace Elements in the Fishery Resource, NOAA Technical Report NMFS SSFR-721, U.S. Department of Commerce, 1978, 313. 8. Kiceniuk, J.W. and Ray, S., Eds., Analysis of Contaminants in Edible Aquatic Resources, General Considerations, Metals, Organometallics, Tainting, and Organics, VCH, New York, 1994, 551. 9. Lall, S.P., Macro and trace elements in fish and shellfish, in Fish and Fishery Products, Composition, Nutritive Properties and Stability, Ruiter A., Ed., CAB International, Wallingford, U.K., 1995, 187. 10. Nunes, M.L. et al., Compositional and nutritional value of fishery products consumed in Portugal, in Seafood Research from Fish to Dish, Luten, J.B., Jacobsen, C., Bekaert, K., Sæbø, A., and Oehlenschläger, J., Eds., Wageningen Academic Publishers, Wageningen, the Netherlands, 2006, 477.
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11. Oehlenschläger, J., Identifying heavy metals in fish, in Safety and Quality Issues in Fish Processing, Bremner H.A., Ed., Woodhead Publishing Ltd., Cambridge, U.K., 2002, 95. 12. Oehlenschläger, J., Marine fish—A source for essential elements?!, in Seafood from Producer to Consumer, Integrated Approach to Quality, Luten, J.B., Børresen T., and Oehlenschläger J., Eds., Elsevier Science B.V., Amsterdam, the Netherlands, 1997, 641. 13. Reilly, C., The Nutritional Trace Metals, Blackwell Publishing, Oxford, 2004, 238. 14. Sidwell, V.D., Chemical and Nutritional Composition of Finfishes, Whales, Crustaceans, Molluscs, and Their Products, NOAA Technical memorandum NMFS F/SEC-11, U.S. Department of Commerce, 1981, 432. 15. Souci, S.W., Fachmann, W. and Kraut, H., Food Composition and Nutrition Tables, MedPharm Scientific Publishers, Stuttgart, Germany, 2008, 1364.
Journal articles 16. Nabrrzyski, M. and Gajewska, R., Content of strontium, lithium and calcium in selected milk products and in some marine smoked fish, Nahrung/Food, 46, 204, 2002. 17. Thurston, C.E., Sodium and potassium content of 34 species of fish, J. Am. Diet. Assoc., 34, 396, 1958. 18. Ranau, R., Oehlenschläger, J., and Steinhart, H., Determination of aluminium in the edible part of fish by GFAAS after sample pretreatment with microwave activated oxygen plasma, Fresen. J. Anal. Chem., 364, 599, 1999. 19. Ranau, R., Oehlenschläger, J., and Steinhart, H., Aluminium content in edible parts of seafood, Eur. Food Res. Technol., 212, 431, 2001. 20. Ballin, U., Kruse, R., and Rüssel-Sinn, H.-A., Ergebnisse der Bestimmung von Gesamtarsen und Arsenobetain in Fischen, Krusten-, Schalen- und Weichtieren, Arch. Lebenmittelhyg., 43, 19, 1992. 21. Borak, J. and Hosgood, H.D., Seafood arsenic: Implications for human risk assessment. Reg. Toxicol. Pharmacol., 47, 204, 2007. 22. Buchet, J.P., Pauwels, J., and Lauwreys, R., Assessment of exposure to inorganic arsenic following ingestion of marine organisms by volunteers, Environ. Res., 66, 44, 1994. 23. De Clerck, R. et al., Arsenic levels in cod, flounder and shrimp caught in Belgian coastal waters (1984–1988), Landbouwtijdschrift, 43, 289, 1990. 24. De Dieter, M. et al., Total and toxic arsenic levels in North Sea fish, Arch. Environ. Contam. Toxicol., 43, 406, 2002. 25. Falconer, C.R. et al., Arsenic levels in fish and shellfish from the North Sea, J. Exp. Mar. Biol. Ecol., 71, 193, 1983. 26. Francesconi, K.A. and Edmonds, J.S., Arsenic species in marine samples, Croat. Chem. Acta, 71, 343, 1998. 27. Francesconi, K.A., Arsenic’s not so bad, J. Environ. Monitor., 6, 94N, 2004. 28. Francesconi, K.A., Toxic metal species and food regulations—Making a healthy choice, Analyst, 132, 17, 2007. 29. Ghidini, S., Delbono, G., and Campanini, G., Cd, Hg and As concentrations in fish caught in the North Adriatic Sea, Vet. Res. Commun., 27 Suppl. 1, 297, 2003. 30. Hirata, S. and Toshimitsu H., Determination of arsenic species and arsenosugars in marine samples by HPLC-ICP-MS, Anal. Bioanal. Chem., 383, 454, 2005. 31. Kennedy, V.S., Arsenic concentrations in some coexisting marine organisms from Newfoundland and Labrador, J. Fish. Res. Board Can., 33, 1388, 1976. 32. Larsen, E.H. et al., Determination of inorganic arsenic in white fish using microwave-assisted alkaline alcoholic sample dissolution and HPLC-ICP-MS, Anal. Bioanal. Chem., 381, 339, 2005. 33. Leah, R.T., Evans, S.J., and Johnson, M.S., Arsenic in plaice (Pleuronectes platessa) and whiting (Merlangius merlangus) from the North East Irish Sea, Mar. Pollut. Bull., 24, 544, 1992. 34. Li, W. et al., A survey of arsenic species in Chinese seafood, Food Chem. Toxicol., 41, 1103, 2003.
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35. Luten, J.B. et al., Identification of arsenobetaine in sole, lemon sole, flounder, dab, crab and shrimps by field desorption and fast atom bombardment mass spectrometry, Chemosphere, 12, 131, 1983. 36. Luten, J.B., Riekwel-Booy, G., and Rauchbaar, A., Occurrence of arsenic in plaice (Pleuronectes platessa), nature of organo-arsenic compound present and its excretion by man, Environ. Health Persp., 45, 165, 1982. 37. Meador, J.P., Ernest, D.W., and Kagley, A., Bioaccumulation of arsenic in marine fish and invertebrates from Alaska and California, Arch. Environ. Contam. Toxicol., 47, 223, 2004. 38. Robberecht, H. et al., Daily dietary arsenic intake in Belgium using duplicate portion sampling and elemental content of various foodstuff s, Eur. Food Res. Technol., 214, 27, 2002. 39. Santa Maria, I. et al., Arsenic levels in Chilean marine species, Bull. Environ. Contam. Toxicol. 37, 593, 1986. 40. Schoof, R.A. et al., A market basket survey of inorganic arsenic in food, Food Chem. Toxicol., 37, 839, 1999. 41. Sloth, J.J., Larsen, E.H., and Julshamn, K., Survey of inorganic arsenic in marine animals and marine certified reference materials by anion exchange high-performance liquid chromatography— inductively coupled plasma mass spectrometry, J. Agric. Food Chem., 53, 6011, 2005. 42. Storelli, M.M. and Marcotrigiano, G.O., Organic and inorganic arsenic and lead in fish from the South Adriatic Sea, Italy, Food Add. Contam., 17, 763, 2000. 43. Izak-Biran, T. and Guinn, V.P., Determination of cesium and potassium in marine species by neutron activation analysis, J. Radioanal. Chem., 55, 61, 1980. 44. Bratakos, M.S., Lazos, E.S., and Bratakos S.M., Chromium content of selected Greek foods, Sci. Total Environ., 290, 47, 2002. 45. Cabrera-Vique, C., Chromium presence in foods and beverages: A review, http://www.foodsciencecentral.com/fsc/ixid12724 46. Lendinez, E. et al., Chromium in basic foods of the Spanish diet: Seafood, cereals, vegetables, olive oils and dairy products, Sci. Total Environ., 278, 183, 2001. 47. Vos, G., Hovens, J.P.C., and Hagel, P., Chromium, nickel, copper, zinc, arsenic, selenium, cadmium, mercury and lead in Dutch fishery products 1977–1984, Sci. Total Environ., 52, 25, 1986. 48. Arafa, E.A. et al., Determination of iodine and bromine in fish samples by radiochemical neutron activation analysis, J. Trace Microprobe Techn., 18, 461, 2000. 49. Dahl, L. et al., The iodine content of Norwegian foods and diets, Pub. Health Nutr. 7, 569, 2003. 50. Haldimann, A. et al., Iodine content of food groups, J. Food Comp. Anal., 18, 461, 2005. 51. Julshamn, K. et al., A preliminary study on tailoring of fillet iodine concentrations in adult Atlantic salmon (Salmo salar L.) through dietary supplementation, Aquacult. Nutr., 12, 45, 2006. 52. Karl, H. and Münkner, W., Iod in Fischen und Fischerzeugnissen, Inf. Fischwirtsch., 45, 115, 1998. 53. Karl, H. and Münkner, W., Jod in marinen Lebensmitteln, Ernährungs-Umschau, 46, 288, 1999. 54. Da Silva, E.G.P. et al., Fast method for the determination of copper, manganese and iron in seafood samples, J. Food Comp. Anal., 21, 259, 2008. 55. Yebra, M.C. and Moreno-Cid, A., On-line determination of manganese in solid seafood samples by flame atomic absorption spectrometry, Anal. Chim. Acta, 477, 149, 2003. 56. Zaijun, L., Jiaomai, P., and Jian, T., Determination of trace molybdenum in vegetable and food samples by spectrophotometry with p-carboxyphenylfluorone, Anal. Bioanal. Chem., 374, 11125, 2002. 57. Bourre, J-M.E. and Paquotte, P.M., Contributions (in 2005) of marine and fresh water products (finfish and shellfish, seafood, wild and farmed) to the French dietary intakes of vitamin D and B12, selenium, iodine and docosahexaenoic acid: Impact on public health, Int. J. Food Sci. Nutr., DOI: 10.1080/09637480701553741, 2007. 58. Canbanero, A.I. et al., Quantification and speciation of mercury and selenium in fish samples of high consumption in Spain and Portugal, Biol. Trace Elem. Res., 103, 17, 2005. 59. Diaz-Alarcon, J.P. et al., Determination of selenium in fresh fish from Southeastern Spain for calculation of daily dietary intake, J. Agric. Food Chem., 42, 334, 1994.
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60. Finley, J.W., Increased intakes of selenium-enriched foods may benefit human health, J. Sci. Food Agric., 87, 1620, 2007. 61. Fox, T.E. et al., Bioavailability of selenium from fish, yeast and selenate: A comparative study in humans using stable isotopes, Eur. J. Clin. Nutr., 58, 343, 2004. 62. McNaughton, S.A. and Marks, G.C., Selenium content in Australian foods: A review of literature values, J. Food Comp. Anal., 15, 169, 2002. 63. Murphy, J. and Cashman, K.D., Selenium content of a range of Irish foods, Food Chem., 74, 493, 2001. 64. Önning, G., Separation of soluble selenium compounds in different fish species, Food Chem., 68, 133, 2000. 65. Ørnsrud, R. and Lorentzen, M., Bioavailability of selenium from raw or cured selenomethionineenriched fillets of Atlantic salmon (Salmo salar) assessed in selenium-deficient rats, Br. J. Nutr., 87, 13, 2002. 66. Pappa, E.C., Pappas, A.C., and Surai, P.F., Selenium consent in selected foods from the Greek market and estimation of the daily intake, Sci. Total Environ., 372, 100, 2006. 67. Plessi, M., Bertellini, D., and Monzani, A., Mercury and selenium content in selected seafood, J. Food Comp. Anal., 14, 461, 2001. 68. Rayman, M.P., The importance of selenium to human health, Lancet, 356, 233, 2000. 69. Sager, M., Selenium in agriculture, food, and nutrition, Pure Appl. Chem., 78, 111, 2006. 70. Satovic, V., Beker, D., and Gumhalter-Karolyi, L., Selenium content in the pilchard of the Adriatic Sea, Eur. Food Res. Technol., 217, 154, 2003. 71. Sirichakwal, P.P. et al., Selenium content of Thai foods, J. Food Comp. Anal., 18, 47, 2005. 72. Ventura, M.G. et al., Selenium content in selected Portuguese foodstuffs, Eur. Food Res. Technol., 224, 395, 2007. 73. Wyatt, C.J. et al., Selenium (Se) in foods in Northern Mexico, their contribution to the daily Se intake and the relationship of Se plasma levels and glutathione peroxidase activity, Nutr. Res., 16, 949, 1996. 74. Das, A.K. et al., Determination of thallium in biological samples, Anal. Bioanal. Chem., 385, 665, 2006. 75. Bu-Olayan, A.H. and Al-Yakoob, S., Lead, nickel and vanadium in seafood: An expose assessment for Kuwaiti consumers, Sci. Total Environ., 223, 81, 1998. 76. Lavilla, I., Vilas, P., and Bendicho C., Fast determination of arsenic, selenium, nickel and vanadium in fish and shellfish by electrothermal atomic absorption spectrometry following ultrasound-assisted extraction, Food Chem., 106, 403, 2008. 77. Myron, D.R., Givand, S.H., and Nielsen, F.H., Vanadium content of selected foods as determined by flameless atomic absorption spectroscopy, J. Agric. Food Chem., 25, 297, 1977. 78. Seixas, S. and Pierce, G.J., Vanadium, rubidium and potassium in Octopus vulgaris (Mollusca: Cephalopoda), Sci. Mar., 69(2), 215, 2005. 79. Sepe, A. et al., Determination of cadmium, chromium, lead and vanadium in six fish species from the Adriatic Sea, Food Addit. Contamin., 20, 543, 2003. 80. Celik, U. and Oehlenschläger, J., Determination of zinc and copper in fish samples collected from Northeast Atlantic by DPSAV, Food Chem., 87, 343, 2004. 81. Celik, U. and Oehlenschläger, J., Zinc and copper content in marine fish samples collected from the eastern Mediterranean Sea, Eur. Food Res. Technol. 220, 37, 2005. 82. Scherz, H. and Kirchhoff E., Trace elements in foods: Zinc content of raw foods—A comparison of data originating from different geographical regions of the world, J. Food Comp. Anal., 19, 420, 2006. 83. Astorga Espana, M.S., Rodriguez Rodriguez, E.M., and Diaz Romero, C., Comparison of mineral and trace element concentrations in two molluscs from the Strait of Magellan (Chile), J. Food Comp. Anal., 20, 273, 2007. 84. Banaoui, A. et al., Trace metal distribution in the mussel Perna perna along the Moroccan coast, Mar. Pollut. Bull., 48, 378, 2004.
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85. Bustamante, P. et al., Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean, Polar Biol., 19, 264, 1998. 86. Cheung, Y.H. and Wong, M.H., Trace metal contents of the Pacific oyster (Crassostrea gigas) purchased from markets in Hong Kong, Environ. Manage., 16, 753, 1992. 87. Gökoðlu, N. and Yerlikaya, P., Determination of proximate composition and mineral contents of blue crab (Callinectes sapidus) and swim crab (Portunus pelagicus) caught off the Gulf of Antalya, Food Chem., 80, 495, 2003. 88. Gokoglu, N., Gokoglu, M., and Yerlikaya, P., Seasonal variations in proximate and elemental composition of pearl oyster (Pinctada radiata, Leach, 1814), J. Sci. Food Agr., 86, 2161, 2006. 89. Gokoglu, N., Yerlikaya, P., and Gokoglu, M., Trace elements in edible tissues of three shrimp species (Penaeus semisulcatus, Parapenaeus longirostris and Paleomon serratus), J. Sci. Food Agr., 88, 175, 2008–07–25. 90. Hsieh, Y-H.P., Leong, F-M., and Barnes, K.W., Inorganic constituents in fresh and processed cannonball jellyfish (Stomolophus meleagris), J. Agric. Food Chem., 44, 3117, 1996. 91. Ichihashi, H. et al., Multielemental analysis of purpleback flying squid using high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS), Environ. Sci. Technol., 35, 3103, 2001. 92. Ichihashi, H. et al., Multi-elemental concentrations in tissues of Japanese common squid (Todarodes pacificus), Arch. Environ. Contam. Toxicol., 41, 483, 2001. 93. Kücükgülmez, A. et al., Proximate composition and mineral content of the blue crab (Callinectes sapidus) breast meat, claw meat and hepatopancreas, Int. J. Food Sci. Technol., 41, 1023, 2006. 94. Mohapatra, A. et al., Trace elemental characterization of some food crustacean tissue samples by EDXRF technique, Aquaculture, 270, 552, 2007. 95. Orban, E. et al., Nutritional and commercial quality of the striped venus clam, Chamelea gallina, from the Adriatic Sea, Food Chem., 101, 1063, 2006. 96. Seixas, S., Bustamante, P., and Pierce, G.J., Interannual patterns of variation in concentrations of trace elements in arms of Octopus vulgaris, Chemosphere, 59, 1113, 2005. 97. Skonberg, D.I. and Perkins, B.L., Nutrient composition of green crab (Carcinus maenus) leg meat and claw meat, Food Chem., 77, 401, 2002. 98. Vernocchi, P. et al., Characterization of Mediterranean mussels (Mytilus galloprovincialis) harvested in Adriatic Sea (Italy), Food Control, 18, 1575, 2007. 99. Vyncke, W. et al., Trace metals in cut through shell (Spisula subtruncata) from Belgian coastal waters, Food Addit. Contam., 16, 1, 1999. 100. Agusa, T. et al., Concentrations of trace elements in muscle of sturgeons in the Caspian Sea, Mar. Pollut. Bull., 49, 789, 2004. 101. Agusa, T. et al., Exposure assessment for trace elements from consumption of marine fish in Southeast Asia, Environ. Pollut., 145, 766, 2007. 102. Anthony, J.E. et al., Yields, proximate composition and mineral content of finfish and shellfish, J. Food Sci., 48, 313, 1983. 103. Begum, A. et al., Selected elemental composition of the muscle tissue of three species of fish, Tilapia nilotica, Cirrhina mrigala and Clarius batrachus, from the fresh water Dhanmondi Lake in Bangladesh, Food Chem., 93, 439, 2005. 104. Burger, J. and Gochfeld, M., Heavy metals in commercial fish in New Jersey, Environ. Res., 99, 403, 2005. 105. Carvalho, M.L., Santiago, S., and Nunes, M.L., Assessment of the essential element and heavy metal content of edible fish muscle, Anal. Bioanal. Chem., 382, 426, 2005. 106. Chen, Y-C. and Chen, M-H., Heavy metal concentrations in nine species of fishes caught in coastal waters off Ann-Ping, S.W. Taiwan, J. Food Drug Anal., 9, 107, 2001. 107. Demirezen, D. and Uruc, K., Comparative study of trace elements in certain fish, meat and meat products, Meat Sci., 74, 255, 2006. 108. El-Faer, M.Z. et al., Mineral and proximate composition of some commercially important fi sh of the Arabian Gulf, Food Chem., 45, 95, 1992.
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109. Engman, J. and Jorhem, L., Toxic and essential elements in fish from Nordic waters, with the results seen from the perspective of analytical quality assurance, Food Addit. Contam., 15, 884, 1998. 110. Fabris, G., Turoczy, N.J., and Stagnitti, F., Trace metal concentrations in edible tissue of snapper, flathead, lobster, and abalone from coastal waters of Victoria, Australia, Ecotoxicol. Environ. Safety, 63, 286, 2006. 111. Gordon, D.T. and Roberts, G.L., Mineral and proximate composition of Pacific Coast fish, J. Agric. Food Chem., 25, 1282, 1977. 112. Gras, N. et al., A study on some trace elements in Chilean seafoods, J. Radioanal. Nucl. Chem., 169, 247, 1993. 113. Güner, S. et al., Proximate composition and selected mineral content of commercially important fish species from the Black Sea, J. Sci. Food Agr., 78, 337, 1998. 114. Huang, W-B., Heavy metal concentrations in the common benthic fishes caught from the coastal waters of Eastern Taiwan, J. Food Drug Anal., 11, 324, 2003. 115. Kalay, M., Ay, Ö., and Canli, M., Heavy metal concentrations in fish tissues from the Northeast Mediterranean Sea, Bull. Environ. Contam. Toxicol., 63, 673, 1999. 116. Karakotsidis, P.A., Zotos, A., and Constantinides, M., Composition of the commercially important Mediterranean finfish, crustaceans, and molluscs, J. Food Comp. Anal., 8, 258, 1995. 117. Krelowska-Kulas, M., Content of some metals in mean tissue of salt-water and fresh-water fish and in their products, Nahrung, 39, 166, 1995. 118. Kwoczek, M. et al., Essential and toxic elements in seafood available in Poland from different geographical regions, J. Agric. Food Chem., 54, 3015, 2006. 119. Maher, W. et al., Characteristics of selenium in Australian marine biota, Appl. Organometal. Chem., 6, 103, 1992. 120. Mormede, S. and Davies, I.M., Trace elements in deep-water fish species from the Rockall Trough, Fish. Res. 51, 197, 2001. 121. Ogunlade, I., Olaofe, O., and Fadare, T., Chemical composition, amino acids and functional properties of selected seafood, J. Food Agric. Environ., 3, 130, 2005. 122. Ruelas-Inzunza, J. and Paez-Osuna, F., Essential and toxic metals in nine fish species for human consumption from two coastal lagoons in the Eastern Gulf of California, J. Environ. Sci. Health Part A, 42, 1411, 2007. 123. Sharif, A.K.M. et al., Determination of arsenic, chromium, mercury, selenium and zinc in tropical marine fish by neutron activation, J. Radioanal. Nucl. Chem., 170, 299, 1993. 124. Sharif, A.K.M. et al., Trace element concentrations in ten species of freshwater fish of Bangladesh, Sci. Total Environ., 138, 117, 1993. 125. Sidoumou, Z. et al., Distribution and concentration of trace metals in tissues of different fish species from the Atlantic coast of Western Africa, Bull. Environ. Contam. Toxicol., 74, 988, 2005. 126. Sivaperumal, P., Sankar, T.V., and Viswanathan Nair, P.G., Heavy metal concentrations in fish, shellfish and fish products from internal markets of India vis-à-vis international standards, Food Chem., 102, 612, 2007. 127. Tahvonen, R. et al., Mineral content in Baltic herring and Baltic herring products, J. Food Comp. Anal., 13, 893, 2000. 128. Tallandini, L. et al., Naturally occurring levels of elements in fishes as determined by PIXE and XRF methods, Nuclear Instr. Meth. Phys. Res., B40/41, 630, 1989. 129. Teeny, F.M. et al., Mineral composition of the edible muscle tissue of seven species of fish from the Northeast Pacific, J. Agric. Food Chem., 32, 852, 1984. 130. Türkmen, A. et al., Heavy metals in three commercially valuable fish species from Iskenderun Bay, Northern East Mediterranean Sea, Turkey, Food Chem., 91, 167, 2005. 131. Devesa, V. et al., Arsenic in cooked seafood products: Study on the effect of cooking on total and inorganic arsenic content, J. Agric. Food Chem., 49, 4132, 2001. 132. Karl, H. et al., Changes of the iodine content in fish during household preparation and smoking, Deutsche Lebensm.-Rundsch., 101, 1, 2005.
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133. Mierke-Klemeyer, S. et al., Retention of health-related beneficial components during household preparation of selenium-enriched African catfish (Clarias gariepinus) fillets, Eur. Food Res. Technol., 227, 827, 2008. 134. Capelo, J.L. et al., Trends in selenium determination/speciation by hyphenated techniques based on AAS or AFS, Talanta, 68, 1442, 2006. 135. http://irmm.jrc.ec.europa.eu/html/CRLs/crl_heavy_metals/legislation/Official_methods_for_the_ determination_of_heavy_metals_in_feed_and_food.pdf 136. Kellner, R. et al., Analytical Chemistry: A Modern Approach to Analytical Science, 2nd edn., John Wiley & Sons, Weinheim, Germany, 2004, 1209.
Chapter 21
Analysis of n-3 and n-6 Fatty Acids Vittorio M. Moretti and Fabio Caprino Contents 21.1 Introduction ................................................................................................................. 377 21.2 Analysis of Total Fatty Acids .........................................................................................379 21.2.1 Lipid Extraction ...............................................................................................379 21.2.2 Preparation of Fatty Acid Derivatives .............................................................. 380 21.2.2.1 Acid-Catalyzed Esterification and Transesterification ..................... 380 21.2.2.2 Diazomethane................................................................................. 382 21.2.2.3 Base-Catalyzed Transesterification .................................................. 382 21.2.3 Gas Chromatographic Analysis ....................................................................... 382 21.2.4 Column........................................................................................................... 383 21.2.5 Mass Spectrometry of Fatty Acid Derivatives .................................................. 384 References ............................................................................................................................... 387
21.1
Introduction
The last two decades have seen an exponential increase of interest in the health effects of n-3 fatty acids from fish oils. Several reviews are available on their physiological roles and functions,1,2 on their tissue distribution in humans,3 on their structural and functional roles in cellular membranes,4 on their role in gestation and parturition,5 inflammation,6 immune response and autoimmunity,7 cortical and retinal developments,8–10 cardiovascular disease,11–13 cancer,14–16 and cellular life span.17 377
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Clinical and epidemiological studies indicated that the consumption of fish and fish oils can prevent certain human cardiovascular conditions.18 These potential healthy properties of marine lipids renewed interest in investigating the lipid content and the fatty acid composition of fish and seafood products around the world. Studies were especially concentrated on the content and the availability of long-chain polyunsaturated fatty acids (PUFAs) that are typical of the aquatic habitats. Examples of PUFA include eicosapentanoic acid (EPA), and docosahexaenoic acid (DHA). The fatty acid composition of fish, shellfish, seafood products, and encapsulated fish oils products have been extensively treated and discussed.19,20 Fish oils and marine lipids are considered to be the main source of n-3 PUFAs in the human diet. They are composed of complex fatty acids, typically an even-carbon chain length from C14 to C24 with 0–6 methylene interrupted double bonds. Marine lipids also contain minor amounts of less common fatty acids with nonmethylene interrupted and branched-chain fatty acids.21 According to the popular shorthand system PUFAs of interest to biochemists, food scientists, and nutritionists can be indicated with the chain length (e.g., 22): number of cis ethylenic bonds (e.g., 6) and number of carbon atoms counted from the terminal methyl group (e.g., n-3). For example, DHA is equivalent to 22:6n-3. n-3 and n-6 fatty acids, in particular linoleic acid (18:2n-6), a-linolenic acid (18:3n-3), EPA (20:5n-3), and DHA (22:6n-3), are generally known as essential fatty acids (EFAs) in vertebrates.22 These fatty acids cannot be completely synthesized de novo in the body and consequently Table 21.1 Pathways of the Biosynthesis of C20 and C22 PUFA from n-3 and n-6 C18 Precursors n-3 Pathway
n-6 Pathway
18:3n-3 (α-linolenic acid)
18:2n-6 (linoleic acid)
↓ Δ6 desaturase
↓ Δ6 desaturase
18:4n-3 (stearidonic acid)
18:3n-6 (γ-linolenic acid)
↓ Elongase
↓ Elongase
20:4n-3
20:3n-6 (dihomo-γ-linolenic acid)
↓ Δ5 desaturase
↓ Δ5 desaturase
20:5n-3 (EPA)
20:4n-6 (arachidonic acid)
↓ Elongase
↓ Elongase
22:5n-3 (docosapentaenoic acid)
22:4n-6 (adrenic acid)
↓ Elongase
↓ Elongase
24:5n-3
24:4n-6
↓ Δ6 desaturase
↓ Δ6 desaturase
24:6n-3
24:5n-6
↓ Peroxisomal oxidation
↓ Peroxisomal oxidation
22:6n-3 (docosaesahexaenoic acid)
22:5n-6
Analysis of n-3 and n-6 Fatty Acids
◾ 379
must be supplied in the diet. Humans and animals cannot interconvert n-3 and n-6 fatty acids. In some, but not in all (e.g., lions and cats23), vertebrate species the long chain–polyene acids of the n-3 and n-6 families are biosynthesized through a combination of elongation and desaturation reactions, starting from a-linolenic and linoleic acids, respectively. These processes are particularly important in animal systems and are required for the production of C20 and C22 PUFAs, which are often termed highly unsaturated fatty acids (HUFAs), as the precursors of biologically active eicosanoids.24 Although these statements also apply to fish, the extent to which they apply quantitatively to a given fish species varies widely. In fish species that cannot perform these conversions, the C20 and C22 HUFAs are dietary EFAs and their C18 homologues do not satisfy EFA requirements in the fish diet.25 The elongation and the desaturation pathways, whereby the C18 is converted to their C20 and C22 counterparts in fish, are reasonably understood and are presented in Table 21.1. For a more extensive information about the classification and the nomenclature of fatty acids and lipids the reader is referred to Fahy et al.,26 Lobb and Chow,27 and Robinson.28
21.2 Analysis of Total Fatty Acids Usually, the analysis of fatty acids in a fish tissue involves mainly three steps: lipid extraction, preparation of fatty acid derivatives, and gas chromatographic (GC) analysis. For decades, GC has been the most applied method for fatty acids analysis.29–32 The success of GC with flame ionization detector (FID) for the analysis of fatty acids is based on the ability of this technique to separate dozens of fatty acids depending on the type and the length of the column, and on the economical accessibility of the GC instrumentation that is actually present in all analytical laboratories. The advent of wall-coated open tubular (WCOT) capillary column, available in a wide range of different stationary phases, has led to an excellent resolution capability of this technique. Specific separation problems of fatty acids connected to specific applications could be solved by the alternative methodologies of sample preparation.33,34
21.2.1
Lipid Extraction
Fish lipids are prone to oxidation, and should be analyzed immediately after sampling to minimize the changes occurring in lipid components. When the immediate extraction is not feasible, the sample should be frozen as soon as possible, possibly in liquid nitrogen or dry ice, and stored in glass containers under nitrogen at −80°C. Both wet fish tissues and organic solvents must not come in contact with any plastic ware, since plasticizers are very easily leached out and could be co-chromatographed with fatty acids causing severe interferences in chromatograms. Such compounds (usually the esters of phthalic acid) are characterized by an abundant base peak at m/z 149 in their mass spectra. Also any source of contamination by mineral oils and detergents should be avoided. It is usually advisable to add an appropriate antioxidant, such as butylated hydroxy toluene (BHT), at 50–100 mg L−1 level to the solvent in which lipids are dissolved. Optimal conditions for sample handling and lipid storage were extensively reviewed by Christie.35 The most cited methods for lipid extraction in research papers are the Bligh and Dyer36 and the Folch et al.37 methods. These methods are based on the use of a chloroform/methanol mixture, with the water content of the matrix as a tertiary component, or with an appropriate addition of water in order to obtain a tertiary system. The food tissue is usually homogenized in the presence of such mixtures using an Ultra-Turrax or a Waring blender. The main differences between the
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two methods are in the solvent/sample ratio and in the chloroform/methanol ratio. In the Bligh and Dyer method, the solvent/sample ratio is 1 part sample to 3 parts 1:2 chloroform/methanol, followed by 1 part chloroform. In contrast, the Folch method employs a ratio of 1 part sample to 20 parts 2:1 chloroform/methanol mixture, followed by the washing of the extract with a weak salt solution. Many modifications of the original procedures have been published38–41 in order to improve lipid extraction in certain food matrices or for particular applications. Iverson et al.42 compared the two methods for total lipid extraction in a broad range of fish and seafood tissues, and found that the Bligh and Dyer method produced lower amount of lipid content in fi sh samples containing >2% lipid when compared to the Folch method. The authors explained this incomplete lipid yield from several standpoints, mainly with the limited solubility of triacylglycerols in the chloroform/methanol mixture (1:2, v/v). To overcome this problem, Christie35 suggested, in the presence of fatty samples, to add a preliminary extraction step with a nonpolar solvent, such as chloroform or diethyl ether, prior to the Bligh and Dyer procedure. As a general consideration, both Bligh and Dyer, and Folch methods produce reliable results when applied to fish samples, if performed respecting the solvent/sample ratio. Unfortunately, in many research papers these methods are modified, but modifications to the procedures are neither described nor validated. Christie has reasonably argued that these extraction methods are often misunderstood and therefore misused.35
21.2.2
Preparation of Fatty Acid Derivatives
Prior to GC analysis, the lipid sample has to be hydrolyzed and fatty acids converted into nonpolar derivatives, usually methyl esters (FAMEs). The formation of FAMEs is normally performed on the lipid sample in the presence of a catalyst dissolved in or mixed with methanol. A heat treatment may be applied to the sample, that may last from few minutes to several hours, depending on the temperature and on which catalyst is used. Several types of acid- or base-catalyzed reactions are suitable for the transesterification of lipids. These reactions have been extensively described by several authors.29,31,32,43–48
21.2.2.1
Acid-Catalyzed Esterification and Transesterification
Three common used acid reagents are hydrogen chloride, sulfuric acid, and boron trifluoride, all mixed or dissolved in methanol. These acidic reagents not only transesterify triacylglycerols and other complex lipids but also esterify free fatty acids (FFAs) present in the lipid sample. Heating of the sample is required to quicken the reaction. Among them, the most frequently used reagent for FAME preparation is 5% anhydrous hydrogen chloride in methanol, which is usually prepared by bubbling dry gaseous hydrogen chloride into dry methanol. The reagent may also be prepared by adding acetyl chloride slowly to a large excess of dry methanol (1:10, v/v) under ice. As a by-product methyl acetate is formed, but it does not interfere with the reaction.43 Th is reagent is reported to have limited stability49 and should not be used if stored for more than 1 week. In a typical procedure, to the fish oil (5–10 mg) tricosanoic acid (23:0) is added as internal standard, the sample is dissolved in 1 mL of toluene in a test tube and methanolic hydrogen chloride (2 mL) is added. The sample is then left overnight at 50°C in a stoppered tube or refluxed for about 2 h. After the addition of a potassium carbonate solution, the FAMEs are extracted thoroughly with hexane. The hexane phase is then dried over anhydrous sodium sulfate.
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With this reagent, all fatty acids are transesterified at the same reaction rate. An inert solvent has to be added to the reaction mixture (toluene) to facilitate the formation of FAMEs, as triacylglycerols, which often represent more than 80%–90% of the total lipid sample, are not soluble in methanolic hydrogen chloride. Among solvents used, toluene, tetrahydrofuran, and methyltert-butyl ether are quite effective. Under given reaction conditions, the effect of added solvent type has been studied.50 The main disadvantages of this derivatization procedure are the relatively long time needed to complete the reaction and that it is not suited for some sensitive functional groups, such as epoxyl, cyclopropane, or cyclopropene rings.43 An alternative acid catalyst is sulfuric acid in methanol. The reagent could be obtained by adding concentrated sulfuric acid to methanol at the concentration of 1%–2%. The reaction times recommended are the same used with methanolic hydrogen chloride, 2 h under reflux or overnight at 50°C. When using sulfuric acid as the catalyst, care should be taken to avoid concentration and temperature higher than those commonly recommended. As sulfuric acid is a strong oxidizing agent, excessive concentration or high temperatures could lead to the destruction of PUFAs of fish oils.48 It is worth noting that PUFAs must always be handled with care and should not be subjected to more vigorous conditions than are necessary. In general, methanolic hydrogen chloride (5%) or methanol–sulfuric acid (1%) is considered to be the best general purpose transesterifying reagent. They transesterify O-acyl lipids efficiently under reflux for 2 h or overnight in a stoppered tube at 50°C. The derivatization procedure has not been reported to cause any isomerization of monounsaturated fatty acids. However, there is some controversy over whether derivatization causes the isomerization of geometrical isomers of conjugated linoleic acid. Kramer et al.51 have evaluated acid and base catalysts in the esterification of milk fat. They concluded that acid-catalyzed methods caused extensive isomerization of conjugated dienes and formed allylic methoxy artifacts, and therefore they did not recommend the use of these reagents for the determination of conjugated linoleic acids in milk fat. Also boron trifluoride in methanol is a powerful transesterifying reagent for fatty acids.52,53 This reagent could be used to transesterify most lipid classes at the concentration of 6%–14%, at reaction temperature from 80°C to 100°C, and time taken for transesterification ranges from 2 to 60 min. A procedure using alkali transesterification and boron trifluoride/methanol has been adopted both by the Association of Official Analytical Chemists (AOAC) and by the American Oil Chemists’ Society (AOCS), and are the recommended official methods for the determination of fatty acids in fish oils.54,55 These methods have two steps. The first step is a brief treatment of the lipid sample with alkali (commonly NaOH in methanol). The second step is the heating of the sample in BF3 –methanol for 2 min. Some criticism on these official methods has been reported by Ackman.56 He suggested to omit the alkali transesterification step, and used equal volumes of BF3 –methanol at 7% concentration and of n-hexane in order to reduce the concentration of the catalyst to 3.5% of the total volume, and heated for 1 h at 100°C in a screw-cap tube flushed with nitrogen. According to Christie,43 the BF3 method has serious drawbacks. The main one is the formation of methoxy artifacts from unsaturated fatty acids by the addition of methanol across the double bond,57 when high concentrations of BF3 in methanol are used. In addition, there is some evidence that the formation of artifacts is more likely when aged reagents are used. Christie, in view of the many side reactions and the high acid content in comparison with other reagents, did not consider to use this method in his own laboratory.43
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Handbook of Seafood and Seafood Products Analysis
Diazomethane
Diazomethane is another methylation reagent.58 Compared to acid catalysts, diazomethane esterifies fatty acids more rapidly, but it has no ability to catalyze the transesterification of lipids. As a consequence, when diazomethane is used for the preparation of FAMEs from lipids, an alkaline hydrolysis step has to precede. Diazomethane is generally prepared in ethereal solution by the action of alkali on nitrosamide in the presence of an alcohol.59 It is potentially explosive and great care must be taken during its preparation, and apparatus with ground-glass joints and strong light must be avoided. Th is reagent has a short life and must be prepared fresh daily. Suitable microapparatus are commercially available for small-scale diazomethane preparation.
21.2.2.3
Base-Catalyzed Transesterification
Compared with acid catalysts, base catalysts transesterify neutral lipids in anhydrous methanol at a much faster speed. However, they are unable to esterify FFAs. Furthermore, the reaction requires more rigid anhydrous conditions because the presence of water leads to the irreversible hydrolysis of lipids. A comprehensive review on this group of reagents has been reported by Bannon et al.60 The most popular, basic transesterifying reagent is 0.5–2.0 M sodium methoxide in anhydrous methanol, which is prepared by dissolving fresh clean sodium in methanol. Other reagents include potassium hydroxide or sodium hydroxide in methanol. The reaction is very rapid. For example using 0.5 M sodium methoxide in dry methanol, phosphoglycerides could be completely transesterified in a few minutes at an ambient temperature.48 Two drawbacks associated with alkaline catalysts are their inability to esterify FFAs and their requirement of more anhydrous condition during the reaction. Therefore, this method cannot be used for any oil if much FFAs are present, often a problem with raw fi sh oils, because these FFAs remain as FFAs. A comparison of the features of the common methods used for transesterification/esterification of fatty acids is presented in Table 21.2.
21.2.3 Gas Chromatographic Analysis The GC analysis of fi sh and marine oils generally requires simultaneous separation and quantitation of 25 or more fatty acids. Obviously, the number of fatty acids detected depends on many factors; mainly the type of column used, separation conditions, sample loading, availability of authentic standards, objective of the study, and on the skill of the analyst, who is required to investigate a larger number of minor and novel fatty acids.21 For routine use, split injection is by far the most practical means of sample introduction. The essential problem of this type of injection is that of achieving a linear split of the injected sample at the column inlet.61 Bayer and Liu have listed many factors that have been claimed to give rise to nonlinear splitting,62 and have studied these factors. In a particularly readable report on this subject Grob 63 has described various injection techniques, and reviewed various existing problems and solutions.64 The commonly used carrier gases in capillary GC are helium and hydrogen. Ackman 29 recommends the use of hydrogen, which is less expensive than helium and at optimum flow rates hydrogen takes 1.5 times less for an analysis.
Analysis of n-3 and n-6 Fatty Acids
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Table 21.2 A General Comparison among Commonly Used Transesterifying/Esterifying Reagents Acid in MeOH Features
HCl
Alkali in MeOH
H2SO4
BF3
NaOCH3
1%–2%
6%–14%
0.2–2 N
KOH
NaOH
In Ether CH2N2
Common concentration
5%
Common reaction temperature
50°C-refluxing
Common reaction time
30 min–2 h 30 min–2 h 2–10 min Seconds–1 h
Esterifying power
Medium
Medium
High
No
No
No
High
Transesterifying power
Low
Low
Low
High
High
High
No
Ease of preparation
No
Yes
No
No
Yes
Yes
No
Potential hazard associated with preparation
Yes
No
Yes
Yes
No
No
Yes
Saponification after reaction
No
No
No
Yes
Yes
Yes
No
Sensitive to water interference
Low
Low
Low
High
High
High
Low
Ambient-refluxing
Ambient Minutes
Source: Modified from Liu, K.-S., J. Am. Oil Chem. Soc., 71(11), 1179, 1994. With permission.
21.2.4
Column
Commercially available WCOT capillary columns are made of capillary tubing (0.05–0.53 mm internal diameter) with a film (thickness 0.1–8.0 mm) of stationary phase uniformly applied to the inside of the capillary wall.65 Basically, the separation of fatty acid methyl esters can be performed on three types of WCOT capillary columns coated with nonpolar, polar, and very polar stationary phases depending on the type of lipid sample to be analyzed and on the objectives of the study.29,31,32 The choice of the stationary phase obviously affects the retention time and the resolution of the analytical method. The use of apolar column, such as DB-5 (5% phenyl 95% dimethyl polysiloxane), leads to a separation profile that is rather different from that obtained with polar columns, with unsaturated fatty acids eluted ahead of saturated fatty acids of the same chain length. The main disadvantage of these columns is the partial overlapping of some unsaturated fatty acids. In fact, 18:2n-6 is not fully resolved from 18:1n-9 and coelutes with 18:3n-3, and this is also true for the corresponding C20 and C22 fatty acids.66 For these reasons, these columns are practically not used for the separation of fish oil fatty acids, although they could have some advantages in GC–mass spectrometry applications (GC–MS), due to their low grade of bleed and their stability at high temperatures.
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Among polar columns, mainly two types of stationary phases of progressive polarity can be chosen for the analysis of FAMEs. In the polyethylene glycol stationary phases (i.e., DB-Wax, Supelcowax-10, Omegawax, AT-FAME), a broad range of fatty acids from C4 to C24 can be separated according to the number of carbon atoms and to the degree of unsaturation. The use of polyethylene glycol columns is widely accepted and these columns are used for the analysis of a wide range of samples, such as vegetable oils, animal fats, and fish and marine oils,67–72 with an excellent separation of n-3 and n-6 fatty acids. On these columns, the separation of geometrical cis and trans isomers cannot be obtained. Therefore, for the separation of complex mixtures of unsaturated FAMEs, containing many positional and geometrical isomers of monoenoic, dienoic, and trienoic fatty acids, as in the partially hydrogenated vegetable or marine oils, an additional resolution is needed. Better resolution and separative performance are obtained using capillary columns coated with 50% (DB-23) to 100% of cyanopropyl polysiloxane phase, such as SP2340,73–75 SP-2380, SP-2560,76,77 CP-Sil 88,77–85 HP-88,86 BPX-70,87 or AT-Silar-10088 (see Table 21.3). It is generally recognized that columns coated with cyanopropyl phase are mandatory for the GC analysis of cis and trans isomers of fatty acids. Both the AOCS Official Method Ce 1h-05,89 developed for the determination of cis, trans, saturated, and monounsaturated PUFAs in vegetable or nonruminant animal oils and fats76 and the AOAC International Official Method of Analysis 996.0690 developed for the determination of total, saturated, and monounsaturated fats in foodstuff s91 recommend the use of this type of high polarity columns for the analysis of FAMEs isomers. As a general rule, an optimal separation of a classic fish oil can be obtained efficiently with polyethylene glycol columns 30 m length × 0.25 mm i.d., as already mentioned. A medium-polarity cyanopropyl column, such as DB-23, is suitable for the analysis of complex mixtures of PUFAs, including n-3 fatty acids, and the partial separation of 18:1 isomers could be obtained. When detailed information on positional and geometrical isomers has to be acquired, the use of a 100-m column coated with 100% of a cyanopropyl phase is recommended. In the GC analysis of fatty acids on a polyethylene glycol column, the key limitation is the incomplete separation of trans-monoenoic from cis-monoenoic fatty acids. These overlaps can be partially overcome by using a 100-m cyanopropyl column operating at 180°C, as demonstrated by Ratnayake et al.76
21.2.5 Mass Spectrometry of Fatty Acid Derivatives GC, coupled to mass spectrometry (GC–MS), is the most powerful technique for the structural analysis of fatty acid mixtures, in particular for the determination of the position of double bonds in isomeric fatty acids.92 As reported above, fatty acids are generally analyzed by GC–FID as methyl ester derivatives. Unfortunately, the structural information obtained from the mass spectra of FAMEs is frequently of limited value. For example, the position of the double bond in the aliphatic chain cannot be determined due to the bond migration that occurs during the ionization.93 To overcome this problem, the analyst has two choices. One of them is the preparation of specific adducts with the double bonds that give a specific fragmentation pattern.94 The second approach is the derivatization of the fatty acid carboxyl group with a reagent containing a nitrogen atom.95 When the derivatized fatty acid is ionized in the mass spectrometer, the nitrogen atom carries the charge and consequently the double bond ionization and the migration are minimized.
Low Medium/high Medium/high Medium/high High High
High High High High
50% cyanopropylphenyl-50% dimethylpolysiloxane
Polyalkylene glycol
Polyalkylene glycol
Polyethylene glycol
Polyethylene glycol
Polyethylene glycol
Polyethylene glycol
Polyethylene glycol
50% cyanopropyl-50% methylpolysiloxane
Polarity
5% diphenyl-95% dimethylpolysiloxane
Phase
DB-23; AT-225
CP-Wax 57 CB
Supelcowax 10; ZEBRON ZB-WAX
Omegawax; FAMEWAX; AT-FAME
HP-INNOWax; Stabilwax; Cp-Wax 52 CB; ZEBRON ZB-WAX
DB-WAX; Rt-Wax
PAG
SPB-PUFA
DB-225; Rtx-225; SPB-225; CP-Sil 43 CB
DB-5; HP-5; Rtx-5 CPSil-8 CB; SPB-5; Equity-5
Column
40°C to 250°C/260°C
20°C to 200°C/225°C
35°C to 280°C
50°C to 280°C
40°C to 260°C/270°C
20°C to 250°C/260°C
30°C to 220°C
50°C to 220°C
40°C to 220°C/240°C
−60°C to 325°C/350°C
Temperature Limit
Table 21.3 Commonly Used Stationary Phases for the GC Analysis of Fatty Acid Methyl Esters
(continued)
Complex mixtures, partial cis/trans separation
Long-chain fatty acid mixture, fish oils
Applications
Analysis of n-3 and n-6 Fatty Acids ◾ 385
High High High High High
80% biscyanopropyl-20% cyanopropylphenylpolysiloxane
88% cyanopropyl 12% methylarylpolysiloxane
90% biscyanopropyl-10% cyanopropylphenylpolysiloxane
100% biscyanopropyl polysiloxane
Polarity
70% biscyanopropyl-30% cyanopropylphenylpolysiloxane
Phase
SP-2560; SP-2340; Rt-2560; CP-Sil 88; AT-SILAR-100
SP-2380; Rt-2330; AT-SILAR-90; BPX-90
HP-88
SP-2330; AT-SILAR-90;
BPX-70
Column
Subambient to 250°C
Subambient to 275°C
0°C to 250°C/260°C
Subambient to 250°C
40°C to 250°C/260°C
Temperature Limit
Applications
Complex mixtures, mandatory for cis/ trans separation in partially hydrogenated fish or vegetable oils
Commonly Used Stationary Phases for the GC Analysis of Fatty Acid Methyl Esters
◾
Table 21.3 (continued)
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Thus, the formation of characteristic fragments ions permits the localization of unsaturated bonds and other functional groups in the hydrocarbon chain. The most common derivatives of this type used for the analysis of unsaturated fatty acids are picolinyl (3-hydroxymethylpyridinyl) esters96 and 4,4-dimethyloxazoline (DMOX) derivatives.97 It is worth considering that the mass spectra derived from either picolinyl and DMOX derivatives do not provide information about the cis/trans configuration of the double bonds. These structural features need to be confirmed by an independent technique. In the mass spectra of the DMOX derivatives, if a double bond is positioned between carbon n and carbon n + 1, then a mass interval of 12 amu between ions corresponding to fragments containing carbon n − 1 and carbon n is usually observed. In the case of the picolinyl esters, when a double bond is reached along the alkyl chain, a mass interval of 26 amu between ions corresponding to fragments containing n − 1 and n + 1 carbons is observed. These methods have been successfully applied to the analysis of trans-18:1 positional isomers in ruminant fat and in PVHO by Mossoba et al.98 and Aro et al.80 Recently Van Pelt and Brenna99 presented a GC–MS/MS (ion trap) method for determining the location of double bonds in PUFA methyl esters. This procedure is based on the chemical ionization of neutral FAMEs in the gas phase with acetonitrile, called by authors “covalent adduct chemical ionization” (CACI). Briefly, acetonitrile under chemical ionization conditions in an ion trap mass spectrometer self-reacts to form the (1-methyleneimino)-1-ethenylium (MIE, m/z 54) that reacts with double bonds of polyunsaturated FAMEs to yield a series of covalent product ions all appearing at (M + 54)+. Collisional dissociations of these ions yield diagnostic fragments permitting unambiguous localization of double bonds.100 Interestingly, this method has been also used in the analysis of positional and geometrical isomers of conjugated linoleic acids.101
References 1. Sinclair, A., Attar-Bashi, N., and Li, D., What is the role of a-linolenic acid for mammals? Lipids 37 (12), 1113–1123, 2002. 2. Trautwein, E., n-3 Fatty acids—Physiological and technical aspects for their use in food, European Journal of Lipid Science and Technology 103 (1), 45–55, 2001. 3. Arterburn, L. M., Hall, E. B., and Oken, H., Distribution, interconversion, and dose response of n-3 fatty acids in humans, American Journal of Clinical Nutrition 83 (6 Suppl), 1467S–1476S, 2006. 4. Valentine, R. C. and Valentine, D. L., Omega-3 fatty acids in cellular membranes: A unified concept, Progress in Lipid Research 43 (5), 383–402, 2004. 5. Allen, K. G. and Harris, M. A., The role of n-3 fatty acids in gestation and parturition, Experimental Biology and Medicine 226 (6), 498–506, 2001. 6. Calder, P. C., N-3 polyunsaturated fatty acids and inflammation: From molecular biology to the clinic, Lipids 38 (4), 343–352, 2003. 7. Harbige, L., Fatty acids, the immune response, and autoimmunity: A question of n-6 essentiality and the balance between n-6 and n-3, Lipids 38 (4), 323–341, 2003. 8. Hoffman, D. R., Birch, E. E., Birch, D. G., and Uauy, R. D., Effects of supplementation with omega 3 long-chain polyunsaturated fatty acids on retinal and cortical development in premature infants, The American Journal of Clinical Nutrition 57 (5 Suppl), 807S–812S, 1993. 9. Salem, N., Jr., Litman, B., Kim, H. Y., and Gawrisch, K., Mechanisms of action of docosahexaenoic acid in the nervous system, Lipids 36 (9), 945–959, 2001. 10. Simopoulos, A. P., Omega-3 fatty acids in health and disease and in growth and development, The American Journal of Clinical Nutrition 54 (3), 438–63, 1991.
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11. Basu, H., Pernecky, S., Sengupta, A., and Liepa, G. U., Coronary heart disease: How do the benefits of w-3 fatty acids compare with those of aspirin, alcohol/red wine, and statin drugs, Journal of the American Oil Chemists’ Society 83 (12), 985–997, 2006. 12. de Lorgeril, M., Salen, P., Martin, J. L., Monjaud, I., Delaye, J., and Mamelle, N., Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: Final report of the Lyon Diet Heart Study, Circulation 99 (6), 779–785, 1999. 13. Psota, T. L., Gebauer, S. K., and Kris-Etherton, P., Dietary omega-3 fatty acid intake and cardiovascular risk, The American Journal of Cardiology 98 (4A), 3i–18i, 2006. 14. Simopoulos, A. P., The importance of the ratio of omega-6/omega-3 essential fatty acids, Biomedicine & Pharmacotherapy 56 (8), 365–379, 2002. 15. Wakai, K., Tamakoshi, K., Date, C., Fukui, M., Suzuki, S., Lin, Y., Niwa, Y. et al., Dietary intakes of fat and fatty acids and risk of breast cancer: A prospective study in Japan, Cancer Science 96, 590–599, 2005. 16. Xia, S. H., Wang, J., and Kang, J. X., Decreased n-6/n-3 fatty acid ratio reduces the invasive potential of human lung cancer cells by downregulation of cell adhesion/invasion-related genes, Carcinogenesis 26 (4), 779–784, 2005. 17. Valencak, T. G. and Ruf, T., N-3 polyunsaturated fatty acids impair lifespan but have no role for metabolism, Aging Cell 6 (1), 15–25, 2007. 18. Kris-Etherton, P. M., Harris, W. S., and Appel, L. J., Fish consumption, fish oil, omega-3 fatty acdis, and cardiovascular disease, Circulation 106, 2747–2757, 2002. 19. Ackman, R., Ratnayake, W., and Macpherson, E., EPA and DHA contents of encapsulated fish oil products, Journal of the American Oil Chemists’ Society 66 (8), 1162–1164, 1989. 20. Ackman, R. G., Fatty acids in fish and shellfish, Fatty Acids Foods Their Health Implications, 3rd edn., 2008, pp. 155–185. 21. Ratnayake, W., Olsson, B., and Ackman, R., Novel branched-chain fatty acids in certain fish oils, Lipids 24 (7), 630–637, 1989. 22. Spector, A., Essentiality of fatty acids, Lipids 34 (0), S1–S3, 1999. 23. Sinclair, A. J., McLean, J. G., and Monger, E. A., Metabolism of linoleic acid in the cat, Lipids 14 (11), 932–936, 1979. 24. Tapiero, H., Nguyen Ba, G., Couvreur, P., and Tew, K. D., Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies, Biomedicine & Pharmacotherapy 56 (5), 215–222, 2002. 25. Sargent, J. R., Tocher, D. R., and Bell, J. G., The lipids, in Fish Nutrition, 3rd edn., Halver, J. E. and Hardy, R. W. (eds.), Academic Press, San Diego, CA, 2002, pp. 181–257. 26. Fahy, E., Subramaniam, S., Brown, H. A., Glass, C. K., Merrill, A. H., Jr., Murphy, R. C., Raetz, C. R. et al., A comprehensive classification system for lipids, Journal of Lipid Research 46 (5), 839–861, 2005. 27. Lobb, K. and Chow, C. K., Fatty acid classification and nomenclature, in Fatty Acids in Food and Their Health Implications, Chow, C. K. (ed.), Marcel Dekker, New York, 2000, pp. 1–15. 28. Robinson, P. G., Common names and abbreviated formulae for fatty acids, Journal of Lipid Research 23 (8), 1251–1253, 1982. 29. Ackman, R. G., The gas chromatograph in practical analyses of common and uncommon fatty acids for the 21st century, Analytica Chimica Acta 465, 175–192, 2002. 30. Brondz, I., Development of fatty acid analysis by high-performance liquid chromatography, gas chromatography, and related techniques, Analytica Chimica Acta 465 (1–2), 1–37, 2002. 31. Eder, K., Gas chromatographic analysis of fatty acid methyl esters, Journal of Chromatography B: Biomedical Sciences and Applications 671 (1–2), 113–131, 1995. 32. Shantha, N. C. and Napolitano, G. E., Gas chromatography of fatty acids, Journal of Chromatography A 624 (1–2), 37–51, 1992.
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33. Ratnayake, W. M., Overview of methods for the determination of trans fatty acids by gas chromatography, silver-ion thin-layer chromatography, silver-ion liquid chromatography, and gas chromatography/ mass spectrometry, Journal of AOAC International 87 (2), 523–539, 2004. 34. Nikolova-Damyanova, B., Lipid analysis by silver ion chromatography, in Advances in Lipid Methodology—Five, Adlof, R. O. (ed.), The Oily Press, Bridgwater, England, 2003, pp. 43–123. 35. Christie, W. W., Lipid extraction, storage and sample handling, in Lipid Analysis, 3rd edn., Christie, W. W. (ed.), The Oily Press, Bridgwater, England, 2003, pp. 91–102. 36. Bligh, E. G. and Dyer, W. J., A rapid method of total lipid extraction and purification, Canadian Journal of Biochemistry and Physiology 37 (8), 911–917, 1959. 37. Folch, J., M. Lees, M., and Stanley, G. H. S., A simple method for the isolation and purification of total lipides from animal tissues Journal of Biological Chemistry 226, 497–509, 1957. 38. Smedes, F. and Askland, T. K., Revisiting the development of the Bligh and Dyer total lipid determination method, Marine Pollution Bulletin 38 (3), 193–201, 1999. 39. Ways, P. and Hanahan, D. J., Characterization and quantification of red cell lipids in normal man Journal of Lipid Research 5, 318–328, 1964. 40. Manirakiza, P., Covaci, A., and Schepens, P., Comparative study on total lipid determination using Soxhlet, Roese-Gottlieb, Bligh & Dyer, and modified Bligh & Dyer extraction methods, Journal of Food Composition and Analysis 14 (1), 93–100, 2001. 41. Smedes, F. and Thomasen, T. K., Evaluation of the Bligh & Dyer lipid determination method, Marine Pollution Bulletin 32 (8–9), 681–688, 1996. 42. Iverson, S., Lang, S., and Cooper, M., Comparison of the bligh and dyer and folch methods for total lipid determination in a broad range of marine tissue, Lipids 36 (11), 1283–1287, 2001. 43. Christie, W. W., Preparation of ester derivatives of fatty acids for chromatographic analysis, in Advances in Lipid Methodology, Christie, W. W. (ed.), The Oily Press, Dundee, Scotland, 1993, pp. 69–111. 44. Knapp, D. R., Handbook of Analytical Derivatization Reactions, Wiley Interscience, New York, 1979. 45. Ackman, R. G., Application of gas-liquid chromatography to lipid separation and analysis: Qualitative and quantitative analysis, Fatty Acids Foods Their Health Implications, 3rd edn., 2008, pp. 47–65. 46. Liu, K.-S., Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials, Journal of the American Oil Chemists’ Society 71 (11), 1179–1187, 1994. 47. Rosenfeld, J. M., Application of analytical derivatizations to the quantitative and qualitative determination of fatty acids, Analytica Chimica Acta 465 (1–2), 93–100, 2002. 48. Christie, W. W., Preparation of derivatives of fatty acids, in Lipid Analysis, 3rd edn., The Oily Press, Bridgwater, England, 2003, pp. 205–224. 49. Kishimoto, Y. and Radin, N. S., A reaction tube for methanolysis; instability of hydrogen chloride in methanol, Journal of Lipid Research 6 (3), 435–436, 1965. 50. Krisnangkura, K. and Simamaharnnop, R., Continuous transmethylation of palm oil in an organic solvent, Journal of the American Oil Chemists’ Society 69 (2), 166–169, 1992. 51. Kramer, J., Fellner, V., Dugan, M., Sauer, F., Mossoba, M., and Yurawecz, M., Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids, Lipids 32 (11), 1219–1228, 1997. 52. Metcalfe, L. D., Schmitz, A. A., and Pelka, J. R., Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis, Analytical Chemistry 38 (3), 514–515, 1966. 53. Bannon, C. D., Craske, J. D., Hai, N. T., Harper, N. L., and O’Rourke, K. L., Analysis of fatty acid methyl esters with high accuracy and reliability. II. Methylation of fats and oils with boron trifluoridemethanol, Journal of Chromatography A 247 (1), 63–69, 1982. 54. AOAC, Official method 969.33, in Official Methods of Analysis of AOAC International, 16th edn., O’Dea, M. F. (ed.), AOAC International, Arlington, MA, 1995.
390 ◾ Handbook of Seafood and Seafood Products Analysis 55. AOCS, Official method Ce 1b-89—Fatty acid composition by GLC marine oils, in Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th edn., American Oil Chemists’ Society, Champaign, IL, 1989. 56. Ackman, R., Remarks on official methods employing boron trifluoride in the preparation of methyl esters of the fatty acids of fish oils, Journal of the American Oil Chemists’ Society 75 (4), 541–545, 1998. 57. Morrison, W. R. and Smith, L. M., Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride—Methanol, Journal of Lipid Research 5 (4), 600–608, 1964. 58. Schlenk, H. and Gellerman, J. L., Esterification of fatty acids with diazomethane on a small scale, Analytical Chemistry 32 (11), 1412–1414, 1960. 59. Glastrup, J., Diazomethane preparation for gas chromatographic analysis, Journal of Chromatography A 827 (1), 133–136, 1998. 60. Bannon, C. D., Breen, G. J., Craske, J. D., Hai, N. T., Harper, N. L., and O’Rourke, K. L., Analysis of fatty acid methyl esters with high accuracy and reliability. III. Literature review of and investigations into the development of rapid procedures for the methoxide-catalysed methanolysis of fats and oils, Journal of Chromatography A 247 (1), 71–89, 1982. 61. Bannon, C. D., Craske, J. D., Felder, D. L., Garland, I. J., and Norman, L. M., Analysis of fatty acid methyl esters with high accuracy and reliability. VI. Rapid analysis by split injection capillary gasliquid chromatography, Journal of Chromatography 407, 231–241, 1987. 62. Bayer, E. and Liu, G. H., New split injection technique in capillary column gas chromatography, Journal of Chromatography A 256, 201–212, 1983. 63. Grob, K., Injection techniques in capillary GC, Analytical Chemistry 66 (20), 1009A–1019A, 1994. 64. Grob, K. and Biedermann, M., The two options for sample evaporation in hot GC injectors: Thermospray and band formation. Optimization of conditions and injector design, Analytical Chemistry 74 (1), 10–16, 2002. 65. Poole, C. F. and Poole, S. K., Separation characteristics of wall-coated open-tubular columns for gas chromatography, Journal of Chromatography A 1184 (1–2), 254–280, 2008. 66. Christie, W. W., Gas chromatographic analysis of fatty acids derivatives, in Gas Chromatography and Lipids: A Practical Guide, Christie, W. W. (ed.), The Oily Press, Bridgewater, MA, 1989. 67. Busetto, M. L., Moretti, V. M., Moreno-Rojas, J. M., Caprino, F., Giani, I., Malandra, R., Bellagamba, F., and Guillou, C., Authentication of farmed and wild turbot (Psetta maxima) by fatty acid and isotopic analyses combined with chemometrics, Journal of Agricultural and Food Chemistry 56 (8), 2742–2750, 2008. 68. Caprino, F., Moretti, V. M., Bellagamba, F., Turchini, G. M., Busetto, M. L., Giani, I., Paleari, M. A., and Pazzaglia, M., Fatty acid composition and volatile compounds of caviar from farmed white sturgeon (Acipenser transmontanus), Analytica Chimica Acta 617 (1–2), 139–147, 2008. 69. Turchini, G. M., Moretti, V. M., Mentasti, T., Orban, E., and Valfre, F., Effects of dietary lipid source on fillet chemical composition, flavour volatile compounds and sensory characteristics in the freshwater fish tench (Tinca tinca L.), Food Chemistry 102 (4), 1144–1155, 2007. 70. Moretti, V. M., Madonia, G., Diaferia, C., Mentasti, T., Paleari, M. A., Panseri, S., Pirone, G., and Gandini, G., Chemical and microbiological parameters and sensory attributes of a typical Sicilian salami ripened in different conditions, Meat Science 66 (4), 845–854, 2004. 71. Paleari, M. A., Moretti, V. M., Beretta, G., Mentasti, T., and Bersani, C., Cured products from different animal species, Meat Science 63 (4), 485–489, 2003. 72. Paleari, M. A., Moretti, V. M., Bersani, C., Beretta, G., and Mentasti, T., Characterization of a lard cured with spices and aromatic herbs, Meat Science 67 (4), 549–557, 2004. 73. Lanza, E. and Slover, H., The use of SP2340 glass capillary columns for the estimation of thetrans fatty acid content of foods, Lipids 16 (4), 260–267, 1981. 74. Lin, K. C., Marchiello, M. J., and Fischer, A. G., Determination of the amount of trans-octadecenoate and trans-9,trans-12-octadecadienoate in fresh lean and fatty tissues of pork and beef, Journal of Food Science 49, 1521–1524, 1984.
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75. Ratnayake, W. M. N. and Bearerogers, J. L., Problems of analyzing C-18 cis-fatty and trans-fattyacids of margarine on the Sp-2340 capillary column, Journal of Chromatographic Science 28 (12), 633–639, 1990. 76. Ratnayake, W. M., Plouffe, L. J., Pasquier, E., and Gagnon, C., Temperature-sensitive resolution of cis- and trans-fatty acid isomers of partially hydrogenated vegetable oils on SP-2560 and CP-Sil 88 capillary columns, Journal of AOAC International 85 (5), 1112–1118, 2002. 77. Ratnayake, W. M. N., Hansen, S., and Kennedy, M., Evaluation of the CP-Sil 88 and SP-2560 GC columns used in the recently approved AOCS official method Ce 1h-05: Determination of cis-, trans-, saturated, monounsaturated, and polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by capillary GLC method, Journal of the American Oil Chemists’ Society 83 (6), 475–488, 2006. 78. Aldai, N., Osoro, K., Barron, L. J. R., and Najera, A. I., Gas-liquid chromatographic method for analysing complex mixtures of fatty acids including conjugated linoleic acids (cis9trans11 and trans10cis12 isomers) and long-chain (n-3 or n-6) polyunsaturated fatty acids. Application to the intramuscular fat of beef meat, Journal of Chromatography A 1110 (1–2), 133–139, 2006. 79. Aro, A., Antoine, J. M., Pizzoferrato, L., Reykdal, O., and van Poppel, G., Trans fatty acids in dairy and meat products from 14 European Countries: The TRANSFAIR study, Journal of Food Composition and Analysis 11, 150–160, 1998. 80. Aro, A., Kosmeijer-Schuil, T., van de Bovenkamp, P., Hulshof, P., Zock, P., and Katan, M., Analysis of C18:1 cis and trans fatty acid isomers by the combination of gas-liquid chromatography of 4,4-dimethyloxazoline derivatives and methyl esters, Journal of the American Oil Chemists’ Society 75 (8), 977–985, 1998. 81. Indurain, G., Beriain, M. J., Goni, M. V., Arana, A., and Purroy, A., Composition and estimation of intramuscular and subcutaneous fatty acid composition in Spanish young bulls, Meat Science 73 (2), 326–334, 2006. 82. Precht, D. and Molkentin, J., Rapid analysis of the isomers of trans-octadecenoic acid in milk fat, International Dairy Journal 6, 791–809, 1996. 83. Wilson, R., Lyall, K., Payne, J., and Riemersma, R., Quantitative analysis of long-chain transmonoenes originating from hydrogenated marine oil, Lipids 35 (6), 681–687, 2000. 84. Wolff, R., Bayard, C., and Fabien, R., Evaluation of sequential methods for the determination of butterfat fatty acid composition with emphasis on trans-18:1 acids. Application to the study of seasonal variations in french butters, Journal of the American Oil Chemists’ Society 72 (12), 1471–1483, 1995. 85. Wolff, R. L. and Bayard, C. C., Improvement in the resolution of individual trans-18/1 isomers by capillary gas-liquid-chromatography—Use of a 100-M Cp-Sil-88 column, Journal of the American Oil Chemists Society 72 (10), 1197–1201, 1995. 86. Delmonte, P. and Rader, J. I., Evaluation of gas chromatographic methods for the determination of trans fat, Analytical and Bioanalytical Chemistry 389 (1), 77–85, 2007. 87. Mjos, S. A., Properties of trans isomers of eicosapentaenoic acid and docosahexaenoic acid methyl esters on cyanopropyl stationary phases, Journal of Chromatography A 1100 (2), 185–192, 2005. 88. Huang, Z., Wang, B., Pace, R. D., and Oh, J. H., Trans fatty acid content of selected foods in an African-American community, Journal of Food Science 71 (6), C322–C327, 2006. 89. AOCS, Official Method Ce 1h-05, in Official Methods and Recommended Practices of the AOCS, 5th edn., Firestone, D. (ed.), AOCS, Champaign, IL, 1999. 90. AOAC International, Official method of analysis 996.06, revised 2001, in Official Methods of Analysis, 18th edn., AOAC Int., Gaithersburg, MD, 2001. 91. House, S. D., Determination of total, saturated, and monounsaturated fats in foodstuff s by hydrolytic extraction and gas chromatographic quantitation: Collaborative study, AOAC International 80 (3), 555–563, 1997. 92. Dobson, G. and Christie, W. W., Mass spectrometry of fatty acid derivatives, European Journal of Lipid Science and Technology 104 (1), 36–43, 2002.
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93. Christie, W. W., Gas chromatography-mass spectrometry methods for structural analysis of fatty acids, Lipids 33 (4), 343–53, 1998. 94. Christie, W. W., Gas chromatography-mass spectrometry and fatty acids, in Gas Chromatography and Lipids: A Practical Guide, Christie, W. W. (ed.), The Oily Press, Ayr, Scotland, 1989, pp. 96–109. 95. Dobson, G. and Christie, W. W., Structural analysis of fatty acids by mass spectrometry of picolinyl esters and dimethyloxazoline derivatives, TrAC Trends in Analytical Chemistry 15 (3), 130–137, 1996. 96. Harvey, D. J., Mass spectrometry of picolinyl and other nitrogen-containing derivatives of fatty acids, in Advances in Lipid Methodology—One, Christie, W. W. (ed.), The Oily Press, Ayr, U.K., 1992, pp. 19–80. 97. Spitzer, V., Structure analysis of fatty acids by gas chromatography—Low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives—A review Progress in Lipid Research 35 (4), 387–408, 1997. 98. Mossoba, M., Yurawecz, M., Roach, J., Lin, H., McDonald, R., Flickinger, B., and Perkins, E., Elucidation of cyclic fatty acid monomer structures. Cyclic and bicyclic ring sizes and double bond position and configuration, Journal of the American Oil Chemists’ Society 72 (6), 721–727, 1995. 99. Van Pelt, C. K. and Brenna, J. T., Acetonitrile chemical ionization tandem mass spectrometry to locate double bonds in polyunsaturated fatty acid methyl esters, Analytical Chemistry 71 (10), 1981–1989, 1999. 100. Lawrence, P. and Brenna, J. T., Acetonitrile covalent adduct chemical ionization mass spectrometry for double bond localization in non-methylene-interrupted polyene fatty acid methyl esters, Analytical Chemistry 78 (4), 1312–1317, 2006. 101. Michaud, A. L., Yurawecz, M. P., Delmonte, P., Corl, B. A., Bauman, D. E., and Brenna, J. T., Identification and characterization of conjugated fatty acid methyl esters of mixed double bond geometry by acetonitrile chemical ionization tandem mass spectrometry, Analytical Chemistry 75 (18), 4925–4930, 2003.
SENSORY QUALITY
IV
Chapter 22
Quality Assessment of Fish and Fishery Products by Color Measurement Reinhard Schubring Contents 22.1 22.2 22.3 22.4
Introduction ..................................................................................................................395 Instrumentation ........................................................................................................... 397 Novel Method of Color Evaluation .............................................................................. 399 Color Measurement on Fish and Fishery Products ....................................................... 400 22.4.1 Aquaculture .................................................................................................... 400 22.4.2 Fish Mince, Surimi, and Surimi-Based Products ............................................. 403 22.4.3 Processing Effect on Color of Fish and Fishery Products ................................. 403 22.4.3.1 Refrigerated and Frozen Storage ..................................................... 403 22.4.3.2 Thermal Processing (Heating and Smoking) ................................... 403 22.4.3.3 High-Pressure Processing ................................................................ 403 22.5 Summary ..................................................................................................................... 407 References ................................................................................................................................417
22.1
Introduction
We perceive the world in which we live by our five senses—vision, hearing, touch, taste, and smell, of which the sense of vision is usually the first used in detecting events and objects around us in the visual world. The process of seeing comprises many cooperating activities, first detected by our eyes 395
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and then interpreted by our brain, recognition of movement and location of object, relationship of objects to their surroundings, the intensity and quality of the light, and the color appearance of object or events in the visual scene [1]. Scientific understanding of the processes involved in determining color appearance has been elucidated in the last two to three centuries. The experiments in mixing colors performed during this time period clearly demonstrated that people with normal color vision must have at least three retinal pigments in their eyes, detecting the short, medium, and long waves of the visible spectrum. The first truly functional system for measuring color as specified by the Commission Internationale de l’Eclairage (CIE) was the so-called CIE 1931 2° visual field system of color measurement. Since then, many improvements have been incorporated into the system to make it almost visually uniform and this research continues [1]. With the development of the computer, complex color measurements and calculations are now routinely used in research and food industry for studies of food functionality, for product ingredient standardization, and for process control. Th ree interacting factors are required for the measurement of the color appearance of any object in a scene. These are an understanding of the human visual process, the effect of light on objects in their environment, and the nature of the materials observed [1]. The CIE system of color measurement transforms the reflection or transmission spectrum of the object into a three-dimensional color space using the spectral power distribution of the illuminant and the color-matching functions of the standard observers. The original 1931 CIE Y, x, y system of color measurement is not visually uniform. Constant hue and chroma are distorted and equal visual distances increase severalfold from purple-red to green. Near-uniform color spaces of practical importance are the Hunter and the CIELUV and CIELAB spaces. The CIE L*a*b* also known as CIELAB has generally replaced the Hunter space in industrial application. The coordinates L*a*b* serve to define the locations of any color in the uniform color space. Color terms can be divided into the subjective and the objective. The subjective terms, i.e., the psychosensorial, are brightness, lightness, hue, saturation, chroma, and colorfulness. Colorfulness is that aspect of visual sensation according to which an area appears to exhibit more or less chromatic color. Although hue is easily understood as that attribute described by color names such as red, green, purple, and the like, the difference between saturation and chroma is less easy to comprehend. Saturation is colorfulness judged in proportion to its brightness, whereas chroma is colorfulness relative to the brightness of its surroundings. A similar difference exists between lightness and brightness. Lightness is relative to brightness. Lightness is unaffected by the level of illumination because it is the proportion of the light reflected, whereas the sensation of brightness increases with an increase in the level of illumination. The objective terms, i.e., the psychophysical, are related to the stimulus and evaluated from spectral power distribution, the reflectance or transmittance of the object, and observer response. They provide the basis for the psychometric qualities that correspond more nearly to those perceived [1]. For CIELAB space the terms are lightness L*, hue h* = tan−1 (a*/b*), and chroma C* = (a*2 + b*2)1/2. CIELAB total color difference can be expressed either as the coordinates of color space or as the correlates of lightness, chroma, and hue.
( ) ( ) ( )
1/2
ΔE * = ⎡ ΔL*2 + Δa *2 + Δb *2 ⎤ ⎣⎢ ⎦⎥ or
( ) ( ) ( )
1/2
ΔE * = ⎡ ΔL*2 + ΔC *2 + ΔH *2 ⎤ ⎢⎣ ⎥⎦
Quality Assessment of Fish and Fishery Products by Color Measurement
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397
Color is the perception that results from the detection of light after it has interacted with an object. The perceived color of an object is affected by three entities: the physical and chemical composition of the object, the spectral composition of the light source illuminating the object, and the spectral sensitivity of the viewer’s eye. As everyone is sensitive to the color of foods, appetite is stimulated or dampened in almost direct relation to the observer’s reaction to color. The color we see clearly indicates the flavor we will taste [2]. For food products, the consumers often assess the initial quality of the product by its color and appearance. In food processing and cooking, color serves as a cue for the doneness of foods and is correlated with changes of aroma and flavor. For example, color or lightness such as the lightness of canned tuna is important for identity and quality grading. In general, color and appearance affect the consumer’s perceptions of other sensory modalities [3]. The appearance of fish and meat products is an essential factor according to which the consumers judge their acceptance [4]. However, the color of food is not stable, in that it changes with decreasing freshness. The appearance of a newly landed fish is unforgettable, in that the interplay of the subtle shades of beautiful colors makes it a joy to behold and irresistible as an item of food. Just a few hours after death, though, it begins to look less obviously attractive, and its now “ordinary” colors are much more familiar to the majority of the public [5].
22.2 Instrumentation Photoelectric color-measuring instruments can be divided into two classes: trichromatic colorimeters and spectrophotometers. Colorimeters are tristimulus (three-filtered) devices that make use of red, green, and blue filters, which emulate the response of the human eye to light and color. In some quality control applications, these tools represent the lowest cost answer. The more modern tristimulus instruments are linked to computers with automatic compensation and the provision of a number of color spaces. A colorimeter uses a light source to light the specimen being measured. The light reflected off the object then passes through the glass filters to simulate the standard observer functions for a particular illuminant. A photodetector beyond each filter then detects the amount of light passing through the filters. These signals are then displayed as X, Y, and Z values. The Chroma Meter CR-300 (Figure 22.1) used for own measurements offers 8 mm diameter measuring area and diff use illumination/0° viewing geometry for a wide variety of applications. The measuring head of the CR-300 uses diff use illumination/0° viewing geometry (specular component included) to provide measurements of a wide variety of surfaces that correlate well with color as seen under diff use lighting conditions. A pulsed xenon arc (PXA) lamp
(a)
(b)
(c)
(d)
Figure 22.1 Selection of instruments for color measurement on fish and fishery products. (a) Color Reader CR-10, (b) HunterLab MiniScan XE Plus, (c) Chroma Meter CR-300, and (d) spectral color meter spectro-pen®.
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inside a mixing chamber provides diff use, uniform lighting over the 8 mm diameter specimen area. Only the light reflected perpendicular to the specimen surface is collected by the opticalfiber cable for color analysis. As illuminant condition D65 and for calibration purposes a white standard CR-A43 were used. Use of the computer software ChromaControl C (release 2.04) allows the easy calculation of hue and chroma. Besides the aforementioned instrument that is widely used for color measurement on fish and fishery products there are further instruments available that can be used for the same purpose. For example, the Color Reader CR-10 (from Konica Minolta) is a very compact, battery-powered, handheld portable colorimeter (tristimulus type) for quick color control (Figure 22.1). At first, simply measure the target and then the sample. After one second the color difference—expressed in L*a*b* and DE * or L*C *H * and DE *—will appear on the LCD screen. The measuring area of 8 mm in addition to the 8/d geometry allows for universal use in a wide variety of applications. All measurements are taken under the conditions of standard illuminant D65 and 10° observer. To get a printout of the readings, the CR-10 can be connected to an external printer. Spectrophotometer is the most accurate instrument for measuring color. It uses a light source to light the specimen being measured. The light reflected by the object then passes to a grating that breaks it into the spectrum. The spectrum falls onto a diode array that measures the amount of light at each wavelength. This spectral data is then sent to the processor where it is multiplied together with data table values for the selected CIE illuminant and the 2° or 10° standard observer functions to the X, Y, and Z values. These are further transformed to the CIELAB values L*, a*, b* by using the following equations: L* = 116 (Y /Yn ) − 16 1/3
1/3 1/3 a * = 500 ⎡(X /X n ) − (Y /Yn ) ⎤ ⎣ ⎦ 1/3 1/3 b * = 200 ⎡− (Y / Yn ) − (Z /Z n ) ⎤ ⎣ ⎦
Own color measurements on fish samples were carried out with the spectral color meter spectropen® (Dr. Lange, Düsseldorf, Germany). This is a colorimeter (Figure 22.1) operating on the spectral method described in DIN 5033 using the 45°/0° circular viewing geometry, i.e., the sample is illuminated with polychromatic light encircling it at an angle of 45°, with the optical unit observing the reflected light from a horizontal angle (0°) toward the sample surface. Spectro-pen is a genuine grating colorimeter measuring the visible spectral range (400–700 nm) at intervals of 10 nm. A 10°-standard observer function and D65 as illuminant were used (light source: polychromatic with tungsten lamp). The PC-software “spectral–QC” allows state-of-the-art data processing. Before measuring each lot the colorimeter was calibrated against a white standard (LZM 224). A widely used spectrophotometer in fish processing and research is the HunterLab MiniScan XE Plus 45/0 LAV spectrophotometer (Figure 22.1) with the special glass or polycarbonate-covered nose. Up to 99 product setups can be stored in memory. Moreover, 999 tristimulus sample readings or 500 spectral data readings can be stored for later recall or output to a computer or printer. Each setup includes color scale, illuminant, observer, indices, standard type, pass/fail tolerances, and sample averaging. Setups can be linked together for easy measurement under different illuminants or color scales. Flexibility is the cornerstone of each HunterLab portable system. Both
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45°/0° and sphere (diff use/8°) geometries are available. Small, large, smooth textured, and many other types of samples can easily be measured. The LCD screen displays numerical and graphical data with automatic storage and retrieval of tristimulus or spectral data. For even greater capability, MiniScan XE Plus can be used with a PC and EasyMatch® QC or EasyMatch® Formulation software. Fish sample that is at least 25 mm thick for steaks, fillets, and patties or 12.5 mm thick for breaded products should be selected as a prerequisite for color measurement. The precision of predicting color parameters from one instrument to another varied with food commodity. The type of sample seemed to have a larger effect on the precision of the measurement than the size of the instrument’s measuring area. The variation from one instrument to another was found to be systematic and can be described by linear regression. The regression can be used to predict color values expected from one instrument with those obtained from another. The precision of prediction will increase with increased homogeneity of the food samples [6]. When instrumental color measurements of raw salmon flesh were performed by one tristimulus filter colorimeter and two spectrophotometers, it was found that the instruments gave different values in absolute terms, based on the sampling conditions. The highest correlation between astaxanthin content and instrumental color reading were obtained for the a* and chroma values of the spectrophotometers when sampling on 1 cm thick cutlets on white background [7]. After a comparison between a tristimulus colorimeter (Minolta CR 200) and two spectrophotometers (CM-508i and CM-2002) it was concluded that reproducible and objective measurements expressed in the CIE system were obtained with all the three instruments. A strong correlation was found between the CR 200 and each of the two spectrophotometers. However, absolute values found with the CR 200, especially for b*, differed from values obtained with the two spectrophotometers [8].
22.3
Novel Method of Color Evaluation
Image features, i.e., color, size, shape, and texture, have been extensively applied in the food industry for quality evaluation and inspection of a wide variety of food. Color features are effective tools for indicating the reconstruction of components of food products during processing. Three different types of color spaces, hardware orientated, human orientated, and instrumental, are generally used for the extraction of color features. Hardware-orientated spaces are preferable for observing small changes in the color of food products during processing. With the remarkable development of computer hardware, it might become realistic to employ the whole image data as input features, which might be an exceptional indicator of food qualities [9]. Very recently, automated image analysis methods have been presented that were able to describe quality properties, such as area of the cutlet, dorsal fat depot, red muscle, fat percentage, and color from a large number of scanned images of rainbow trout cutlets. It is also evident that it is possible to produce the images of cutlets of adequate quality for image analysis using a simple flatbed scanner [7]. For this purpose, no elaborate lighting regime is necessary [10]. An image-acquisition system was recently presented that allows the obtaining of digital images in L*a*b* color units for each pixel of the digital RGB image. Five models were built that were able to measure color in L*a*b* units and simultaneously measure the color of each pixel on the target surface. This is not the case with conventional colorimeters. The best results were achieved with the quadratic and neural network model [11]. A more easy, performable method has been proposed that uses a combination of digital camera, computer, and graphic software to measure and analyze the surface color of food products [12]. Measuring color, particularly in the L*a*b* space, provides a better statistical discrimination between the groups of fish studied than sensory analysis. In fact, although in agreement with the
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results of the panel, the colorimetric method can distinguish all of the groups in terms of the mean color and the heterogeneity of color. The very high degree of precision of these results provides an understanding of the effect of the drying processes on the color of the different samples [13]. However, it became obvious that the determination of only one quality attribute was not sufficient. Correct classification based on experimental variables measured by discriminant function analysis was poor for color data alone, acceptable for electronic nose data alone, and excellent with these data combined [14]. Combining the data from the various sensors improves the estimate of the freshness of fish [15]. To demonstrate this, color, texture, and electronic nose measurements were selected and their calibrated outputs combined to construct an Artificial Quality Index (AQI) [16]. It was reported that machine vision is able to differentiate and quantify color distributions in fish samples with uneven color. In the case of fresh tuna, the hue values offered less variability and more monotonous changes with storage time. Color of fresh tuna exposed to 4% CO for 48 h remained unchanged with refrigerated storage time. The color of control sample did change substantially and turned brown [17]. A new index, named Entire Color Index (ECI), was developed to express h* and C *, which as combined variables cannot be considered separately. ECI was calculated as ECIi = C * cos (hi − hmean). In all species there was a remarkable dorsoventral gradient in mean L* and h*, with the ventral area being statistically significant and brighter than the dorsal one. ECI value was species specific but did not show any statistically significant dorsoventral gradient, with the exception of Pagrus pagrus. Storage time affected L* and h* only in the dorsal skin area. However, the effect of storage on ice was better reflected in mean ECI value, which showed a marked decrease from day 3 to day 7 in both the dorsal and the ventral skin area. It is concluded that the results provide data for a nonsubjective determination of skin color pattern and show that ECI offers a good index of the actual color in a meaningful and objective way [18].
22.4 Color Measurement on Fish and Fishery Products In contrast to warm-blooded meat, reports on color measurements taken on fish and/or fishery products are harder to find in books dealing with the color of foods [1,19–24]. Color measurement on fresh meat [1] is explained as a typical example for muscle foods, modeling color stability is discussed on fresh beef [25]. Therefore, the aim of this chapter is to give an overview on the importance and application of color measurement on fish and fishery products. The following main areas of application of color measurements are considered: aquaculture, fish mince, surimi, and surimi-based products, processing effect on color of fish and fishery products (refrigerated and frozen storage, thermal processing, heating and smoking), and high-pressure processing.
22.4.1
Aquaculture
For a long time color measurements performed in this field have been dominated by those taken on farmed Atlantic salmon. Here, traditionally color scales were used by experienced inspectors and are still in use together with up-to-date instrumentation for color measurements [26]. At present, besides the still important salmonides other fish species used for farming are subject to color measurement as well as the comparison between farmed species and their wild counterparts. Table 22.1 provides a brief review of the literature under the aspect of aquaculture.
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Table 22.1 Color Measurements in the Field of Aquaculture Task
Effects on Color
References
Spawning migration and color in chum salmon
L increased while a, b, and a/L decreased with increasing maturity
[27]
Muscle carotenoid content and color of rainbow trout
Increased pigmentation caused increase in C* and reduced h* and L*
[28]
Carotenoid concentration in diet
Increased concentration led to increased a*, b*, and C*, and decreased L* and h*
[29]
Influence of rearing temperature
Arctic char reared at 10°C had higher a* than those reared at 15°C
[30]
Influence of feeding on pigmented diets on color of Arctic char muscle
Deposition of carotenoids in flesh resulted in decrease in L and increase in a and b
[31]
Color differences between farmed and wild fish
Wild and farmed pikeperch were very similar in L* and a*, b* value of wild fish was significantly lower
[32]
Influence of location at which measurements were taken on rainbow trout
Effect was highly significant on L*, C*, and h*. Order found was: data from tail > data from head part > data from middle part
[33]
Effect of dietary fat level on flesh color of Atlantic salmon
a* and b* were higher in salmon fed high fat diets than in those fed medium fat diets
[34]
Effect on flesh color of commercial and experimental slaughtering techniques
Bleeding in ice slurry, whole body electrical treatment, and percussion were compared. Fish killed by electricity had higher a* and lower L* values. No differences in b* were among samples
[35]
Influence of muscle activity on color in Atlantic salmon
Electrically stimulated fish had significant higher a* and C* than stressed and rested fish, but there were almost no difference between groups in L* and h*
[36]
Influence of slaughtering under high- and low-stress conditions
In farmed Atlantic salmon were no significant differences found in L*, but a* and b* were higher under high-stress
[37]
Comparison in fillet color of diploid and triploid fish
Color of salmon triploids reared in seawater was lower in L* and higher in a*. However, shi drum triploids were higher in L* than diploids. After cooking difference disappeared
[38,39]
(continued)
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Table 22.1 (continued)
Color Measurements in the Field of Aquaculture
Task
Effects on Color
References
Influence of rigor mortis on fillet color of Atlantic salmon
Pre-rigor fillet cuts improved color characteristics (lower L*, higher a*, and b* values) when compared to post-rigor
[40]
Effect of pre-rigor filleting of wild and farmed cod on lightness
Time of filleting did not affect L* of the cut surface measured on day 10 after slaughter
[41]
Influence of duration photoperiods on skin color of gilthead sea bream
Only L* differed significantly between treatments. Fish reared under permanent light displayed highest values
[42]
Diet supplementation with two carotenoids on skin color
Shrimp shell meal diets enhanced reddish h* and C* values. L* of red porgy skin was not influenced
[43]
Rearing conditions of European catfish fed natural (NF) and formulated (FF) feed
Fish were reared on NF in earthen ponds and on FF in recirculating systems. Only a* was significantly influenced, being lower in fish fed FF
[44]
Farming conditions on color of raw fillets of European catfish
The shorter the farming time, the lighter the fillets, a high water temperature produced yellower and greener fillets
[45]
Comparison of farmed and wild yellow perch
Farmed yellow perch had higher L* and wild perch higher a*
[46]
Effect of diets supplemented with astaxanthin and saponified red chili extract on rainbow trout
Raw fillets showed decrease in L*, h*, and C* and increase in a* and b* as diet feeding time increased. Frozen fillets showed decrease in L* and h* and increase in a*, b*, and C*. During smoking L* and a* did not change and the other values increased
[47]
Influence of cage color and light environment on skin color of Australian snapper
Skin L* value was much higher in fish held in white than in black cages but was not affected by light environment
[48]
Comparison of crustacean meals with fish meal diet on color of salmon
Highest L* was obtained for fish meal diet and significant lower L* were found for crustacean meal diets
[49]
Rearing conditions of wild perch fed natural (NF) and farmed perch fed formulated (FF) feed
a* was higher and b* was lower in wild perch, both differences were significant. L* was not different
[50]
Feasibility of adding astaxanthin as source of red color directly during processing of pastes
Linear regression relationships were found for CIE L*, a*, h*, and astaxanthin content (Ax, mg/kg): L* = −0.37Ax + 76, a* = 0.56Ax − 2.7, h* = −1.53Ax + 98
[51]
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22.4.2
◾
403
Fish Mince, Surimi, and Surimi-Based Products
The second major area in which color measurements are frequently used is characterized by the comminution of fi sh muscle and steps to restructure the comminuted muscle to fi sh muscle analogs mainly by heating. The gel formation necessary to achieve the goal is dependent on the functionality of muscle proteins and is supported by the addition of substances that are able to support gel formation. Most of these technological steps include characteristic color changes of fi sh muscle. Therefore, it is no wonder that color measurements have been applied at an early stage. In the three relevant textbooks dealing with surimi [52–54], color measurements on surimi gels are highlighted or treated in special chapters [54]. Whiteness (WS), as an index for the general appearance of a surimi gel, has been introduced and can be calculated as Whiteness = L* − 3b* or whiteness = 100 − [(100 − L*)2 + a *2 + b *2 ]0.5 Table 22.2 displays briefly a number of relevant papers that characterize the importance of performing instrumental color measurements in this area to evaluate the quality of products.
22.4.3 Processing Effect on Color of Fish and Fishery Products 22.4.3.1 Refrigerated and Frozen Storage Chilling and freezing are two processing steps for short- and long-term preservation of newly caught fish that influence the color of skin and flesh to a large extent. Therefore, as briefly shown in Tables 22.3 and 22.4, numerous papers on color measurement have been published.
22.4.3.2 Thermal Processing (Heating and Smoking) As early as 1968, a paper has been published on instrumental color measurement taken on radiation-sterilized pre-fried cod and halibut patties. The breaded pre-fried products were irradiated at 4.5 Mrad, stored for 12 months at 22°C, and evaluated by color reflectance measurement. Pre-fried patties became lighter in color after irradiation. The halibut patties were lighter than the cod patties initially, after irradiation and after storage [123]. The research on colorimetry of salmon has dealt mainly with the color of the canned product. An overview on early activities in this respect has been given in [127]. Since then the effects of thermal processing on color of fish have been extensively investigated. Results of these researches are displayed in Table 22.5.
22.4.3.3
High-Pressure Processing
Besides traditional technologies, the modern ones are more and more on the agenda of fi sh processing. An example of the practical use of minimal processing in the form of the application of high-pressure processing can be seen in the processing of oysters. HP-treated oysters had higher L values than untreated oysters; the magnitude of changes increased with treatment
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Table 22.2 Color Measurements on Surimi and Surimi Products Task
Color Effects
References
Order fish mince according to species, processing parameters, and color differences
Color matching of fish mince provide a good basis for assessing practical limits of grading for color
[55]
Washing of fish mince
Resulted in higher L and lower a values
[56]
Treating of fish mince by H2O2
L of fish mince increased with increasing levels of H2O2. Soaking of fish mince from flaps in H2O2 decreased a and b values
[57,58]
Adding of hydrocolloids to fish mince and observing color changes during frozen storage
During 3 months at −18°C L generally decreased slightly, accompanied by increases in a and b
[59]
Minced sardine flesh washed with different solutions (sodium bicarbonate and water)
L* of washed mince with 0.5% NaHCO3 increased markedly, while in all treatments a* decreased and b* increased in water-washed mince
[60]
Washing of catfish frame mince and influence of storage
L values increased whereas a and b values decreased after washing. During storage at 5, 0, and −20°C L, a, and b remained unchanged
[61]
Influence of refrigerated and frozen storage on surimi color
L* and b* taken on Pacific whiting surimi did not change during storage up to 5 d but decreased at day 7. a* did not change throughout storage. After freezing, surimi showed only slight changes in L*, a*, b* and remained stable during 2 months of frozen storage
[62]
Numbers of washing steps in preparing surimi from channel catfish mince
Significant differences in L, a, b were found between gels prepared with washed and unwashed surimi only after the first wash. There were no additional changes after two or three washes
[63]
Gels prepared from surimi of different fish species with or without protease inhibitors
L* of cooked gels without inhibitors was lowest for pollock and highest for arrowtooth flounder (AF) surimi. The a* indicated a more greenish hue. The b* of AF was highest indicating more yellow hue
[64]
Effect of adding nonmuscle proteins as gelation aid to horse mackerel surimi
The L* of kamaboko increased within 0.5 h after cooking and then decreased slightly. The a* decreased and b* increased gradually. Addition of liquid egg white to kamaboko increased its L* but reduced a* and b*
[65]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.2 (continued)
◾
405
Color Measurements on Surimi and Surimi Products
Task
Color Effects
References
Addition of several hydrocolloids in different concentrations (0.5%–4.0%) to blue whiting muscle mince
Gel color was virtually unaffected by the presence of different hydrocolloid concentration in the formulation
[66]
Improvement of surimi color of dark-fleshed fish species
Raising water or oil content significantly increased L* and decreased b* of the gels. Potato starch decreased b* but not L*. In general, adding water, oil, or titanium dioxide was considered as effective to whiten the color of surimi gels
[67]
Color of freeze-dried surimi powder of three marine fish species from Malaysian waters were checked
Highest L was found for threadfin bream, followed by purple-spotted bigeye and lizardfish. Similar results were found for a and b
[68]
Different washing treatments at acidic and alkaline pH areas for kamaboko production from sardine regarding their effect on color of final product
Washing resulted in higher L* and whiteness index of protein concentrates. Further improvement of both parameters achieved during heating. Lowest a* reached by alkaline washing at pH 11.5. pH modification also influenced b* value
[69]
Increase of whiteness of small-scale mud carp gel
Extensive washing increased whiteness. Setting temperature had no effect on color
[70]
Low-salt restructured fish products from Mexican flounder
Using transglutaminase or whey protein concentrate (WPC) decreased L* while a* and b* increased in comparison to control
[71]
Effect of addition of amidated low methoxyl pectin to Mexican flounder
Addition of pectin increased significantly L* and b* of the gels whereas a* increased only slightly
[72]
Effect of quercitin on fish gels fortified with ω-3 fatty acids
L* is reduced and b* is almost doubled by addition of quercitin compared to fish gels containing only fish oil
[73]
Comparison of surimi prepared by pH shifting (alkaline, AL; acidic, AC) with conventionally washed surimi (CW)
CW surimi gels exhibited highest whiteness due to higher L* and lower a* and b* values. AC surimi gels were lower in whiteness than AL surimi gels
[74]
Influence of moisture content on surimi color of different fish species
L* and whiteness increased with higher moisture content, changes in a* and b* were not consistent
[75]
(continued)
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Table 22.2 (continued)
Color Measurements on Surimi and Surimi Products
Task
Color Effects
References
Extraction and recovery of Atlantic croaker proteins with the pH-shift process
Isolate pastes had higher L* than surimi pastes, surimi gel had higher L* than isolate, b* was highest in acid-aided gel
[76]
Color changes of mince, surimi, and kamaboko from horse mackerel with different washing methods were compared
L* of mince increased rapidly with ozonized water treatment, but slowly with alkaline solution treatment. Similar changes of L* in surimi and kamaboko were observed. a* of mince decreased during washing. Decrease in b* of surimi and kamaboko was seen. Whiteness of washed mince, surimi, and kamaboko increased with increase in washing time for all three methods and similar patterns were also observed for L*. L* and whiteness of surimi were lower than those of washed mince
[77]
Protein recovery from processing by-products via solubilization/precipitation
Gels from proteins recovered from acidic treatments were whiter and less yellow than their basic counterparts
[78]
Surimi containing ω-3 fatty acids from algal oil was prepared by addition of oil-in-water emulsions or bulk oil
All surimi treatments containing algal oil had an increase in L*, a*, and b*. The largest change in color in surimi with algal oil was in b*
[79]
Influence of various ohmic heating conditions on surimistarch gels
Surimi-starch gels showed a decrease in L*, b*, and whiteness when starch concentration increased. Increase in moisture caused an increase in whiteness. No clear influence on color of applied voltage observed
[80]
Effects of cysteine proteinase inhibitor (CPI) containing fraction from chicken plasma on autolysis inhibition and color of Pacific whiting surimi
No differences in whiteness were observed between samples with and without CPI addition. CPI could be used in surimi seafood without affecting the color of finished products
[81]
Effect of white grape dietary fiber concentrate (WGDF) against hemoglobin-mediated oxidation of washed cod mince, with and without 10% added herring oil. Changes in a* used as an indicator of lipid oxidation
During ice storage, control samples without Hb did not show changes in a* both with and without herring oil added. Samples with 2% and 4% added WGDF did not show significant changes in a* during storage both with and without added herring oil
[82]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.2 (continued)
◾
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Color Measurements on Surimi and Surimi Products
Task
Color Effects
References
Influence of processing temperatures (93°C, 85°C, and 75°C) and times (0–120 min) on color of surimi seafood
High processing temperatures and longer processing times deteriorate the color of surimi seafood and are not recommended
[83]
Influence on color of surimi from Alaska pollock (AP), pork leg, and chicken breast (CB) prepared by washing with water two or four times
AP surimi had higher L* and whiteness than other samples, CB surimi were lower in L*. a* and b* were lower in AP surimi, CB surimi were higher in a* and b*
[84]
Inclusion of various levels of a microbial transglutaminase or a carrageenan additive to restructured hake products with or without dietary fiber with regard to its effect on color
Concerning a* and b* values, there was a consistent increasing trend with growing carrageenan levels. Higher carrageenan contents made the fish products containing Swelite redder and yellower and inclusion of Fibruline increased a*
[85]
Feasibility of obtaining fish-restructured products acceptable for consumers determined by mixing striped mullet and Mexican flounder
Gels showed higher darkness and redness in the following order: striped mullet (dark-fleshed), mixture (1:1) of both species, Mexican flounder (white-fleshed)
[86]
Influence on color of using microbial transglutaminase (MTGase) or WPC as binders in preparation low-salted restructured fish products from striped mullet
In low-salt gels, MTGase and WPC had no effect on L*, WPC increased a*, MTGase, and WPC increased b*. The level of salt did not affect the color in restructured products (no changes in L* observed)
[87]
Color changes of surimi seafood under electron beam (e-beam) and heat
L* and a* were not affected by e-beam treatment, L* and a* increased when heat treated. Gradual decrease in b* for e-beam, but increase in b* in heat-treated samples
[88]
pressure. HP-induced changes in color generally imported a cooked, more voluminous, and juicy appearance to the raw oyster tissue [148]. Further results on the influence of color by treating fi sh with high pressure are shown in Table 22.6.
22.5
Summary
The overview given here indicates the importance of color measurement in the evaluation of quality and safety in fish processing. One of the advantages is the fact that the measuring technique is not expensive and can therefore be applied on a wide scale.
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Table 22.3
Color Measurements on Refrigerated Products
Task
Color Effects
References
Influence of storage condition
RSW-stored ocean perch kept their skin color better than iced fish. a values for RSW fish supported by CO2 were higher. L and b did not change significantly
[89]
Treatment by antioxidants to preserve skin color during refrigerated and frozen storage
Thornyhead rockfish skin color was generally improved by treatment. After 4 months the control had lost red color. After 8 d of refrigerated storage treated samples maintained more skin redness than control which a* decreased
[90,91]
Effect of MAP on color of Pacific red snapper during storage at 2°C compared with samples stored in air
L decreased over time, a was lower after 7 d and no difference in b between treatments. After 7 d ΔE higher in MAP and after 21 d ΔE lower in MAP than in air
[92]
Color changes during iced storage early postmortem
Immediately after fish has been caught in demersal fish L* in fillets increased along body axis from head to tail while in pelagic fish L* decreased in same direction. a* appears to decrease in both demersal and pelagic fish fillets while b* increased in demersal and decreased in pelagic species along body axis from head to tail
[93]
Color changes during iced storage of ray
Slight reduction in L while a and b hardly underwent any variation. On cooked muscle higher L and b were found that did not change during storage
[94]
Influence of ice type used for storage on color of fish (herring, sardine, and horse mackerel)
Color measurements on the skin of iced stored fish performed on board did not show any significant differences between specimens stored in flake ice and those stored in slurry ice
[95–97]
Influence of CO2-modified MAP on color of mackerel fillets stored at −2°C for 21 d
L and b increased significantly at day 14, a increased between day 7 and 21. After cooking L, a, and b of CO2-modified MAP fillet were not significantly different
[98]
Channel catfish fillet strips stored in CO2 environment
Strips were stored for 4 weeks at 2°C and 8°C under aerobic, 25% CO2 and 80% CO2, in CO2 packed samples L increased and a decreased
[99]
Influence of tumbling catfish fillets containing 2-methylisoborneol in citric acid solution (0%–2%)
Fish tumbled in 2% acid showed highest L*, followed by 0.5% acid, water-treated fish and then untreated sample. Fish tumbled with water showed lowest a* and b*
[100]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.3 (continued)
◾ 409
Color Measurements on Refrigerated Products
Task
Color Effects
References
Evaluation of color changes of king salmon stored in air packs at 0°C for 22 d
Frame sides had significantly higher a* and b* and lower L* than skin sides. L* did not change with time. a* and b* did not vary with harvest method. a* at skin side decreased over time
[101]
Evaluation of quality changes of farmed halibut fed by diets only differing in fat content stored in ice for 26 d
L* and b* of cutlets increased significantly during first 6 d of storage. Negative a* decreased between day 4 and 6. No further changes in color after day 6
[102]
Effect of MAP on color changes of gutted farmed sea bass when stored at 3°C for up to 9 d
Six different atmospheres (with increasing CO2 content) were used for storage. Only a* and b* were influenced by storage time. L* was independent on time
[103]
Comparison of color of sea bass fillets stored in MAP or air
No difference emerged in whiteness among the differently preserved fillets
[104]
Effect of fasting on color of ordinary muscle in full-cycle cultured bluefin tuna during chilled storage
L*, a*, and b* of post-fasting group were lower than for pre-fasting group throughout storage
[105]
Influence of ice storage of sardine and mackerel muscles on redness index (RI) (a*/b* ratio)
RI of iced-stored mackeral and sardine muscles decreased when storage time increased. RI of washed mince was lower than that of unwashed mince and decreased during first 6 d of iced storage
[106]
Comparison of color changes in ordinary and dark muscle of yellowtail during iced storage
a* value in dark muscle was higher than in ordinary muscle at day 0 and decreased during 2 d of ice storage. b* tended to increase in both muscle types throughout storage whereas L* changes only slightly. Changes in color tones in both muscles during 4 d of ice storage were different for the different fish species investigated
[107]
Evaluation of color changes in two batches (different areas, sizes, and rearing conditions) of Senegalese sole during 28 d of iced storage
SS-1 lot had significantly higher L*, a*, and b* than the SS-2 lot until rejection point by the sensory panel. L* and a* hardly changed throughout storage in both batches whereas b* for the SS-2 lot increased during storage
[108]
Influence of ice storage of bigeye snappers on color of resulting surimi
Gel whiteness decreased markedly as the storage time increased. Gels prepared from headed and gutted fish had slightly higher whiteness than those produced from whole fish
[109]
(continued)
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Handbook of Seafood and Seafood Products Analysis
Table 22.3 (continued)
Color Measurements on Refrigerated Products
Task
Color Effects
To study potential skin and fillet color differences at the point of slaughter, during rigor mortis, and after ice storage for 7 d, at a time when the fish are typically available to consumers. Skin color (CIE L*, a*, b*, and related values) was determined by a Minolta Chroma Meter. Roche Salmo FanTM Lineal and Roche Color Card values were determined by a computer vision method and a sensory panel
Table 22.4
The changes in skin and fillet color of anesthetized and exhausted Atlantic salmon were determined. Perimortem handling stress initially affected several color parameters of skin and fillets. Significant transient fillet color changes also occurred in the pre-rigor phase and during the development of rigor mortis. The color change patterns during storage were different for the two groups of fish. These differences were relatively small and probably could not be spotted by consumers
References [26]
Color Measurements on Frozen Products
Task
Color Effects
References
Effect of frozen storage at −18°C up to 1 year on color of weakfish fillet
L, a, and b for all product forms did not change significantly between months of harvest and during storage
[110]
Frozen storage (3 and 6 months at −20°C and −80°C) of vacuumpacked fillets of rainbow trout
Increasing L*, a*, b*, and decreasing h*. Color characteristic from different parts of fillet differed significantly
[111]
Browning progress during frozen storage and after thawing of yellowtail dark muscle
Ratio b*/a* was used as indicator to follow the browning progress. When b*/a* > 0.5, only slight browning. When b*/a* > 0.8, product becomes not merchantable
[112]
Evaluation of dietary oil source Peruvian fish oil (PO), rapeseed oil (RO), capelin oil (CO), soybean oil (SO) diet, and frozen storage on flesh color of Atlantic salmon
L* and b* of fish fed CO diet was lower than the others and a* of fish fed PO diet was different from those of fish fed RO and SO diets in raw fillet. Important increases in L*, a*, b*, or C* were observed during frozen storage
[113]
Investigation of seasonal development of color of fillets of Norwegian spring-spawning herring
Fillets were lighter, less red, and yellower at the anterior compared to the posterior end. They tended to become darker and redder with progress of season
[114]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.4 (continued)
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411
Color Measurements on Frozen Products
Task
Color Effects
References
Effect of long-term frozen storage up to 13 months at different temperatures (−10°C, −20°C, −30°C) on color of individually packed fish fillets
Only during storage at −10°C and somewhat lower at −20°C a noticeable increase in L* was observed. At −30°C L* did not change. b* behaved comparable to L*
[115,116]
Effectiveness of different cryoprotectants in stabilizing color of restructured trout products during frozen storage
After 6 months of frozen storage control and sodium lactate-treated products were lighter and yellower than carbohydrate-treated products. Samples did not differ in a* after 6 months. After each freeze/thaw cycle control and sodium lactate-treated raw products were lighter than carbohydrate-treated. a* did not change with increasing number of FT cycles. Carbohydratetreated products were generally less red and yellow compared to control and lactate-treated products
[117,118]
Preventing color changes in frozen-stored minced muscle of Atlantic mackerel
DE 18 maltodextrin or the combination of sucrose/sorbitol slowed down color changes. b and a were respectively lower and higher in treated samples than in control. Only slight differences between samples were found in L
[119]
Influence on whiteness (WS) of natural actomyosin (NAM)
WS of NAM extracted from frozen fillets and was less than that of NAM extracted from fresh fish. When whole frozen fish was used for NAM extraction WS was less compared with NAM from frozen fillet. WS decreased in the presence of aldehydes
[120]
Impact of freezing temperature (−10°C, −25°C, −40°C, −55°C, or −70°C) on color of farmed Atlantic cod fillet
Regression analysis showed curvilinear relationship between freezing temperature and L*. Fillets frozen at −10°C had higher L* than those frozen at −70°C. L* were similar for fillets frozen at −25°C to −55°C
[121]
Fading phenomenon of farmed steelhead fillet during frozen storage
The increases in expressible fluid correlated positively with fading (L*) and negatively with a*
[122]
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Handbook of Seafood and Seafood Products Analysis
Table 22.5 Color Measurements on Heat-Treated Products Task
Color Effects
References
Influence of species and formulation on color of AP, catfish, and redfish during thermal processing
L increased for catfish but decreased for redfish and AP after thermal processing, a and b increased in general during canning, formulation of canned products had no effect on color
[124]
Effects of tripolyphosphate dips on color of thermally processed mullet
Only b was significantly influenced by treatment, also when fresh and frozen fish in using for canning were compared
[125]
Discoloration in thermally processed blue crab meat
Blue crab meat became darker with increasing heating process. Meat at bottom of a can was darker than that on top
[126]
Effect of heating of Pacific chum salmon in water (60°C–100°C) for 0–40 min
Increase in processing temperature or time increased L*, but decreased a* and b* of muscle
[127]
Effect of cooking of aquacultured fish fillets (pacu, rainbow trout, hybrid striped bass, catfish, and tilapia)
Cooking increased L and decreased a and b. Strongest change in L observed in catfish and least change in hybrid striped bass
[128]
Effect of baking and cold smoking on color of cultured and wild salmon
Baking and smoking caused a* to decrease, while L* increased. No color differences between wild and farmed salmon. Color of raw, baked, and smoked rainbow trout flesh was related to carotenoid concentration of raw flesh
[129,130]
Relation of color and color stability of smoked fillets during chill storage to duration of frozen storage prior to smoking of fillets
Smoked fillets from fish fed lower level of astaxanthin had significantly higher L, lower a and b compared to smoked products from fish fed higher level. Smoked products from fish fed high fat level had higher L and lower a and b than smoked fillets from fish fed diet with the lowest fat level. Only small changes in color during chill storage
[131]
Color changes in smoked rainbow trout fed diet supplemented with canthaxanthin in combination with different lipid levels and the effect of different packaging conditions
Smoke-curing lead to a decrease of L* and an increase of h* more marked in fish fed the diet with high lipid level. Use of MAP for the packaging of fillets lead to maintain the color of the flesh in comparison with packaging under vacuum or under air
[132]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.5 (continued)
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413
Color Measurements on Heat-Treated Products
Task
Color Effects
References
Investigation of effects of salting (injection salting vs. dry salting), smoking temperature (20°C vs. 30°C), and storage (chilled storage vs. no storage) on surface color of cold smoked salmon fillets
Higher increase in b* and C* and higher h* and ΔE* of dry-salted than injection-salted fillets. No influence on L* and a* by salting method. Drop in a* was higher after smoking fillets at 20°C than at 30°C, ΔE* was higher when fillets were smoked at 30°C than at 20°C
[133]
Comparison of color of sliced salmon sampled in a French hypermarket from fish originally grown in Norway, Scotland, and Ireland
There was a significant effect of country of origin on color, with the Irish having higher values for a*, b*, C*, and h* than the Norwegian with the Scottish in-between
[134,135]
Effects of cold-smoking temperature (range 21.5°C– 29.9°C) and dietary oil sources (pure Peruvian fish oil (FO) or pure SO supplements) on color characteristics
Only b* values exhibited correlations with temperature. Dietary oil source only had significant effects on a* and C* during storage. Salmon fed diets with FO were slightly redder than salmon fed SO. FO-group had C* values higher than SO-group
[136]
Effects of tripolyphosphate (STPP) in brine on smoke adsorption and color of cold smoked mullet
L, reflecting smoke adsorption, was lightest for control fillets, followed by 5% salt treatment, and darkest for 5% and 10% STPP both with 5% salt
[137]
Comparison of steam blanching, water blanching, and MW-heating applied to desalted cod
Thermally treated cod was lighter and little yellower. Heat induced changes in cod were a little smaller than in MW-heated cod
[138]
Color analysis of skinless catfish fillets from steamtreated catfish and control
No differences in L, a, b, and whiteness were found between steam-treated and control fillets
[139]
Effects of sous vide cooking on color of fish/sauce packs
Sous vide cooking caused a loss of L, a, and b. Sauce color lightened as indicated by a rise in L/b values
[140]
Influence of irradiation on color of rainbow trout muscle
Significant difference in L* at the time before and after exposure. a* was identical and b* decreased
[141]
Assessment of influence of heating on rainbow trout muscle color by heated fish cutlets at temperatures in the range 30°C–70°C
L* most influenced by heating increased linearly up to 60°C without further changes. a* did not change markedly whereas b* slightly increased at higher temperatures
[142]
(continued)
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Handbook of Seafood and Seafood Products Analysis
Table 22.5 (continued)
Color Measurements on Heat-Treated Products
Task
Color Effects
References
Variability in color of smoked salmon samples
Values obtained on 114 smoked salmon with L* (45.4–61.8), a* (13.4–34.2), and b* (16.9–33.7) reflect their variability in color
[143]
Evaluation of kinetics of reactions leading to changes in salmon color during thermal processing
When heating, muscle color is characterized by rapid whitening followed by slow browning. Whitening occurred within the first 10 min with L* increased to a maximum and a* and b* decreased to a minimum. During browning, changes of L*, b*, and ΔE* followed a zero-order reaction
[144]
Effect of fat content and fillet shape on color of smoked Atlantic salmon
Smoked salmon had lower L* and a* and higher b* compared with raw salmon. Ice-stored salmon displayed higher change due to smoking in L* and lower change of a* and b* with increasing fat content. The change in L*, a*, and b* of frozen stored salmon showed no correlation with raw material characteristics. Changes in color from raw to smoked products are affected by variations in fat content only when fresh material is used
[145]
Influence on color of cold smoked Atlantic salmon by freezing the raw fish before smoking and by freezing the finished product after smoking
L*, a*, and b* increased significantly due to freezing before and after smoking. Freezing before smoking had a significant effect on L*: fish frozen whole and fish frozen as fillets had higher L* than freshly smoked fillets. Freezing before smoking, either as whole fish or as fillets, also increased a* and b*. Freezing of fillets after smoking increased average a* and b* values in fillets that were either stored fresh or frozen after smoking, respectively. Increases in a* and b* were much larger than increase in L*
[146]
Color changes in skinless mahimahi fillet portions either treated with filtered smoke (FS) or left untreated for 24 h, followed by either aerobic storage at 4°C for 8 d or freezing for 30 d (−25°C) followed by thawing and aerobic storage at 4°C for 8 d
Treating mahimahi fillets with FS increased a* in muscle and stabilized it during frozen storage. Redness did decay rapidly on cold storage for both defrosted and fresh filtered-smoke-treated products, and reached initial (pre-smoking) redness levels in 2 d
[147]
Quality Assessment of Fish and Fishery Products by Color Measurement
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415
Table 22.6 Color Measurements on Fish Treated by High Pressure Task
Color Effects
References
Pressure-treatment of pelagic and demersal species from different fishing grounds using same conditions
Color changes measured on demersal fishes caused by pressure treatment resulted in marked increase of L* and in decreases of both a* and b*. High-pressure treatments (higher than 150–200 MPa, 5 min) resulted in a cooked appearance of pollock, cod, tuna, mackerel, salmon trout, carp, plaice, and anglerfish. Only octopus retained a raw appearance till 400–800 MPa
[149,150]
High pressure (100–300 MPa) applied (for 0–30 min) to fresh seafood to control enzymerelated texture
L and b increased progressively with amount of pressure applied and duration of pressure application while a was reduced. ΔE indicated no color difference between sample treated up to 200 MPa for 10 min
[151]
Changes in color of turbot fillets during frozen storage at −20°C after pressure shift freezing (PSF) and air blast freezing (ABF)
PSF resulted in overall increase in L* and b* and decrease in a*. Frozen storage did not particularly modify color parameters of PSF fillets. ABF did not give a cooked aspect after thawing to the turbot fillets. During the storage of ABF fillets, b* increased significantly
[152]
Influence of high-pressureassisted thawing (PAT) on color of fillets from redfish, cod, rainbow trout, whiting, haddock, and salmon
In raw fillets, color changes (high ΔE*) were mainly caused by a strong increase in L*. Smaller changes were monitored for a* (decrease) and b* (increase). After heat treatment, the influence of high pressure on color was much smaller. ΔE* between cooked fillets previously thawed either by highpressure treatment or conventionally, varied from negligible to significant
[153]
Color changes in carp muscle exposed to high pressures of 50–500 MPa/10 min
Carp muscles lost their transparency, L increased parallel with an increase of pressurization at room temperature
[154]
Effect of high-pressure treatment (up to 500 MPa, 5 min) on color of sea bass fillets after 0, 7, and 14 d of refrigerated storage
Nonpressurized fillets showed an increase in L* for refrigerated storage time of 7 d, followed by decrease in L* after 14 d of storage. a* and b* remained constant during storage. Regardless of pressure level, application of pressure on fillet increased L*
[155]
High pressure to obtain cod sausage with added chitosan
After pressure treatment L* increased markedly, whereas b* decreased slightly
[156] (continued)
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Handbook of Seafood and Seafood Products Analysis
Table 22.6 (continued)
Color Measurements on Fish Treated by High Pressure
Task
Color Effects
References
Effect of high-pressure treatments at 400 and 600 MPa (1 and 5 min) on color of heat-induced fish gels obtained from arrowtooth flounder fish paste
Thermal treatments of pressure-treated and control samples were assigned to kamaboko (90°C), setting (40°C/90°C) and modori (60°C/90°C). L* ranged from 76.6 to 81.7, a* were from −1.37 to −0.69 and b* varied from 6.0 to 10.9. Modori samples had lowest L*
[157]
Comparison of AP and Pacific whiting surimi gels containing potato starch and/or egg white (400 and 650 MPa for 10 min at 20°C) with heat-induced gels (90°C, 40 min)
Pressure treatment improved whiteness (WS) of surimi gels as compared with heat-treated surimi gels, whereas additives did not. At 400 MPa WS was 10% higher than heated gels. At 650 MPa WS increased 8%
[158]
Effect on color of minced albacore muscle treated with high hydrostatic pressure at 275 and 310 MPa for 2–6 min
L* increased with pressure giving a lighter product, a* decreased and b* increased giving a light yellow/grayish hue, cooked appearance product
[159]
Effect of combined application of high-pressure treatment and modified atmospheres on color of fresh Atlantic salmon
L* of salmon increased with increasing intensity and time of pressurization. High-pressure treatment (150 MPa, 60 min or 200 MPa, 10 min) resulted in an opaque product (L* > 70). A product with L* > 70 or a* < 13 is unacceptable
[160]
Changes in whiteness of tilapia gels obtained by combined hydrostatic pressure (200 MPa) and setting (50°C) treatments
The whiteness, which is related to the degree of protein denaturation, was highest in the gel formed by cooking
[161]
Effects of PSF and/or PAT on color of sea bass muscle were evaluated and compared with conventional (air-blast) frozen (AF) and thawed (AT) samples
L* did not change when the muscle was treated by AF/AT, but showed a very important increase in the high-pressure-treated systems (AF/PAT, PSF/AT, and PSF/PAT), being still larger in the case of the PSF/PAT. b* increased in all highpressure treated systems. a* did not present modifications due to application of high pressure. After cooking, all systems showed similar appearance
[162]
Investigation of the effect of high-pressure treatment on color of rainbow trout and mahimahi during cold storage for 6 d
L* for rainbow trout increased as pressure increased and did not change after 3 d storage. L* increased after 6 d storage compared to the other 2 d. a* decreased after pressurization. For mahimahi L* increased slightly with increasing pressure, during storage L* differed slightly. As pressure and storage time increased a* decreased. b* increased over the control for all pressures tested
[163]
Quality Assessment of Fish and Fishery Products by Color Measurement Table 22.6 (continued)
◾
417
Color Measurements on Fish Treated by High Pressure
Task Effect of different levels of pressure treatment on the color of vacuum-packed cold-smoked dolphinfish (Coryphaena hippurus) and on the subsequent chilled storage at 5°C of the final product
Color Effects L* was lower in fillets than in slices and increased with pressure, especially in slices. a* and b* in fillets increased with pressure. During chilled storage for 70 d, L* was quite stable in pressure-treated and untreated smoked slices. L* in pressurized sample was higher than in untreated batch. During chilled storage a* increased in both batches. Pressurization brought about a slight increase in b* and values held relatively steady during chilled storage. b* increased in the unpressurized batch during storage
References [164]
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16. Di Natale, C., Data fusion in MUSTEC: Towards the definition of an Artificial Quality Index, in: Quality of Fish from Catch to Consumer: Labelling, Monitoring & Traceability, Luten, J.B., Oehlenschläger, J., and Olafsdottir, G., Eds., Wageningen Academic Publishers, Wageningen, the Netherlands, 2003, p. 273. 17. Balaban, M.O., Kristinsson, H.G., and Otwell, W.S. Evaluation of color parameters in a machine vision analysis of carbon monoxide-treated fish. Part I: Fresh tuna, J. Aquat. Food Prod. Technol., 14, 5, 2005. 18. Pavlidis, M., Papandroulakis, N., and Divanach, P., A method for the comparison of chromaticity parameters in fish skin: Preliminary results for coloration pattern of red skin Sparidae, Aquaculture, 258, 211, 2006. 19. Pearson, A.M. and Dutson, T.R., Quality Attributes and Their Measurement in Meat, Poultry and Fish Products, Blackie Academic & Professionals, Glasgow, U.K., 1994. 20. Hutchings, J.B., Food Color and Appearance, Aspen Publishers, Gaithersburg, MD, 1999. 21. Hutchings, J.B., Food Colour and Appearance, Blackie Academic & Professionals, Glasgow, U.K., 1994. 22. Hutchings, J.B., Expectations and the Food Industry: The Impact of Color and Appearance, Kluwer Academic/Plenum Publishers, Dordrecht, the Netherlands, 2003. 23. Kress-Rogers, E., Instrumentation and Sensors for the Food Industry, Butterworth Heinemann, Oxford, U.K., 1993. 24. Kress-Rogers, E. and Brimelow, C.J.B., Instrumentation and Sensors for the Food Industry, 2nd edn., Woodhead Publishing, Cambridge, U.K., 2001. 25. Jakobsen, M. and Bertelsen, G., Modelling colour stability in meat, in Colour in Food: Improving Quality, MacDougall, D.B., Ed., Woodhead Publishing., Cambridge, U.K., 2002, p. 233. 26. Erikson, U. and Misimi, E., Atlantic salmon skin and fillet color changes effected by perimortem handling stress, rigor mortis, and ice storage, J. Food Sci., 73, C50, 2008. 27. Reid, R.A. et al., Structural and chemical changes in the muscle of chum salmon (Oncorhynchus keta) during spawning migration, Food Res. Int., 26, 1, 1993. 28. Choubert, G., Blanc, J.-M., and Courvalin, C., Muscle carotenoid content and colour of farmed rainbow trout fed astaxanthin or canthaxanthin as affected by coking and smoke-curing procedures, Int. J. Food Sci. Technol., 27, 277, 1992. 29. Hatlen, B., Jobling, M., and Bjerkeng, B., Relationships between carotenoid concentration and colour of fillets of Arctic charr, Salvelinus alpinus (L.), fed astaxanthin, Aquacult. Res., 29, 191, 1998. 30. Ginés, R. et al., Effects of rearing temperature and strain on sensory characteristics, texture, colour and fat of Arctic charr (Salvelinus alpinus), Food Qual. Pref., 15, 177, 2004. 31. Shahidi, F., Synowiecki, J., and Penney, R.W., Pigmentation of Arctic char (Salvelinus alpinus) by dietary carotenoids, J. Aquat. Food Prod. Technol., 2, 99, 1993. 32. Jankowska, B. et al., A comparison of selected quality features of the tissue and slaughter yield of wild and cultivated pikeperch Sander lucioperca (L.), Eur. Food Res. Technol., 217, 401, 2003. 33. Choubert, G., Blanc, J.-M., and Valle, F., Colour measurement, using the CIELCH colour space, of muscle of rainbow trout, Oncorhynchus mykiss (Walbaum), fed astaxanthin: Effects of family, ploidy, sex, and location of reading, Aquacult. Res., 28, 15, 1997. 34. Bjerkeng, B. et al., Quality parameters of the flesh of Atlantic salmon (Salmo salar) as affected by dietary fat content and full-fat soybean meal as a partial substitute for fish meal in the diet, Aquaculture, 157, 297, 1997. 35. Morzel, M., Sohier, D., and van de Vis, H., Evaluation of slaughtering methods for turbot with respect to animal welfare and flesh quality, J. Sci. Food Agric., 83, 19, 2003. 36. Roth, B., Slinde, E., and Arildsen, J., Pre or post mortem muscle activity in Atlantic salmon (Salmo salar). The effect on rigor mortis and the physical properties of flesh, Aquaculture, 257, 504, 2006. 37. Kiessling, A. et al., Texture, gaping and colour of fresh and frozen Atlantic salmon flesh as affected by pre-slaughter iso-eugenol or CO2 anaesthesia, Aquaculture, 236, 645, 2004. 38. Bjørnevik, M. et al., Temporal variation in muscle fibre area, gaping, texture, colour and collagen in triploid and diploid Atlantic salmon (Salmo salar L), J. Sci. Food Agric., 84, 530, 2004.
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39. Segato, S. et al., Effect of triploidy on quality traits of shi drum (Umbrina cirrosa L.) until the second rearing year, Aquacult. Res., 38, 59, 2007. 40. Skjervold, P.O. et al., Properties of salmon flesh from different locations on pre- and post rigor fillets, Aquaculture, 201, 91, 2001. 41. Kristoffersen, S. et al., Effects of pre-rigor filleting on quality aspects of Atlantic cod (Gadus morhua L.), Aquacult. Res., 37, 1556, 2006. 42. Ginés, R. et al., The effects of long-day photoperiod on growth, body composition and skin colour in immature gilthead sea bream (Sparus aurata L.), Aquacult. Res., 35, 1207, 2004. 43. Kalinowski, C.T. et al., Effect of different carotenoid sources and their dietary levels on red porgy (Pagrus pagrus) growth and skin colour, Aquaculture, 244, 223, 2005. 44. Jankowska, B. et al., Slaughter value and flesh characteristics of European catfish (Silurus glanis) fed natural and formulated feed under different rearing conditions, Eur. Food Res. Technol., 224, 453, 2007. 45. Hallier, A. et al., Influence of farming conditions on colour and texture of European catfish (Silurus glanis) flesh, J. Sci. Food Agric., 87, 814, 2007. 46. González, S. et al., Composition of farmed and wild yellow perch (Perca flavescens), J. Food Comp. Anal., 19, 720, 2006. 47. Arredondo-Figueroa, J.L. et al., Color of raw, frozen, and smoked fillets of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with astaxanthin and saponified red chilli (Capsicum annuum) extracts, J. Aquat. Food Prod. Technol., 16, 35, 2007. 48. Doolan, B.J. et al., Effect of cage colour and light environment on the skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801), Aquacult. Res., 38, 1395, 2007. 49. Suontama, J. et al., Protein from Northern krill (Thysanoessa inermis), Antarctic krill (Euphausia superba) and the Arctic amphipod (Themisto libellula) can partially replace fish meal in diets to Atlantic salmon (Salmo salar) without affecting product quality, Aquacult. Nutr., 13, 50, 2007. 50. Jankowska, B. et al., Slaughter yield, proximate composition, and flesh colour of cultivated and wild perch (Perca fluviatilis L.), Czech J. Anim. Sci., 52, 260, 2007. 51. Osterlie, M. et al., Influence of added astaxanthin level and color on flavor of pastes of rainbow trout, J. Aquat. Food Prod. Technol., 10, 65, 2001. 52. Lanier, T.C. and Lee, C.M., Surimi Technology, Marcel Dekker, New York, 1992. 53. Park, J.W., Surimi and Surimi Seafood, Marcel Dekker, New York, 2000. 54. Park, J.W., Surimi and Surimi Seafood, 2nd edn., Marcel Dekker, New York, 2005. 55. Young, K.W. and Whittle, K.J., Colour measurement of fish minces using Hunter L, a, b values, J. Sci. Food Agric., 36, 383, 1985. 56. Eid, N., Dashti, B., and Sawaya, W., Sub-tropical fish by-catch for surimi processing, Lebensm.-Wiss. u.-Technol., 24, 103, 1991. 57. Brown, P., Rasco, B.A., and Borhan, M., Color removal from the dark muscle of Alaskan pollock (Theragra chalcogramma) fillets and minces using peroxide, J. Aquat. Food Prod. Technol., 2, 125, 1993. 58. Himonides, A.T., Taylor, K.D.A., and Knowles, M.J., The improved whitening of cod and haddock flaps using hydrogen peroxide, J. Sci. Food Agric., 79, 845, 1999. 59. da Ponte, D.J.B., Roozen, J.P., and Pilnik, W., Effects of additions on the stability of frozen stored minced fillets of whiting. II. Various anionic and neutral hydrocolloids, J. Food Qual., 8, 183, 1985. 60. Barrero, M. and Bello, R.E., Characterisation of sardine minced flesh (Sardinella aurita) washed with different solutions, J. Aquat. Food Prod. Technol., 9, 105, 2000. 61. Suvanich, V., Marshall, D.L., and Jahncke, M.L., Microbiological and color quality changes of channel catfish frame mince during chilled and frozen storage, J. Food Sci., 65, 151, 2000. 62. Pipatsattayanuwong, S., Park, J.W., and Morrissey, M.T., Functional properties and shelf life of fresh surimi from Pacific whiting, J. Food Sci., 60, 1241, 1995. 63. Kim, J.M. et al., Surimi from fillet frames of channel catfish, J. Food Sci., 61, 428, 1996.
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64. Reppond, K.D., Wasson, D.H., and Babbitt, J.K., Properties of gels produced from blends of arrowtooth flounder and Alaska pollock surimi, J. Aquat. Food Prod. Technol., 2, 83, 1993. 65. Chen, H.H., Effect of non-muscle protein on the thermogelation of horse mackerel surimi and the resultant cooking tolerance of kamaboko, Fisheries Sci., 66, 783, 2000. 66. Pérez-Mateos, M. and Montero, P., Contribution of hydrocolloids to gelling properties of blue whiting muscle, Eur. Food Res. Technol., 210, 383, 2000. 67. Hsu, C.-K. and Chiang, B.-H., Effects of water, oil, starch, calcium carbonate and titanium dioxide on the colour and texture of threadfin and hairtail surimi gels. Int. J. Food Sci. Technol., 37, 387, 2002. 68. Huda, N., Abdullah, A., and Babji, A.S., Functional properties of surimi powder from three Malaysian marine fish. Int. J. Food Sci. Technol., 36, 401, 2001. 69. Karayannakidis, P.D. et al., The effect of initial wash at acidic and alkaline pHs on the properties of protein concentrate (kamaboko) products from sardine (Sardina pilchardus) samples, J. Food Eng., 78, 775, 2007. 70. Yongsawatdigul, J., Piyadhammaviboon, P., and Singchan, K., Gel-forming ability of small scale mud carp (Cirrhiana microlepis) unwashed and washed mince as related to endogenous proteinases and transglutaminase activities, Eur. Food Res. Technol., 223, 769, 2006. 71. Ramírez, J.A. et al., Production of low-salt restructured fish products from Mexican flounder (Cyclopsetta chittendeni) using microbial transglutaminase or whey protein concentrate as binders, Eur. Food Res. Technol., 223, 341, 2006. 72. Uresti, R.M. et al., Effect of amidated low methoxyl pectin on the mechanical properties and colour attributes of fish mince, Food Technol. Biotechnol., 41, 131, 2003. 73. Pérez-Mateos, M. et al., Quercetin properties as a functional ingredient in omega-3 enriched fish gels fed to rats, J. Sci. Food Agric., 85, 1651 2005. 74. Pérez-Mateos, M. and Lanier, T.C., Comparison of Atlantic menhaden gels from surimi processed by acid or alkaline solubilization, Food Chem., 101, 1223, 2007. 75. Reppond, K.D. and Babbitt, J.K., Gel properties of surimi from various fish species as affected by moisture content, J. Food Sci., 62, 33, 1997. 76. Kristinsson, H.G. and Liang, Y., Effect of pH-shift processing and surimi processing on Atlantic croaker (Micropogonias undulates) muscle proteins, J. Food Sci., 71, C304, 2006. 77. Chai, P.P. and Park, J.W., Physical properties of fish proteins cooked with starches or protein additives under ohmic heating, J. Food Qual., 30, 783, 2007. 78. Chen, Y.-C. and Jaczynski, J., Protein recovery from rainbow trout (Oncorhynchus mykiss) processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives, J. Agric. Food Chem., 55, 9079, 2007. 79. Park, Y. et al., Incorporation and stabilization of omega-3 fatty acids in surimi made from cod, Gadus morhua, J. Agric. Food Chem., 52, 597, 2004. 80. Pongviratchai, P. and Park, J.W., Electrical conductivity and physical properties of surimi-potato starch under ohmic heating, J. Food Sci., 72, E503, 2007. 81. Rawdkuen, S. et al., Effect of cysteine proteinase inhibitor containing fraction from chicken plasma on autolysis and gelation of Pacific whiting surimi, Food Hydrocoll., 21, 1209, 2007. 82. Sanchez-Alonso, I. et al., Inhibition of hemoglobin-mediated oxidation of regular and lipid-fortified washed cod mince by a white grape dietary fiber, J. Agric. Food Chem., 55, 5299, 2007. 83. Shie, J.S. and Park, J.W., Physical characteristics of surimi seafood as affected by thermal processing conditions, J. Food Sci., 64, 287, 1999. 84. Jin, S.-K. et al., Effect of muscle type and washing times on physico-chemical characteristics and qualities of surimi, J. Food Eng., 81, 618, 2007. 85. Cardoso, C., Mendes, R., and Nunes, M.L., Effect of transglutaminase and carrageenan on restructured fish products containing dietary fibres, Int. J. Food Sci. Technol., 42, 1257, 2007. 86. Ramírez, J.A. et al., Low-salt restructured fish products using low-value fish species from the gulf of Mexico, Int. J. Food Sci. Technol., 42, 1039, 2007.
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87. Ramírez, J.A. et al., Low-salt restructured products from striped mullet (Mugil cephalus) using microbial transglutaminase or whey protein concentrate as additives, Food Chem., 102, 243, 2007. 88. Jaczynski, J. and Park, J.W., Physicochemical properties of surimi seafood as affected by electron beam and heat, J. Food Sci., 68, 1626, 2003. 89. Longard, A.A. and Regier, L.W., Color and some composition changes in ocean perch (Sebastes marinus) held in refrigerated sea water with and without carbon dioxide, J. Fish. Res. Board. Can., 31, 456, 1974. 90. Wasson, D.H., Reppond, K.D., and Kandianis, T.M., Antioxidants to preserve rockfish color, J. Food Sci., 56, 1564, 1991. 91. Li, S.J. et al., Color stability and lipid oxidation of rockfish as affected by antioxidant from shrimp shell waste, J. Food Sci., 63, 438, 1998. 92. Gerdes, D.L. and Santos Valdez, C., Modified atmosphere packaging of commercial Pacific red snapper (Sebastes entomelas, Sebastes flavidus, or Sebastes goodei), Lebensm.-Wiss. u.-Technol., 24, 256, 1991. 93. Schubring, R., Determination of fish freshness by instrumental colour measurement, Fleischwirtschaft Int., 2, 26, 1999. 94. Pastoriza, L. and Sampedro, G., Influence of ice storage on ray (Raja clavata) wing muscle, J. Sci. Food Agric., 64, 9, 1994. 95. Schubring, R. and Meyer, C., Ice storage of fish, new aspects: Comparison between flake ice and stream ice—Part I: Sardine (Sardina pilchardus), Dtsch. Lebensm.-Rundsch., 102, 405, 2006. 96. Schubring, R. and Meyer, C., Iced storage of fish, new aspects: Comparison between flake ice and stream ice—Part II: Horse mackerel (Trachurus trachurus), Dtsch. Lebensm.-Rundsch., 102, 508, 2006. 97. Schubring, R. and Meyer, C., Iced storage, new aspects: Comparison between flake ice and stream ice—Part III: Herring (Clupea harengus), Dtsch. Lebensm.-Rundsch., 103, 203, 2007. 98. Hong, L.C. et al., Quality of Atlantic mackerel (Scomber scombrus L.) fillets during modified atmosphere storage, J. Food Sci., 61, 646, 1996. 99. Silva, J.L. and White, T.D., Bacteriological and colour changes in modified atmosphere-packaged channel catfish, J. Food Protect., 57, 715, 1994. 100. Forrester, P.N. et al., Treatment of catfish fillets with citric acid causes reduction of 2-methylisoborneol, but not musty flavour, J. Food Sci., 67, 2615, 2002. 101. Fletcher, G.C. et al., Spoilage of rested harvested King salmon (Oncorhynchus tshawytscha), J. Food Sci., 68, 2810, 2003. 102. Guillerm-Regost, C. et al., Quality characterization of farmed Atlantic halibut during ice storage, J. Food Sci., 71, S83, 2006. 103. Torrieri, E. et al., Influence of modified atmosphere packaging on the chilled shelf life of gutted farmed bass (Dicentrarchus labrax), J. Food Eng., 77, 1078, 2006. 104. Poli, B.M. et al., Sensory, physical, chemical and microbiological changes in European sea bass (Dicentrarchus labrax) fillets packed under modified atmosphere/air or prepared from whole fish stored in ice, Int. J. Food Sci. Technol., 41, 444, 2006. 105. Nakamura, Y.-N. et al., Effect of fasting on physical/chemical properties of ordinary muscles in fullcycle cultured Pacific bluefin tuna Thunnus orientalis during chilled storage, Fisheries Sci., 72, 1079, 2006. 106. Chaijan, M. et al., Changes of pigments and color in sardine (Sardinella gibbosa) and mackerel (Rastrelliger kanagurta) muscle during iced storage, Food Chem., 93, 607, 2005. 107. Sohn, J.-H. et al., Lipid oxidations in ordinary and dark muscles of fish: Influences on rancid off-odor development and color darkening of yellowtail flesh during ice storage, J. Food Sci., 70, S490, 2005. 108. Tejada, M. and De las Heras, C., Sensory changes in farmed Senegalese sole (Solea senegalensis) during ice storage, Food Sci. Technol. Int., 13, 117, 2007. 109. Benjakul, S. et al., Gel-forming properties of surimi produced from bigeye snapper, Priacanthus tayenus and P. macracanthus, stored in ice, J. Sci. Food Agric., 82, 1442, 2002.
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110. Waters, M.E., Chemical composition and frozen storage stability of weakfish, Cynoscion regalis, Mar. Fish. Rev., 45, 27, 1983. 111. No, H.K. and Storebakken, T., Color stability of rainbow trout fi llets during frozen storage, J. Food Sci., 56, 969, 1991. 112. Hiraoka, Y. et al., Preventive method of color deterioration of yellowtail dark muscle during frozen storage and post thawing, Fisheries Sci., 70, 1130, 2004. 113. Regost, C., Jakobsen, J.V., and Rørå, A.M.B., Flesh quality of raw and smoked fillets of Atlantic salmon as influenced by dietary oil sources and frozen storage, Food Res. Int., 37, 259, 2004. 114. Hamre, K., Lie, O., and Sandnes, K., Development of lipid oxidation and flesh colour in frozen stored fillets of Norwegian spring-spawning herring (Clupea harengus L.). Effects of treatment with ascorbic acid, Food Chem., 82, 447, 2003. 115. Schubring, R., Instrumental colour, texture, water holding and DSC measurements on frozen cod fillets (Gadus morhua) during long term storage at different temperatures, Dtsch. Lebensm.-Rundsch., 100, 247, 2004. 116. Schubring, R., Changes in texture, water holding capacity, colour, and thermal stability of frozen cod (Gadus morhua) fillets: Effect of frozen storage temperature, Dtsch. Lebensm.-Rundsch., 101, 484, 2005. 117. Jittinandana, S., Kenney, P.B., and Slider, S.D., Cryoprotectants affect physical properties of restructured trout during frozen storage, J. Food Sci., 70, C35, 2005. 118. Jittinandana, S., Kenney, P.B., and Slider, S.D., Cryoprotectants preserve quality of restructured trout products following freeze thaw cycling, J. Muscle Foods, 16, 354, 2005. 119. Rodríguez-Herrera, J.J. et al., Possible role for cryostabilizers in preventing protein and lipid alterations in frozen-stored minced muscle of Atlantic mackerel, J. Agric. Food Chem., 54, 3324, 2006. 120. Chaijan, M. et al., The effect of freezing and aldehydes on the interaction between fish myoglobin and myofibrillar proteins, J. Agric. Food Chem., 55, 4562, 2007. 121. Mørkøre, T. and Lilleholt, R., Impact of freezing temperature on quality of farmed Atlantic cod, J. Texture Stud., 38, 457, 2007. 122. Ozbay, G., Spencer, K., and Gill, T.A., Investigation of protein denaturation and pigment fading in farmed steelhead (Onchorhychus mykiss) fillets during frozen storage, J. Food Process. Preserv., 30, 208, 2006. 123. Sinnhuber, R.O. et al., Radiation sterilization of pre-fried cod and halibut patties, J. Food Technol., 22, 1570, 1984. 124. Paredes, M.D.C. and Baker, R.C., Physical, chemical and sensory changes during thermal processing of three species of canned fish, J. Food Process. Preserv., 12, 71, 1988. 125. English, P.M. et al., Effects of tripolyphosphate dips on the quality of thermally processed mullet (Mugil cephalus), J. Food Sci., 53, 1319, 1988. 126. Requena, D.D. et al., Detection of discoloration in thermally processed blue crab meat, J. Sci. Food Agric., 79, 786, 1999. 127. Bhattacharya, S., Choudhury, G.S., and Studebaker, S., Color changes during thermal processing of Pacific chum salmon, J. Aquat. Food Prod. Technol., 3, 39, 1994. 128. Pullela, S.V. et al., Quality comparison of aquacultured pacu (Piaractus mesopotamicus) fillets with other aquacultured fish fillets using subjective and objective sensorial traits, J. Aquat. Food Prod. Technol., 9, 65, 2000. 129. Skrede, G. and Storebakken, T., Instrumental colour analysis of farmed and wild Atlantic salmon when raw, baked and smoked, Aquaculture, 53, 279, 1986. 130. Skrede, G. and Storebakken, T., Characteristics of color in raw, baked and smoked wild and prereared Atlantic salmon, J. Food Sci., 51, 804, 1986. 131. Jensen, C. et al., Effect of dietary levels of fat, a-tocopherol and astaxanthin on colour and lipid oxidation during storage of frozen rainbow trout (Oncorhynchus mykiss) and during chill storage of smoked trout, Z. Lebensm. Unters. Forsch. A, 207, 189, 1998.
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132. Quinones, A., Choubert, G., and Gomez, R., Colour stability of smoked muscle rainbow trout: Packaging effect, Sci. Aliments, 23, 132, 2003. 133. Birkeland, S., Haarstad, I., and Bjerkeng, B., Effects of salt-curing procedure and smoking temperature on astaxanthin stability in smoked salmon, J. Food Sci., 69, FEP198, 2004. 134. Rørå, A.M.B., Monfort, M.C., and Espe, M., Effects of country of origin on consumer preference of smoked salmon collected in a French hypermarket, J. Aquat. Food Prod. Technol., 13, 69, 2004. 135. Espe, M. et al., Quality of cold smoked salmon collected in one French hypermarket during a period of 1 year, LWT—Food Sci. Technol., 37, 627, 2004. 136. Rørå, A.M.B. et al., Quality characteristics of farmed Atlantic salmon (Salmo salar) fed diets high in soybean or fish oil as affected by cold-smoking temperature, LWT—Food Sci. Technol., 38, 201, 2005. 137. Antoine, F.R. et al., Phosphate pretreatment on smoke adsorption of cold smoked mullet (Mugil cephalus), J. Aquat. Food Prod. Technol., 9, 69, 2000. 138. Fernandez-Segovia, I. et al., Structure and color changes due to thermal treatments in desalted cod, J. Food Process. Preserv., 27, 465, 2003. 139. Bal’a, M.F.A., Podalak, R., and Marshall, D.L. Microbial and color quality of fillets obtained from steam-pasteurized deheaded and eviscerated whole catfish, Food Microbiol., 17, 625, 2000. 140. Fagan, J.D. and Gormley, T.R., Effect of sous vide cooking, with freezing, on selected quality parameters of seven fish species in a range of sauces, Eur. Food Res. Technol., 220, 299, 2005. 141. Dvorák, P., Kratochvíl, B., and Grolichová, M., Changes o f colour and pH in fish musculature after ionizing radiation exposure, Eur. Food Res. Technol., 220, 309, 2005. 142. Schubring, R., Comparative study of the DSC pattern, color, texture and water-binding capacity of rainbow trout muscle during heating, J. Food Process. Preserv., 32, 190, 2008. 143. Cardinal, M. et al., Sensory characteristics of cold-smoked Atlantic salmon (Salmo salar) from European market and relationships with chemical, physical and microbiological measurements, Food Res. Int., 37, 193, 2004. 144. Kong, F. et al., Kinetics of salmon quality changes during thermal processing, J. Food Eng., 83, 510, 2007. 145. Mørkøre, T. et al., Fat content and fillet shape of Atlantic salmon: Relevance for processing yield and quality of raw and smoked products, J. Food Sci., 66, 1348, 2001. 146. Rørå, A.M.B. and Einen, O., Effects of freezing on quality of cold-smoked salmon based on the measurements of physiochemical characteristics, J. Food Sci., 68, 2123, 2003. 147. Kristinsson, H.G., Danyali, N., and Ua-Angkoon, S., Effect of filtered wood smoke treatment on chemical and microbial changes in mahi mahi fillets, J. Food Sci., 72, C016, 2007. 148. Cruz-Romero, M. et al., Effects of high pressure treatment on physicochemical characteristics of fresh oysters (Crassostrea gigas), Innov. Food Sci. Emerg. Technol., 5, 161, 2004. 149. Schubring, R. et al., Off shore high pressure treatment of freshly caught fish, in: Innovations in Traditional Foods, Fito, P. and Toldra, F., Eds., Elsevier, London, 2005, p. 1213. 150. Matser, A.M. et al., Effects of high pressure on colour and texture of fish, High Press. Res., 19, 109, 2000. 151. Ashie, I.N.A. and Simpson, B.K., Application of high hydrostatic pressure to control enzyme related fresh seafood texture deterioration, Food Res. Int., 29, 569, 1996. 152. Chevalier, D. et al., Effect of pressure shift freezing, air-blast freezing and storage on some biochemical and physical properties of turbot (Scophthalmus maximus), LWT—Food Sci. Technol., 33, 570, 2000. 153. Schubring, R. et al., Impact of high pressure assisted thawing on the quality of fillets from various fish species, Innov. Food Sci. Emerg. Technol., 4, 257, 2003. 154. Sequeira-Munoz, A. et al., Physicochemical changes induced in carp (Cyprinus carpio) fillets by high pressure processing at low temperature, Innov. Food Sci. Emerg. Technol., 7, 13, 2006. 155. Chéret, R. et al., Effects of high pressure on texture and microstructure of sea bass (Dicentrarchus labrax L.) fillets, J. Food Sci., 70, E477, 2005.
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156. López-Caballero, M.E. et al. A functional chitosan-enriched fish sausage treated by high pressure, J. Food Sci., 70, M166, 2005. 157. Uresti, R.M. et al., Restructured products from arrowtooth flounder (Atheresthes stomias) using highpressure treatments, Eur. Food Res. Technol., 220, 113, 2005. 158. Tabilo-Munizaga, G. and Barbosa-Cánovas, G.V., Color and textural parameters of pressurized and heat-treated surimi gels as affected by potato starch and egg white, Food Res. Int., 37, 767, 2004. 159. Ramirez-Suarez, J.C. and Morrissey, M.T., Effect of high pressure processing (HPP) on shelf life of albacore tuna (Thunnus alalunga) minced muscle. Innov. Food Sci. Emerg. Technol., 7, 19, 2006. 160. Amanatidou, A. et al., Effect of combined application of high pressure treatment and modified atmospheres on the shelf life of fresh Atlantic salmon. Innov. Food Sci. Emerg. Technol., 1, 87, 2000. 161. Hwang, J.-S., Lai, K.-M., and Hsu, K.-C., Changes in textural and rheological properties of gels from tilapia muscle proteins induced by high pressure and setting. Food Chem., 104, 746, 2007. 162. Tironi, V., LeBail, A., and de Lamballerie, M., Effects of pressure-shift freezing and pressure-assisted thawing on sea bass (Dicentrarchus labrax) quality, J. Food Sci., 72, C381, 2007. 163. Yagiz, Y. et al., Effect of high pressure treatment on the quality of rainbow trout (Oncorhynchus mykiss) and mahi mahi (Coryphaena hippurus), J. Food Sci., 72, C509, 2007. 164. Gómez-Estaca, J., Gómez-Guillén, M.C., and Montero, P., High pressure eff ects on the quality and preservation of cold-smoked dolphinfi sh (Coryphaena hippurus) fi llets, Food Chem., 102, 1250, 2007.
Chapter 23
Instrumental Texture Isabel Sánchez-Alonso, Marta Barroso, and Mercedes Careche Contents 23.1 Introduction ................................................................................................................. 425 23.2 Selection of the Test Procedure and Sample Preparation .............................................. 426 23.3 Instrumental Texture ................................................................................................... 427 23.3.1 Kramer Test .................................................................................................... 427 23.3.2 Warner–Bratzler Test ....................................................................................... 427 23.3.3 Puncture Test ................................................................................................... 428 23.3.4 Tension Analysis .............................................................................................. 429 23.3.5 Compression Test ............................................................................................ 429 23.3.6 Texture Profile Analysis.................................................................................... 430 23.3.7 Viscoelastic Methods ....................................................................................... 431 23.3.8 Stress Relaxation Test .......................................................................................431 23.3.8.1 Creep Test ....................................................................................... 431 23.3.8.2 Small Amplitude Oscillatory Test ................................................... 432 23.4 Texture Measurement for Quality Assessment ............................................................. 432 23.5 Conclusion ................................................................................................................... 433 References ............................................................................................................................... 433
23.1
Introduction
The consumption quality of fish and fish products depend largely on their texture characteristics. It can be considered that the complex structural configurations, which comprise contractile muscle fibers, are largely responsible for these characteristics. Fish muscle fibers are generally much 425
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shorter than in their counterparts mammals and birds. Instead of being connected to tendons, they are bound together, one cell deep with their ends attached to sheets of connective tissue called myocommata which separate one block of cells from another. On heating, the myocommata which are composed largely of the protein collagen are broken down releasing the myotomes, which are the characteristic flakes of cooked fish fillets.1 The texture of fish can be affected by many factors. These include species differences, biological condition of the fish, methods of catch or slaughter, storage and processing conditions, or culinary treatments. In particular, after the death of the fish, a number of changes associated with the onset and resolution of rigor mortis occur, so that before rigor the muscle is soft and elastic, in rigor, muscle becomes hard due to the contraction of the fibers forming the actomyosin complex, and with its resolution, the muscle becomes soft and less elastic. During frozen storage, one of the frequently used preservation methods, fish muscle may suffer from a series of unwanted changes in texture, especially in some lean species. These changes result in a hard and dry product, and it has been shown that this process is accompanied by myofibrillar proteins aggregation. Szczesniak 2,3 has reviewed the complexity of texture in foods, the perception of texture, and the consumer attitudes toward this property, as well as the most complete sensory method, the Sensory Texture Profile.2,3 Nonoral methods commonly performed in the fish sector by pressing parts of the fish body with the finger are of high importance for fish inspection, and have been incorporated as parameters that comprise the Quality Index Method (QIM).4 Alongside with the development of sensory texture, many instrumental methods have been developed for measuring the textural properties of foods5,6 and fish in particular.7–9 They have been classified in three groups5: (i) fundamental, when well-defined rheological properties are measured; (ii) empirical, when instrumental parameters correlate with texture measured by sensory tests; (iii) imitative, which are those tests that resemble the conditions to which the food material is subjected in practice. Most of the reported data on fish flesh texture for quality assessment are based on mechanical tests that are empirical or imitative. This chapter will describe the different instrumental texture measurements as applied in fish and some fish products with examples on their use in different types of seafood products and conditions.
23.2 Selection of the Test Procedure and Sample Preparation The selection of the type of test and operating conditions depend on the material and purpose of the study, within the restrictions imposed by the geometry, structure, and fragility of the fish and fish products. For example, different tests may be more suitable depending on the type of sample (e.g., pieces of fish muscle, squid mantle, and surimi gels). The purpose of the study may have an additional effect on the selection of the test procedure or even the instrument of choice. For example, whereas for quality control, a fast, simple, and nondestructive method is required, for other applications this may not be necessary. It also depends on the properties to be measured, e.g., overall properties or of some structural parts (connective tissue vs. myotomes). In general terms, the overall size and shape of the fish is important, especially when the analyses are to be performed in the whole specimen, normally with nondestructive purposes. The operational time for a given test may be also important in order to avoid ageing of the material. For example, some rheological tests in which the fish sample is under a given deformation for a long time may be only suited for some applications. The size and shape characteristics of the fish and fish products often make the preparation of homogeneous samples a difficult task. When analyses are to be performed in fish muscle, its complex structure in terms of organization and orientation of the fibers and connective tissue, as well
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as the fragility of the overall structure are factors to be taken into account. Some of the methods (e.g., Kramer or puncture tests) may render different results depending on the orientation of the fibers. Another example constitutes the effect of cooking for some of the measurements, such as compression. In some species, myotomes will slide past each other with even a gentle pressure exerted. This can lead to some measurement errors. These aspects will be discussed within each of the methods in the next section.
23.3
Instrumental Texture
The most used methods for the analysis of seafood products are the Kramer, Warner–Braztler, puncture, tensile and compression tests, texture profile analysis (TPA), and viscoelastic methods such as stress relaxation, creep, and small amplitude oscillatory measurements.
23.3.1
Kramer Test
It can be measured using the Kramer shear-compression cell. It was originally developed by Kramer et al.10 The standard cell is a multibladed fi xture: the upper part holds 10 blades, and the lower part or cell, where the food sample is placed, is a case with slots to guide the blades. These pass through the food, and the experiment stops when they have completely run through the sample. During the test, complex combinations of compression, extrusion, shear, and friction occur.5 Several factors affecting the performance of the cell including cell volume,11 and number and thickness of the blades12 have been investigated. There are many examples in the literature using, besides the original 10-blade cell,13–15 some modified cells, the most popular being the stable micro systems (SMS) device with five blades with a plexiglas wall in the front part, which allows to see the development of the analysis.16–18 For fish fillets, analysis can be performed both, perpendicular or parallel to the lengthwise of the sample, but the perpendicular setup gives better results.19 Few authors report the orientation of the sample,13,20 despite its importance. Samples of homogeneous structure can be prepared as parallelepipeds but in many cases, e.g., with fish fillets, the thickness can be controlled only to a certain extent. An alternative is to flake the cooked fillets by hand and to place them as a uniform layer in the Kramer cell.21,22 Other authors have chosen to dice the fish muscle and evenly spread a fi xed amount of it in a random fashion.14,23 The parameters usually measured include maximum force per sample weight, slope, and energy of the force–deformation curve. Although in the laboratory a highly linear relationship between maximum force and sample weight has been shown for surimi, this could not be found for fillets. Thus, as suggested previously based on food products other than fish,5 it is advised to use a constant weight of sample for the analysis. A wide variety of conditions, which affect the overall texture of fish, can be successfully assayed with the Kramer test.7 However, sample preparation is time consuming, and there is a relatively high amount of material needed to perform the test, and therefore, it may be unsuitable for some applications.
23.3.2 Warner–Bratzler Test This test was developed by Warner24 and Bratzler.25 It is performed with a device that consists of a blade with two cutting edges forming an angle of 60°, which penetrates another device with a slot.
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The blade cuts the sample like a guillotine and is subjected to a combination of tension, compression, and shearing.5 Usually the parameter measured is the maximum force exerted during the shearing. However, in raw fish, there are two peaks in the force–deformation curve which can give distinct information, so that, following the method proposed by Möller in beef samples,26 the first peak is attributed to the muscle fibers and the second, which is sharper and larger, to the connective tissue27–30. There are several methods similar to the Warner–Bratzler (W–B) test. One of them uses a Fish Shearing Device (FSD) that consists of a blade, which cuts the sample as it traverses a rectangular or circular device.30 Moreover, in this test it is important to take into account how the fibers are oriented with respect to the blade,7 and also the relationship between diameter or cross-sectional area of the test samples.5 One of the problems of W–B is the difficulty of performing the test with small pieces of fish muscle. The shearing takes place in a localized area of the muscle, causing distortion of the muscle fibers.13 Another drawback is that the cell needs frequent dismounting for cleaning and frequent calibration.31 Nevertheless, in studies where different instrumental methods have been compared, the shear test has been considered slightly more sensitive for some applications than, for example, TPA.32,33
23.3.3 Puncture Test Puncture test is exerted with a plunger that is pushed into the fish sample. When the punch begins to penetrate into the food there is a change in slope called the yield point, which marks the instant when the punch begins to break the food.5 Force at the point of rupture, slope and energy of the force–deformation curve, or the depth of penetration over a constant time are the parameters measured.7 In this test, the sample is subjected to a combination of compression and shearing in proportion to the area and perimeter of the cross-section of the plunger. Plungers with different area/perimeter ratios can be used to obtain the compression and shear coefficients, by plotting the puncture force measured at the yield point (F)/area (or F/perimeter) versus 1/diameter (or diameter).34 One of the requirements of this test is that the sample size should be much larger than the punch. In thin samples such as some fish fillets, there is a risk of compressing them against the support plate, so that the test is a combination of puncture and compression, or even full compression.5 Sample orientation has an effect on the test when fish fillets are analyzed. They have been measured perpendicularly35 or parallelly36 to the orientation of the muscle fibers and regarded as an estimation of fish firmness or cohesiveness respectively.37 Moreover, when the measurement is performed in conditions so that there is no direct contact of the puncture probe with the myocommata, the measurement has been interpreted as an estimation of the fibers blocks toughness.38 The “punch and die test”39 is a variation of the puncture test, suitable for cases when the sample is thin. In this case, the support plate contains a hole whose diameter is about the same size as the punch diameter. The parameters measured are maximum shear stress, which is related to the maximum force generated by the punch, stiffness, related to the initial slope of the force– deformation curve, and strain at failure, which depends on the deformation of the sample when the punch force is maximum.39,40 For a given punch diameter, these parameters are dependent on the sample thickness and volume, which are taken into account for obtaining normalized data.39 Johnson et al.41 applied this test both in flakes extracted from the fillet and in the fish fillet. The puncture test is the most popular gel measurement technique used in the industry for evaluating surimi quality. The recorded peak force at break and the depth of penetration (deformation) often are multiplied together to give the gel strength that is used in the Japanese grading
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standards.42 Since surimi gels with different texture properties may render the same gel strength, it has been proposed that force and deformation values should be expressed individually to indicate the gel functionality of surimi.43
23.3.4 Tension Analysis For tension analysis samples are prepared in strips or dumb-bell shaped samples. It is performed by holding them with two parallel clamps, one of which is fi xed, and the other moves away at a constant rate.5 The measured parameters are maximum force or tensile strength and energy. The force deformation curves can be corrected to true stress–strain relationship. The overall stiffness and the hardening index can be calculated from the constant and slope of the linear curve.44 A drawback of this method is that during the tension test, there is a risk of scattering of the point of rupture. Slipping or premature breaking in the clamps can also occur.5 This test is widely applied in materials such as squid mantle.44–46
23.3.5 Compression Test Compression analysis is performed upon application of uniaxial compression force, generally, between two parallel flat surfaces. If the force applied is insufficient to damage the sample, it can be considered a nondestructive test.5 The compression can be exerted to a given distance, percentage deformation, or at a set force. For a true compression test, the probe should be much larger than the sample. From the force–deformation curves, the slope, degree of deformation produced by a set force, and energy calculated by the area under the force–deformation curve, can be calculated.7 As in other tests the force–time curves can be transformed to true stress–strain relationships.47 These authors proposed for fish fillets, the use of a compressive deformability modulus derived from the true stress–strain relationships, which is representative of a material’s overall resistance to deformation. There are a number of parameters that have a high influence on the force–deformation curves. These include the deformation rate, the friction at the contact surfaces, and the physical dimensions of the samples.47–49 The latter is important for fish and fish products, since making homogeneous samples can be a problem due to the special size and shape characteristics of these food samples. In this sense, caution should be taken with interpretation of data when preparing samples with different height to diameter ratio, for example when measuring the compressive properties along the fish fillet. Another factor to be considered is the influence of the shape of the contact bodies. This is well documented for agricultural products such as cereal grains50,51 in which several methods have been described for the calculation of the apparent modulus of elasticity taking into account the shape of these contact bodies. Several parameters such as compression speed, deformation level, and thickness of the material have been assayed in fish muscle, in order to choose the best condition for the study of factors/ technologies affecting the quality of fish.31,35,52 In cooked fish samples the myotomes can fall apart when compressed in the texturometer53 and due to this, some authors do not consider the compression test suitable in cooked products.13 The solution of coating the machine surfaces with an abrasive material has been adopted in some cases.54 In many applications, the methodological conditions do not comply with the requirements for a true compression test. In fish, they are usually designed to resemble the “finger test.” In these
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cases, sample geometries, which are larger than the probe compressing the food, are used, often performing the test directly in the fillets or the whole fish, with varying and sometimes small, thickness along the sample being measured. In many cases, spherical probes are used. It can be considered that the forces derive from compression, and shear5,55 and their contribution may vary when the dimensions of the samples (e.g., thickness, shape of the contact surface) are not constant. Nevertheless, there are examples in the literature in which good correlations with the finger test have been found. This is the case in whole fish or fish fillets when studying the effect of ice storage, feeding strategy, and slaughtering method of rainbow trout (Oncorhynchus mykiss).56 Compression test is a good method for the measurement of the overall resistance to deformation. A problem depicted by Hyldig and Nielsen9 for this test was the difficulty of comparing the results among authors in part due to the fact that conditions of analysis are often not reported, and also because, as discussed previously, many methodological factors can have an influence on the measurements.
23.3.6
Texture Profile Analysis
TPA is an imitative test57,58 in which the sample is compressed twice, mimicking the action of the jaw. Szczesniak59 classified the textural terms for solids and semisolid foods, and this was the starting point to the development of a profiling method of texture applicable to both instrumental57 and sensory60 measurements, which was soon adapted to the Instron Universal Testing Machine.61 The force–deformation curve is analyzed to determine a number of texture parameters, five measured and two calculated. They were originally defined as hardness, cohesiveness, elasticity, adhesiveness, brittleness, chewiness, and gumminess.57 Nowadays, the most used physical definitions of the mechanical texture attributes are62,63: ◾ Hardness is defined as “the peak force during the first compression cycle” (first bite) in Newtons. ◾ Cohesiveness is defined as “the ratio of the positive force area during the second compression portion to that during the first compression, excluding the areas under the decompression portion in each cycle.” Cohesiveness is dimensionless. ◾ Springiness (originally called elasticity) is defined as “the height that the food recovers during the time that elapses between the end of the first bite and the start of the second bite.” Results are expressed commonly in mm. ◾ Adhesiveness is defined as “the negative force area for the first bite, representing the work (N × mm) necessary to pull the plunger away from the food sample.” ◾ Fracturability (originally called brittleness) is defined as “the force in Newtons at the first significant break in curve during the probe’s first compression.” Not all products fracture but when they do fracture the force falls off. ◾ Gumminess is defined as “the product of hardness × cohesiveness” in Newtons. Gumminess only applies to semisolid products. ◾ Chewiness is defined as “the product of gumminess × springiness” (which is equivalent to hardness × cohesiveness × springiness). Chewiness measured a work (N × mm) and only applies for solid products. Fish and fish products including surimi gels have been analyzed using TPA. Breene64 and Pons and Fiszman55 reviewed the TPA analysis in terms of instrumentation, testing conditions, terminology,
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and the time evolution of the main concepts. This test has been applied for fish and fish products in very different experimental conditions. As in the compression test, the size of the compressing units together with the sample size affects the type of forces measured. For the analysis of whole fish, fillets, and mince products, both flat-ended plungers and spherical probes65,66 have been used. The compression percentages also varied depending on whether the test was aimed to be performed in destructive or nondestructive conditions. The effect of different depths of compression and percentages of deformation has also been analyzed.32 In general, it is considered that hardness correlates very well with sensory assessment; however, other parameters such as springiness and cohesiveness render low correlations.2 It is a very popular method but in fish muscle it has been actually questioned the need of a double compression for most applications9 since hardness and fracturability are obtained from the first compression cycle.
23.3.7 Viscoelastic Methods Fish can be considered as a viscoelastic solid and as such, exhibits some of the elastic properties, characteristic of solids, and some flow properties, characteristic of liquids. In a viscoelastic solid, there is an instantaneous deformation upon initial application of the force, and the material continues to deform while the force is being applied. When the rheological properties are only dependent on time, the material displays linear viscoelastic behavior. Nonlinear viscoelasticity is shown when the rheological parameters depend also on other parameters such as magnitude of the stress applied. Most foods behave as linear viscoelastic in a short range of strain.5,67
23.3.8 Stress Relaxation Test In this test, the sample is subjected to a sudden deformation and the force required to hold the deformation constant is measured as a function of time. This test provides several parameters such as relaxation times, viscous, and elastic moduli, as well as the “degree of solidity” of the food. The relaxation curves are usually fitted to nonlinear regression, with two or three exponential terms.67,68 Stress relaxation tests in seafood products have mostly been used in surimi gels, but other applications include fish myofibrillar protein films,69 fish muscle,70–76 fish protein concentrate,77 and squid mantle.78,79 Stress relaxation test has been applied in whole cod (Gadus morhua) stored in ice and frozen stored hake (Merluccius capensis and paradoxus) with the aim of developing a nondestructive method that could correlate with sensory texture changes.80,81 It was shown that stress relaxation is easy to perform, required little sample preparation, and could be used for quality assessment because good agreement was found between the parameters extracted from stress relaxation and nonoral, low deformation sensory texture parameters.
23.3.8.1
Creep Test
In this test, a given stress is applied to the sample, and the displacement required to hold it constant is measured as the function of time. When the stress is removed, the recovery over time is recorded.67 Creep test has been mostly used for the study of the rheological behavior of gels82 but there are also examples of the use of this method in fish muscle.83,84
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A portable instrument was developed by Botta85,86 to determine a texture index for raw Atlantic cod (G. morhua) fillets. The test, which can be considered to be based on a fast creep test, rendered very good agreement with sensory texture measurements performed by trained assessors. Nesvadba87 measured the creep compliance with a new hand-held prototype for cod (G. morhua) and concluded that it is possible to construct a hand-held portable instrument for determining the texture of fish in industrial settings.
23.3.8.2 Small Amplitude Oscillatory Test In the dynamic tests, a food sample is subjected to a small sinusoidal oscillating strain or deformation varying harmonically along the time at fi xed or changeable frequency.88,89 The sample is usually placed between a cone and plate or parallel plates mounted in a controlled stress rheometer. The cone or plate is made to oscillate about a central point with a sinusoidal angular velocity at low amplitude while the shear stress is measured.5 The experimental shear stress–time curve can be separated into two components: an in phase or real component (G¢) associated with the storage of energy associated to elastic behavior and an out of phase or imaginary component (G²) associated with the loss of energy due to viscous behavior. The phase angle (d) between stress and strain can be obtained. Perfectly elastic or viscous materials would render d = 0 or 90°, respectively.67,87,90 Surimi and other protein gels are normally quite elastic so values of d are less than 10°.43 Nesvadba87 proposed the use of an oscillatory test for the study of fish freshness and frozen storage. In addition, glass transition temperature in abalone has been studied using this test.91,92 However, most applications are related to the gelation process of surimi.43,82,93,94 These gelation studies are usually performed at a constant frequency (less than 1 Hz) with varying time and/or temperature.95
23.4
Texture Measurement for Quality Assessment
Along with other applications of the use of instrumental texture analysis, the quality classification, or prediction based on some of the aforementioned instrumental methods has been a subject of interest in several seafood products applications. One of the approaches has been to classify frozen fish into several quality categories according to their Kramer, Warner–Bratzler, and puncture tests parameters. Thus, in frozen stored hake (Merluccius spp.), samples can be classified by this procedure into different time–temperature conditions.96 In some fish species, frozen storage time at a given temperature can be regarded as an estimation of their quality. A linear regression model with time as the variable to be predicted, and the parameters from the aforementioned tests as independent variables, can be performed. This was the approach used in frozen hake stored at −20°C for up to 2 years97 with good results. Instrumental texture analysis has been used in combination with other methods to assess different quality features of fish, such as electronic nose and colormeter measurements. The method has been termed Artificial Quality Index (AQI),98,99 and it is based on the same principles as those of the sensory method named QIM. Thus, data from the different instrumental equipment have been calibrated with their corresponding sensory attributes from the QIM. For this AQI, the stress relaxation test has been the method of choice, rendering good results for ice-stored cod.80,100 The AQI concept could be applied in small, portable instruments,87 and it can be as accurate and precise as the QIM.99 Thus, the replacement of trained sensory panels by a combination of instrumental methods that mimic human senses is a promising approach. The concept was designed
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using ice stored cod as the model species, and later it was applied in sardine during refrigerated storage with good results.101
23.5
Conclusion
There is a wide range of methods to analyze the instrumental texture of fish and fish products. For those products in which the integrity of the fish muscle is maintained, its complex structure imposes some restrictions to the application of some of the tests. Except for quality control, in which it is needed to perform simple and fast tests, for most applications the combination of more than one method can be of great value.
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16. Sánchez-Alonso, I. and Borderías, A.J. Technological effect of red grape antioxidant dietary fibre added to minced fish muscle. Int. J. Food Sci. Technol., 43, 1009, 2008. 17. Sánchez-Alonso, I., Hají-Maleki, R., and Borderías, A.J. Wheat fiber as a functional ingredient in restructured fish products. Food Chem., 100, 1037, 2007. 18. Sánchez-Alonso, I., Solas, M.T., and Borderías, A.J. Physical study of minced fish muscle with a white-grape by-product added as ingredient. J. Food Sci., 72, E94, 2007. 19. Taylor, R.G., Fjaera, S.O., and Skjervold, P.O. Salmon fillet texture is determined by myofibermyofiber and myofiber-myocommata attachment. J. Food Sci., 67, 2067, 2002. 20. McKenna, D.R., Nanke, K.E., and Olson, D.G. The effects of irradiation, high hydrostatic pressure, and temperature during pressurization on the characteristics of cooked-reheated salmon and catfish fillets. J. Food Sci., 68, 368, 2003. 21. Kramer, D.E. and Peters, M.D. Effect of pH and prefreezing treatment on the texture of yellowtail rockfish (Sebastes flavidus) as measured by the Ottawa Texture Measuring System. J. Food Technol., 16, 493, 1981. 22. Botta, J.R., Bonnell, G., and Squires, B.E. Effect of method of catching and time of season on sensory quality of fresh raw Atlantic cod (Gadus morhua). J. Food Sci., 52, 928, 1987. 23. Krivchenia, M. and Fennema, O. Effect of cryoprotectants on frozen whitefish fillets. J. Food Sci., 53, 999, 1988. 24. Warner, K.F. Progress report of the mechanical test for tenderness of meat. Ann. Am. Proc. Soc. Anim. Prod., 21, 114, 1928. 25. Bratzler, L.J. Determining the tenderness of meat by use of the Warner-Bratzler method. Proceedings of the 2nd Reciprocal Meat Conference, Kansas City, 117, 1949. 26. Moller, A.J. Analysis of Warner-Bratzler shear pattern with regard to myofibrillar and connective tissue components of tenderness. Meat Sci., 5, 247, 1981. 27. Montero, P. and Borderias, J. Influence of age on muscle connective tissue in trout (Salmo irideus). J. Sci. Food Agric., 51, 261, 1990. 28. Montero, P. and Borderias, J. Behavior of myofibrillar proteins and collagen in hake (Merluccius merluccius) muscle during frozen storage and its effect on texture. Z. Lebensm. Unters. Forsch., 190, 112, 1990. 29. Montero, P. and Borderias, J. Influence of myofibrillar proteins and collagen aggregation on the texture of frozen hake muscle. In: Quality Assurance in the Fish Industry. Huss, H.H., Jakobsen, M., and Liston, J., Eds. Elsevier Science Publishers B.V.: Amsterdam, the Netherlands, 149, 1992. 30. Chamberlain, A.I., Kow, F., and Balasubramaniam, E. Instrumental method for measuring texture of fish. Food Aust., 45, 439, 1993. 31. Chung, S.L. and Merritt, J.H. Physical measures of sensory texture in thawed sea scallop meat. Int. J. Food Sci. Technol., 26, 207, 1991. 32. Veland, J.O. and Torrissen, O.J. The texture of Atlantic salmon (Salmo salar) muscle as measured instrumentally using TPA and Warner-Brazler shear test. J. Sci. Food Agric., 79, 1737, 1999. 33. Sigurgisladottir, S., Hafsteinsson, H., Jonsson, A., Lie, O., Nortvedt, R., Thomassen, M., and Torrissen, O. Textural properties of raw salmon fillets as related to sampling method. J. Food Sci., 64, 99, 1999. 34. Bourne, M.C. Method for obtaining compression and shear coefficients of foods using cylindrical punches. J. Texture Stud., 5, 459, 1975. 35. Orban, E., Sinesio, F., and Paoletti, F. The functional properties of the proteins, texture and the sensory characteristics of frozen sea bream fillets (Sparus aurata) from different farming systems. Lebensm.-Wiss. u-Technol., 30, 214, 1997. 36. Ando, M., Toyohara, H., Shimizu, Y., and Sakaguchi, M. Validity of a puncture test for evaluating change in muscle firmness of fish during ice storage. Nippon Suisan Gakkaishi, 57, 2341, 1991. 37. Izquierdo-Pulido, M.L., Hatae, K., and Haard, N.F. Nucleotide catabolism and changes in texture indices during ice storage of cultured sturgeon, Acipenser transmontanus. J. Food Biochem., 16, 173, 1992.
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38. Morzel, M., Heapes, M.M., Reville, W.J., and Arendt, E.K. Textural and ultrastructural changes during processing and storage of lightly preserved salmon (Salmo salar) products. J. Sci. Food Agric., 80, 1691, 2000. 39. Segars, R.A., Hamel, R.G., Kapsalis, J.G., and Kluter, R.A. A punch and die test cell for determining the textural qualities of meat. J. Texture Stud., 6, 211, 1975. 40. Sawyer, F.M., Cardello, A.V., Prell, P.A., Johnson, E.A., Segars, R.A., Maller, O., and Kapsalis, J. Sensory and instrumental evaluation of snapper and rockfish species. J. Food Sci., 49, 727, 1984. 41. Johnson, E.A., Peleg, M., Sawyer, F.M., Segars, R.A., and Cardello, A. Mechanical methods of measuring textural characteristics of fish flesh. In: Refrigeration Science and Technology. I.I.F./I.I.R.: Boston, MA, 103, 1981. 42. Park, J. and Morrissey, M. The need for developing uniform surimi standards. In: Quality Control & Quality Assurance for Seafood. Sylvia, G., Shriver, A. L., and Morrissey, M., Eds. Oregon Sea Grant: Corvallis, OR, pp. 64–71, 1993. 43. Kim, Y.J., Park, J.W., and Yoon, W.B. Rheology and texture properties of surimi gels. In: Surimi and Surimi Seafood, 2nd edn., Park, J.W., Ed. 6000 Broken Sound Parkway NW, Suite 300: Boca Raton, FL 33487-2742; CRC Press: Boca Raton, FL, pp. 491–582, 2005. 44. Kuo, J.D., Peleg, M., and Hultin, H.O. Tensile characteristics of squid mantle. J. Food Sci., 55, 369, 1990. 45. Collignan, A. and Montet, D. Tenderizing squid mantle by marination at different pH and temperature levels. Lebensm.-Wiss. u-Technol., 31, 673, 1998. 46. Kagawa, M., Matsumoto, M., Yoneda, C., Mitsuhashi, T., and Hatae, K. Changes in meat texture of three varieties of squid in the early stage of cold storage. Fisheries Sci., 68, 783, 2002. 47. Johnson, E.A., Segars, R.A., Kapsalis, J.G., Normand, M.D., and Peleg, M. Evaluation of the compressive deformability modulus of fresh and cooked fish flesh. J. Food Sci., 45, 1318, 1980. 48. Calzada, J.F. and Peleg, M. Mechanical interpretation of compressive stress-strain relationships of solid foods. J. Food Sci., 43, 1087, 1978. 49. Chu, C.F. and Peleg, M. The compressive behavior of solid food specimens with small height to diameter ratios. J. Texture Stud., 16, 451, 1985. 50. Arnold, P.C. and Mohsenin, N.N. Proposed techniques for axial compression tests on intact agricultural products of convex shape. Trans. ASAE, 14, 78, 1971. 51. Mohsenin, N.N. Contact stresses between bodies in compression. In: Physical Properties of Plant and Animal Materials, Vol. 1. Structure, Physical Characteristics and Mechanical Properties. Mohsenin, N.N., Ed. Gordon & Breach Science Publishers Inc.: New York, pp. 278–308, 1970. 52. Weinberg, Z.G. and Angel, S. Behavior of a formed fish product under the stress-relaxation text. J. Food Sci., 50, 589, 1985. 53. Hatae, K., Yoshimatsu, F., and Matsumoto, J.J. Role of muscle-fibers in contributing firmness of cooked fish. J. Food Sci., 55, 693, 1990. 54. Feinstein, G.R. and Buck, E.M. Relationship of texture to pH and collagen content of yellowtail flounder and cusk. J. Food Sci., 49, 298, 1984. 55. Pons, M. and Fiszman, S.M. Instrumental texture profile analysis with particular reference to gelled systems. J. Texture Stud., 27, 597, 1996. 56. Faergemand, J., Ronsholdt, B., Alsted, N., and Borresen, T. Fillet texture of rainbow-trout as affected by feeding strategy, slaughtering procedure and storage post-mortem. Water Sci. Technol., 31, 225, 1995. 57. Szczesniak, A.S., Brandt, M.A., and Friedman, H.H. Development of standard rating scales for mechanical parameters of texture and correlation between the objective and the sensory methods of texture evaluation. J. Food Sci., 28, 397, 1963. 58. Friedman, H.H., Whitney, J.E., and Szczesniak, A.S. The texturometer-A new instrument for objective texture measurement. J. Food Sci., 28, 390, 1963. 59. Szczesniak, A.S. Classification of textural characteristics. J. Food Sci., 28, 385, 1963. 60. Brandt, M.A., Skinner, E.Z., and Coleman, J.A. Texture Profile Method. J. Food Sci., 28, 404, 1963. 61. Bourne, M.C. Texture profile of ripening pears. J. Food Sci., 33, 223, 1968.
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62. Bourne, M.C. Texture profile analysis. Food Technol., 32, 62, 1978. 63. Bourne, M.C. Food Texture and Viscosity: Concept and Measurement, Academic Press: New York, 1982. 64. Breene, W. Application of texture profile analysis to instrumental food texture evaluation. J. Texture Stud., 6, 53, 1975. 65. Morkore, T. and Einen, O. Relating sensory and instrumental texture analyses of Atlantic salmon. J. Food Sci., 68, 1492, 2003. 66. Carbonell, I., Duran, L., Izquierdo, L., and Costell, E. Texture of cultured gilthead sea bream (Sparus aurata): Instrumental and sensory measurement. J. Texture Stud., 34, 203, 2003. 67. Mohsenin, N.N. Some basic concepts of rheology. In: Physical Properties of Plants and Animals Materials, Vol. 1. Structure, Physical Characteristics and Mechanical Properties. Mohsenin, N.N., Ed. Gordon & Breach Science Publishers Inc.: New York, pp. 88–173, 1970. 68. Peleg, M. Characterization of stress relaxation curves of solid foods. J. Food Sci., 44, 277, 1979. 69. Cuq, B., Gontard, N., Cuq, J.L., and Guilbert, S. Rheological model for the mechanical properties of myofibrillar protein-based films. J. Agric. Food Chem., 44, 1116, 1996. 70. Iso, N., Mizuno, H., Saito, T., Ohzeki, F., and Yang, L.C. Studies on the rheological properties of heated carp meats. Bull. Jpn. Soc. Sci. Fish., 50, 349, 1984. 71. Iso, N., Mizuno, H., Saito, T., Ohzeki, F., and Wang, Z. Studies on the rheological properties of the heated yellowtail meat. Bull. Jpn. Soc. Sci. Fish., 50, 2061, 1984. 72. Iso, N., Mizuno, H., Saito, T., Mochizuki, Y., Ishii, K., Okunuki, H., and Miyata, K. The relationships between the rheological properties and the freshness of fish meat. Bull. Jpn. Soc. Sci. Fish., 53, 1231, 1987. 73. Kimura, H., Saito, T., Mizuno, H., Ogawa, H., Mochizuki, Y., Suyama, Y., and Iso, N. The rheological properties of salted jellyfish during cooking and dipping in water. Bull. Jpn. Soc. Sci. Fish., 57, 463, 1991. 74. Xin, G., Ogawa, H., Tashiro, Y., and Iso, N. Rheological properties and structural changes in raw and cooked abalone meat. Fisheries Sci., 67, 314, 2001. 75. Xin, G., Tashiro, Y., and Ogawa, H. The correlation between rheological properties and characteristic values of structure for steamed abalone meat. Food Sci. Technol. Res., 8, 304, 2002. 76. Xin, G., Tashiro, Y., and Ogawa, H. Rheological properties and structural changes in steamed and boiled abalone meat. Fisheries Sci., 68, 499, 2002. 77. Okazaki, E., Tsukada, K., Kanna, K., and Suzuki, T. Changes of properties of meat-textured fishprotein concentrate during frozen storage. Bull. Jpn. Soc. Sci. Fish., 50, 307, 1984. 78. Mochizuki, Y., Mizuno, H., Ogawa, H., Ishimura, K., Tsuchiya, H., Fukuzawa, M., and Iso, N. Rheological properties of cuttlefish and squid raw meat. Fisheries Sci., 60, 555, 1994. 79. Mochizuki, Y., Mizuno, H., Ogawa, H., Ishimura, K., Tsuchiya, H., and Iso, H. Changes of rheological properties of cuttlefish and squid meat by heat treatment. Fisheries Sci., 61, 680, 1995. 80. Herrero, A.M., Heia, K., and Careche, M. Stress relaxation test for monitoring post mortem textural changes of ice-stored cod (Gadus morhua). J. Food Sci., 69, FEP178, 2004. 81. Herrero, A.M. and Careche, M. Stress relaxation test to evaluate textural quality of frozen stored Cape hake (M. capensis and M. paradoxus). Food Res. Int., 38, 69, 2005. 82. Campo, L. and Tovar, C.A. Influence of the starch content in the viscoelastic properties of surimi gels. J. Food Eng., 84, 140, 2008. 83. Hatae, K., Nakayama, T., Matsui, Y., Shimada, A., and Matsumoto, J.J. Creep compliance behaviours of raw fish muscles in five species. Bull. Jpn. Soc. Sci. Fish., 54, 1595, 1988. 84. Yoshioka, K., and Yamamoto, T. Changes of ultrastructure and the physical properties of carp muscle by high pressurization. Fisheries Sci., 64, 89, 1998. 85. Botta, J.R. Instrument for nondestructive texture measurement of raw Atlantic cod (Gadus morhua) fillets. J. Food Sci., 56, 962, 1991. 86. Botta, J.R. Physical methods of evaluating freshness quality. In: Evaluation of Seafood Freshness Quality. VCH Publishers Inc.: Weinheim, Germany, pp. 35–63, 1995.
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87. Nesvadba, P. Quality control by instrumental texture measurements. In: Seafoods—Quality, Technology and Nutraceutical Applications. Alasavar, C., Taylor, T., Eds. Springer: Berlin, pp. 43–57, 2002. 88. Rao, M.A. Phase transitions, food texture and structure. In: Texture in Food, Vol. 1: Semi-Solid Foods. Brian, M., McKenna, Eds. CRC Press: Boca Raton, Boston, New York, Washington, DC, pp. 36–62, 2003. 89. Barnes, H.A., Hutton, J.F., and Walters, K. An Introduction to Rheology. Elsevier: Amsterdam, 1993. 90. Gunasekaran, S. and Ak, M.M. Dynamic oscillatory shear testing of foods—Selected applications. Trends Food Sci. Technol., 11, 115, 2000. 91. Rahman, M.S., Kasapis, S., Guizani, N., and Amri, O.S. State diagram of tuna meat: Freezing curve and glass transition. J. Food Eng., 57, 321, 2003. 92. Sablani, S.S., Kasapis, S., Rahman, M.S., Al Jabri, A., and Al Habsi, N. Sorption isotherms and the state diagram for evaluating stability criteria of abalone. Food Res. Int., 37, 915, 2004. 93. Ma, L., Grove, A., and Barbosa-Cánovas, G.V. Viscoelastic characterization of surimi gel: Effects of setting and starch. J. Food Sci., 61, 881, 1996. 94. Yoon, W.B., Gunasekaran, S., and Park, J.W. Characterization of thermorheological behavior of Alaska Pollock and Pacific Whiting surimi. J. Food Sci., 69, 338, 2004. 95. Hamann, D.D., Purkayastha, S., and Lanier, T.C. Applications of thermal scanning rheology to the study of food gels. In: Thermal Analysis of Foods. Harwalker, V.R. and Ma, C.Y., Eds. Elsevier, Barking: Essex, U.K., pp. 306–332, 1990. 96. Barroso, M. Careche, M., Barrios, L., and Borderias, A.J. Frozen hake fillets quality as related to texture and viscosity by mechanical methods. J. Food Sci., 63, 793, 1998. 97. Herrero, A.M. and Careche, M. Prediction of frozen storage time of Cape hake (Merluccius capensis and Merluccius paradoxus) by instrumental methods. J. Sci. Food Agric., 86, 2128, 2006. 98. Di Natale, C. Data fusion in MUSTEC: Towards the definition of an artificial quality index. In: Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Luten, J.B., Oehlenschager, J., and Olafsdottir, G., Eds. Wageningen Academic Publishers: Wageningen, the Netherlands, pp. 273–282, 2003. 99. Olafsdottir, G., Nesvadba, P., Di Natale, C., Careche, M., Oehlenschager, J., Tryggvadottir, S.V., Schubring, R., Kroeger, M., Heia, K., Esaiassen, M., Macagnano, A., and Jorgensen, B.A. Multisensor for fish quality determination. Trends Food Sci. Technol., 15, 86, 2004. 100. Careche, M., Tryggvadottir, S.V., Herrero, A., Lägel, B., Petermann, U., Schubring, R., and Nesvadba, P. Instrumental methods for measuring texture of fish. In: Quality of Fish from Catch to Consumer: Labeling, Monitoring and Traceability. Luten, J.B., Oehlenschager, J., Olafsdottir, G., Eds. Wageningen Academic Publishers: Wageningen, the Netherlands, pp. 189–200, 2003. 101. Macagnano, A., Careche, M., Herrero, A., Paolesse, R., Martinelli, E., Pennazza, G., Carmona, P., D’Amico, A., and Di Natale, C. A model to predict fish quality from instrumental features. Sens. Actuators B: Chem., 111, 293, 2005.
Chapter 24
Aroma John Stephen Elmore Contents 24.1 Introduction ................................................................................................................. 440 24.2 Reasons for Studying Fish and Seafood Aroma ............................................................ 440 24.2.1 Identification of Those Compounds, Which Are Important in Desirable Cooked Fish and Seafood Flavor ..................................................................... 440 24.2.2 Identification of Compounds That Give Undesirable Aroma and Flavor to Cooked and Uncooked Fish and Seafood........................................................ 441 24.2.3 Measuring the Effect of Pre- and Postslaughter Treatments ............................ 441 24.3 Sample Preparation ...................................................................................................... 442 24.4 Aroma Extraction Methods .......................................................................................... 442 24.4.1 Solvent Extraction ........................................................................................... 442 24.4.2 Steam Distillation and Vacuum Steam Distillation ......................................... 443 24.4.3 Simultaneous Distillation/Extraction .............................................................. 443 24.4.4 High Vacuum Distillation/Solvent-Assisted Flavor Evaporation ...................... 445 24.4.5 Headspace Analysis ......................................................................................... 446 24.4.6 Adsorption ...................................................................................................... 446 24.4.7 Solid-Phase Microextraction ........................................................................... 447 24.4.8 Analysis of Trimethylamine Oxide Breakdown Products ................................ 449 24.4.9 Analysis of Geosmin and 2-Methylisoborneol ................................................. 449 24.5 Separation and Identification of Aroma Components................................................... 449 24.6 Quantification of Aroma Components ..........................................................................450 24.7 Detection of Components of Sensory Significance ........................................................451 24.8 The Electronic Nose ......................................................................................................452 24.9 Future Developments ....................................................................................................453 References ................................................................................................................................453 439
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24.1
Introduction
Numerous species of fish and shellfish are eaten across the world and, for many of these species, the chemical compounds that contribute to their aroma have been studied. These compounds are generally of low molecular weight (less than 300 Da), volatile, and present at very low concentrations (parts per million or less); most are of limited solubility in water. It is often necessary to characterize fish and seafood aroma, for reasons which will be described later in the chapter. In order to do so, one would extract the volatile material from the food matrix, concentrate it, and attempt to separate and identify the individual components. By far the most widely used technique for the separation and identification of aroma compounds in extracts is gas chromatography–mass spectrometry (GC–MS), and unless stated otherwise, it can be assumed that, in all examples discussed in this chapter, GC–MS was used. As far as possible, the extract should contain only the volatile components, in the same relative proportions as in the food itself, without the introduction of artifacts. However, to obtain such an extract is an extremely difficult task, and so numerous complementary extraction techniques exist, which allow the flavor chemist to obtain a complete knowledge of those compounds that are present in fish aroma. The components which are responsible for fish aroma are present in extremely small quantities, compared with the major constituents, of which water is the most abundant. A number of isolation techniques exist, all based on utilizing the physical properties of the aroma compounds to separate them from the food matrix and from water. The most widely used isolation techniques for the analysis of fish and seafood aroma will be described in this chapter. Often, a detailed analysis of individual compounds may not be necessary. An analytical technique which provides a “fingerprint” of the sample under study may be all that is required; for example, to distinguish fish from different species, or to determine if a piece of fish is of sufficient freshness. Electronic noses are detectors, which use gas sensor arrays or mass spectrometers, the latter sometimes known as MS-noses, to discriminate between samples, without the need for an extraction or separation. The use of electronic noses to discriminate between fish samples will also be discussed in this chapter.
24.2 Reasons for Studying Fish and Seafood Aroma Studies on fish and seafood aroma can broadly be divided into three areas: 1. Identification of those compounds, which are important in desirable cooked fish and seafood flavor; 2. Identification of compounds, which give undesirable aroma and flavor to cooked and uncooked fish and seafood; 3. Examination of how different pre- and postslaughter treatments may affect aroma volatiles.
24.2.1
Identification of Those Compounds, Which Are Important in Desirable Cooked Fish and Seafood Flavor
Aroma extract dilution analysis (AEDA), which will be described later in the chapter, is a technique, which is often used to identify the important aroma compounds in food. For example, (Z)-1,5-octadien-3-one, (E,Z)-2,6-nonadienal, and methional are all potent odorants of fresh,
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boiled trout, salmon, and cod, while propionaldehyde, acetaldehyde, and methional are important in boiled salmon and cod, and (E,E)-2,4-decadienal is important in boiled cod alone [1–3]. In studies on cooked shellfish, 2-acetyl-1-pyrroline, having a nutty, popcorn-like odor, 2,3-butanedione (sour, creamy), 2-methyl-3-furanthiol (cooked rice, meaty), methional (salty, soy sauce-like), and 2-acetyl-2-thiazoline (also nutty, popcorn-like) all contribute to the desirable aromas of cooked crab, lobster, crayfish, prawn, and shrimp [4–7].
24.2.2 Identification of Compounds That Give Undesirable Aroma and Flavor to Cooked and Uncooked Fish and Seafood Several compounds contribute to off-flavors in fish. Off-flavors can be formed through deterioration of fish on storage or may be present in the fish, as a result of their environment [8]. Trimethylamine oxide (TMAO) is produced in most species of marine fish and shellfish during digestion and breaks down enzymatically on storage to give trimethylamine (TMA), dimethylamine, and formaldehyde [9]. High TMA levels lead to low sensory scores, and TMA measurement has often been used to monitor spoilage. Fish and seafood are high in polyunsaturated fatty acids, and lipid oxidation readily occurs in both chilled and frozen fish. Lipid-derived compounds implicated in undesirable fishy aromas in both cooked and uncooked fish and seafood include 2,4,7-decatrienal, (Z)-3-hexenal, (Z,Z)-3,6nonadienal, (Z)-4-heptenal, and 1-octen-3-one [6,8,10]. Methods for measuring freshness quality will be discussed in a later chapter. Two compounds often implicated in the spoilage of freshwater fish through environmental contamination are geosmin and 2-methylisoborneol. Both compounds are microbial metabolites readily absorbed by fish, and both compounds contribute musty, earthy off-flavors. Other common taints are a garlic off-flavor in prawns and an iodoform off-flavor in prawns and shrimps [11]. The iodoform taint is caused by high levels of bromophenols; at lower levels these compounds contribute to desirable marine flavors in prawns [12].
24.2.3
Measuring the Effect of Pre- and Postslaughter Treatments
The effects of different dietary lipids on the aroma volatiles of turbot [13], brown trout [14,15], tench [16], and carp [17] have been studied. Different types of algae affect the volatiles from oysters [18] and bromophenol levels in green grouper [19]. Aroma compounds from wild and farmed fish have also been compared for ayu [20], Atlantic salmon [21], gilthead sea bream [22,23], trout [24], turbot [25], and prawns [12]. The effect of refrigerated and/or frozen storage on aroma compounds, other than TMA, has been widely studied, in fish, such as cod [2,3,10,26,27], trout [1,10], salmon [3,28], whiting [26], tuna [29], whitefish [30], Antarctic krill [31], hake [32], sardine [33–35], and mackerel [26,36], as well as fish oils, such as menhaden, sardine, and bonito oil [37]. Aroma composition may be affected by canning [37–45], smoking [46–56], salting [57], fermentation [58–64], drying [65], pickling [66], and irradiation [67]. Fish sauce is an important ingredient in Asian cuisine, produced from a mixture of fish and salt that has been allowed to ferment for a period of greater than 6 months at 30°C–35°C [68]. Fish sauce has a distinctive aroma, which has been widely studied [69–73]. The use of seafood processing waste has attracted attention. The water used to boil, for example, crab and crayfish, and the inedible material remaining after processing, can be used to prepare seafood flavor extracts [74–77].
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Sample Preparation
There are a number of factors that need to be considered, when analyzing fish and seafood aroma. The meat from fish is relatively homogeneous, compared to that of meat, where the presence of adipose tissue may influence how the meat is cooked. Even so, one consideration may be whether to cook the fish whole, or as a fillet, with or without skin attached, or to reduce variation by mincing the fish and forming a patty from the muscle. Grilling, boiling, pressure-cooking, baking, and frying are some of the cooking processes, which could be used. The sample could be cooked for a constant time or to a constant internal temperature. A well-done fish will have a different aroma profile to a lightly cooked fish, and the degree of cooking should be considered before analysis commences. It is important to employ reproducible methods when cooking the sample. This may be difficult when grilling or frying. Pressure-cooking, although not as commonly used in the kitchen as some of the other methods, has an advantage in that there is no sample loss and temperature control is straightforward. When analyzing a cooked sample, it is important to extract the sample as soon after cooking as possible. Otherwise off-flavors may be generated when it is reheated. There is no reason why the extraction cannot be carried out at room temperature, although at higher temperatures the amount of volatile material extracted will increase. Heated food is normally eaten at around 60°C. Variation within the sample should be minimized by chopping, or mincing, or homogenization and appropriate replication should be performed, in order that effective comparison between treatments may be achieved. Large variations exist in all naturally occurring foodstuffs, and at least four analyses per treatment would always be appropriate when studying seafood.
24.4 Aroma Extraction Methods The reasons for choosing a particular aroma extraction method have been discussed in detail [78]. If accurate quantification of one particular aroma compound is required, the extraction method should be selected to maximize the extraction of that particular compound, without generating that compound in situ. However, if a full aroma profile is required, another extraction method or methods may be more suitable. Some extraction techniques only provide enough extract for one analysis, whereas others provide a liquid sample, which can be used for several different experiments.
24.4.1
Solvent Extraction
Direct extraction of fish and seafood with an organic solvent is of limited use, because the extract will contain much nonvolatile matter, particularly lipid. However, supercritical fluid extraction (SFE) with supercritical carbon dioxide can be a useful technique for the extraction of aromas. Its solvating qualities can be altered by changing the pressure or temperature at which the extraction takes place, and under ideal conditions supercritical carbon dioxide exhibits a strong affinity for most aroma compounds, while most nonvolatile constituents are insoluble. The ease of removal of the solvent, after extraction, to give a concentrated aroma extract, is another attractive feature of SFE. Aro et al. used SFE to extract semivolatile compounds from Baltic herring; the compounds were analyzed by supercritical fluid chromatography [79]. Another way SFE can be used is to remove off-notes from tuna fish oil [80].
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24.4.2 Steam Distillation and Vacuum Steam Distillation Steam distillation finds application in the analyses of volatiles from beverages and high watercontent foods, although it is less applicable to fats and oils. It has the disadvantage that the large quantities of aqueous distillate require further extraction with a solvent, to separate the volatiles from the water. Concentration of the extract is then necessary. The formation of artifacts may also be a problem. The technique has been used to examine the odor of Antarctic krill [81], the components of cuttlefish oil [82], and the effect of irradiation on shrimp volatiles [67]. If steam distillation is performed under high vacuum, less sample degradation occurs. For example, Triqui and Reineccius [83,84] performed steam distillation of anchovy volatiles under vacuum at around 57°C. They extracted the distillate with dichloromethane and then concentrated the extract by microdistillation. A similar technique was used to examine the character-impact compounds of raw oyster [18,85,86]. The vacuum was strong enough to perform the extraction at room temperature and volatiles were condensed in a flask at 2°C, which was connected to three liquid nitrogen-cooled cold traps. Again, the distillate was dissolved in dichloromethane and then concentrated. This method was also to examine the aroma compounds in brown trout [14,87], turbot [13], and mussels [88,89].
24.4.3
Simultaneous Distillation/Extraction
One of the most widely used techniques in aroma analysis combines steam distillation with solvent extraction in a Likens–Nickerson apparatus (Figure 24.1), which was first reported in 1964 for the extraction of hop oil [90]. The extracting solvent is immiscible with and less dense than water. Upon heating, volatile compounds in the steam are transferred to the solvent and both liquids condense. The glassware is constructed so that both solvent and water are returned to their starting vessels. After an extraction time of 1–12 h, the extract is collected and dried, either using anhydrous sodium sulfate, or by freezing and decanting the solvent from the ice. The extract is then concentrated before analysis to a volume of approximately 0.1 mL; a low-boiling extracting solvent is therefore desirable, so that it can be removed without substantial losses of compounds of interest. In addition, the solvent should be of high purity, so that impurities do not become major chromatographic peaks, when the extract is concentrated. Appropriate solvents, which have been widely used, are pentane, diethyl ether, or a combination of the two. Solvents denser than water, e.g., dichloromethane, could be used in a modified apparatus. Numerous authors have used simultaneous distillation/extraction (SDE) at atmospheric pressure to analyze aroma compounds in fish and seafood. In particular, extraction using diethyl ether has been used to analyze carp [91], smoked salmon [53,54], krill [92,93], shrimp [62], clam [94], crab [95], fermented fish pastes [58], and fish sauce [71], while extraction using dichloromethane has been used to study turbot [24], mackerel [96], crab [77,97], scallops [98–100], mussels [101,102], squid [99], prawns [99,103], and phenolic compounds in smoked herring [52]. Morita et al. used SDE with dichloromethane to examine the volatile compositions of 16 different saltwater and freshwater fish species, including tuna, cod, carp, swordfish, mackerel, eel, and flounder [104]. SDE has been widely used for the analysis of bromophenols in fish and seafood. The extracted bromophenols have been usually analyzed by GC–MS [11,12,105–108], although high-performance liquid chromatography (HPLC) method with UV detection has also been developed [109]. SDE has several advantages to the other commonly used extraction techniques. Efficient stripping of volatiles from foods allows quantitative recoveries to be achieved for many
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Coolant
Condenser
Extracting solvent Sample in water
Water bath
Heating mantle
Figure 24.1 Likens–Nickerson apparatus for simultaneous steam distillation/extraction.
compounds [110]. The aroma extract is obtained in a solvent; therefore, many injections can be performed from one extraction. Hence, one sample could provide material for GC, GC–MS, and for quantitative GC–olfactometry (GC–O) techniques, such as AEDA [111]. Fractionation of the extract can be carried out, resulting in increased separation of the components in the extract, facilitating the identification of minor components. For example, Kubota et al. [112] separated the extract from cooked shrimps into neutral and basic fractions. The basic fraction was high in nitrogen-containing compounds, as these compounds become more water-soluble at high pH, due to protonation of the nitrogen. The neutral fraction contained mainly sulfur-containing compounds. As with all aroma extraction techniques, SDE has drawbacks. When the extract is concentrated, by distilling off the solvent, low-boiling volatile compounds can be lost. These compounds include 2-butanone, 2-pentanone, 2- and 3-methylbutanal, diacetyl, 1-propanol, and 1-penten3-ol, which are often present at high levels in headspace extracts of fish and seafood. Artifacts can
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be formed as a result of the high temperatures used. In addition, volatiles can be generated when samples are overcooked during extraction, e.g., through enhanced lipid oxidation [113]. If SDE is carried out under reduced pressure, thermal degradation of labile components can be diminished. By maintaining the system under a static vacuum, extraction at room temperature is possible. Vacuum SDE with dichloromethane has been used to examine lobster [4,114], blue crab [77], and tuna fish sauce [115]. An excellent discussion of SDE is available [110].
24.4.4
High Vacuum Distillation/Solvent-Assisted Flavor Evaporation
Because SDE may lead to artifact formation and overcooking, a high vacuum transfer technique was developed in the early 1990s, as a way of removing the volatile aroma compounds from solvent extracts of food materials. A series of cold traps was used to collect the volatile material, after it had sublimed. This technique was shown to be more efficient than SDE for high-boiling polar compounds, such as furaneol. Dichloromethane at room temperature has been used to extract volatile compounds from ripening anchovy [116,117] and uncooked hake [32], while diethyl ether under reflux has been used for boiled trout [2] and carp [118]. There were numerous drawbacks with the technique and a robust alternative, known as solvent-assisted flavor evaporation (SAFE) was developed, to supersede it (Figure 24.2). Although high vacuum transfer and SAFE are similar techniques in principal, greater thermal control and a more compact arrangement of the glassware means that SAFE is more efficient than high vacuum transfer, resulting in higher yields of high-boiling and polar compounds [119]. It can also be used directly on the food, with no need of a solvent extraction step, producing an extract with typical aroma. Hence, the time of extract preparation can be substantially reduced. No work on fish aroma using SAFE has so far been published. Dropping funnel To vacuum pump
Cold trap
Cold trap Water bath
Figure 24.2
Solvent-assisted flavor evaporation apparatus.
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Handbook of Seafood and Seafood Products Analysis
Headspace Analysis
Commercial automated headspace analyzers are often used for routine analysis of volatile compounds. These allow temperature control of the sample and may include agitation. The headspace sample vial may be pressurized before injection, to improve reproducibility and increase sensitivity. Automated systems have been used for the analysis of sea bream [22], mackerel [36], canned pink salmon [38,39,44,120], and tuna [121]. However, manual headspace analysis was used to characterize the key aroma components of boiled salmon, cod, and trout [1–3], and those of salted-dried white herring [57]. A concentrated headspace extract can be obtained by passing a stream of inert gas (nitrogen or helium) over the sample and condensing the volatiles in a series of traps cooled by ice, solid carbon dioxide, or liquid nitrogen. Extraction of the condensate with a small amount of a suitable solvent provides an aroma extract suitable for chromatographic analysis. Alternatively, the condensed volatiles can be swept directly into the injection port of a gas chromatograph. The latter technique was used to examine spoilage in prawns and also to measure the low-boiling volatile compounds, hydrogen sulfide [41] and ethanol [42], in tuna. Ethanol was also measured in canned salmon, using a similar technique [122], although automated headspace analysis appears to be the most appropriate methodology for ethanol quantification [123,124]. Both hydrogen sulfide and ethanol are regarded as indicators of spoilage in fish [125]. Yasuhara and Shibamoto [126] trapped collected volatiles from nine species of seafood, including mackerel, red salmon, and squid, in a cysteamine solution, which converted the aldehydes present into thiazolidines. The thiazolidines were then extracted in dichloromethane and concentrated for analysis. This method was particularly useful for measuring formaldehyde and acetaldehyde, which are usually too volatile to be measured by conventional aroma analysis techniques. The theory and practice of static headspace analysis have been well discussed [127].
24.4.6
Adsorption
Headspace aroma volatiles can also be collected on suitable adsorbent materials, the most widely used of which is Tenax TA, a porous polymer resin based on 2,6-diphenylene oxide. These materials readily adsorb volatiles while having little affinity for water, making them particularly useful in the analysis of samples with high water content. In a typical collection, purified inert gas sweeps the volatiles from the sample flask into a small tube containing from 10 to 200 mg of the adsorbent (Figure 24.3), which is usually called a “trap.” Typically at least five volumes of headspace should be collected from the sample vessel. Adsorbed volatiles can be heat desorbed directly onto a gas chromatographic column by placing the trap in a specially modified injection port, thus avoiding loss of components or unnecessary dilution. Cooling the front of the column cryogenically or with Peltier cooling during this desorption will avoid any loss in chromatographic resolution. Tenax TA has been use for the analysis of boiled crab [128], fresh smelt [129], fresh ayu [20], steamed clam [94], and fish sauce [69,72,73]. Forerunners of Tenax TA include Tenax GC, which has been used to analyze cooked salmon [21], roasted dried squid [130], and fresh and fermented shrimp [60]. The aromas of fresh and canned sea urchin gonads were compared by trapping on graphitized carbon black, followed by microwave desorption [43]. Solvent desorption, rather than heat desorption, from the trap with diethyl ether, followed by concentration, has been used to prepare aroma extracts of whitefish [131,132], smelt, perch, pike, rainbow trout, cod, sole, haddock [132], salmon [133], and oysters [134], using Tenax GC; and
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Carrier gas in
SVL union
Glass-lined steel trap containing 85 mg Tenax TA
Water bath
Figure 24.3 Headspace adsorption on Tenax TA.
lobster with Tenax TA [4]. Ishizaki et al. collected shrimp volatiles on Tenax TA, using a pump, and then desorbed the trap with diethyl ether [135]. Automated devices exist for headspace adsorption, followed by thermal desorption in a dedicated injection port. These have been used to analyze raw sardine [35], canned salmon [40], cooked catfish [136–138], baked herring [139], raw oyster [86,140], pasteurized crab [141], and boiled crayfish [141,142]. Where samples have been extracted manually, multitrap autosamplers can be used to heat desorb the volatile compounds, allowing round-the-clock GC–MS analysis. Aroma volatiles in fresh [143,144], cooked [145], and smoked [143] salmon were measured using Tenax TA, followed by automated thermal desorption, as were fresh cod, saithe, mackerel, redfish [144], and ripened cod’s roe [146], while the same apparatus with Tenax GR was used to examine salted herring during ripening [147] and off-flavor development in herring oil [148]. Headspace adsorption on Tenax is a desirable technique because it is sensitive, and extracts a wide boiling point range of volatile compounds; artifact formation is minimal, as extraction is carried out under inert gas flow. However, a dedicated injection system for traps may be expensive, especially if automated extraction is desired. Normally only one GC analysis is obtained from each extraction, unless solvents are used to desorb the contents of the traps.
24.4.7 Solid-Phase Microextraction A very popular and simple to use technique, introduced in the early 1990s [149], solid-phase microextraction (SPME) uses a small fused silica fiber, coated in an adsorbent material, mounted inside a syringe-like device (Figure 24.4). The needle is pushed through a septum and the fiber is exposed to the headspace above the food or beverage sample, which is sealed in a suitable container. Volatile compounds are adsorbed onto the fiber and, at the end of the extraction, the fiber can be removed from the sample vessel and directly desorbed into the split/splitless injector of a gas chromatograph. The injector of the GC contains a very narrow quartz liner, which helps to focus
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(a)
(b)
Figure 24.4 Solid phase microextraction: (a) fiber inside syringe barrel; and (b) fiber exposed during extraction.
the volatile compounds at the front of the GC column. Alternatively, cryofocusing of the aroma volatiles on the front of the column can be performed, to prevent peak broadening. SPME can be automated and its ease of use, relatively low cost, and affinity for a large range of compounds has meant that it has become a widely used technique for the isolation of aroma volatiles [150]. Numerous different stationary phases have been used as coatings for SPME fibers. Coatings may be absorptive—volatiles are bound to the surface of the fiber, or adsorptive—volatiles are trapped within pores in the stationary phase [151]. Popular absorptive phases include polydimethylsiloxane (PDMS), while Carboxen and divinylbenzene (DVB) are adsorptive phases. Fibers containing a mixture of stationary phases are commonly used. Virtually every available type of SPME fiber has been used for the analysis of fish, and several workers have compared the performances of two or more fibers. A PDMS fiber was used to examine the effects of storage on yellowfin tuna [29], while a polyacrylate fiber was found to be most suitable for comparing smoked with unsmoked black bream and rainbow trout [48], and also for analyzing smoked cod and swordfish [49]. Using a PDMS/DVB fiber, Song et al. [152] discovered that the aroma of uncooked hepatic tissue was stronger than that of uncooked muscle for carp, flounder, mackerel, and skipjack. Both a PDMS/DVB fiber and a PDMS fiber were used to examine the aroma of fresh scallops [153], while DVB/Carboxen/PDMS fibers have been used to examine sardine freshness [33,34]. Carboxen/PDMS fibers have been used to monitor king salmon [28], whiting, cod, and mackerel spoilage [26], and measure aroma compounds in sea bream, chum salmon, mackerel, sardine, tuna, prawn, and shrimp [154].
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Using the technique known as in-fiber derivatization, low-molecular weight aldehydes were measured in raw pollock by SPME [155]. The aldehydes in the fish reacted with the stationary phase of the fiber, to give a derivative, which, in the case of formaldehyde and acetaldehyde, was more amenable to GC–MS analysis than their underivatized equivalents. In-fiber derivatization has also been used to measure formaldehyde in 12 fish species, including cod, haddock, tuna, and trout [156]. Like headspace adsorption on Tenax, SPME is a desirable technique because it is sensitive, and extracts a wide boiling point range of volatile compound. Again artifact formation is minimal. Its big advantage over headspace adsorption is that it can be used with any GC. It is also the easiest aroma extraction technique to use, requiring little training. Method development is straightforward and a choice of stationary phases means that the extraction can be tailored, in order to maximize extraction of desired compounds. Like headspace adsorption with thermal desorption, only one GC analysis is obtained from each extraction.
24.4.8 Analysis of Trimethylamine Oxide Breakdown Products As stated earlier in the chapter, TMA measurement is regularly used to monitor spoilage, and TMA is often reported as a component of fish aroma. For example, Chung and Cadwallader [5] reported that TMA gave a fishy note to boiled crab aroma, while Milo and Grosch [10] reported that TMA gave an amine-like aroma to cod, which increased during frozen storage. TMA and other TMAO-breakdown products are often measured alone, to provide an indicator of fish freshness, using techniques that are not appropriate for the analysis of other aroma compounds. These techniques will be described in a later chapter. Several workers have used SPME to analyze volatile amines. Chan et al. [157] used a Carboxen/DVB/PDMS fiber for the extraction of volatile amines from homogenized fillets of mangrove snapper and freshwater grouper. Li et al. [158] used an amine-selective SPME fiber to study TMA levels in ground chub fillet stored under different conditions.
24.4.9 Analysis of Geosmin and 2-Methylisoborneol 2-Methylisoborneol and geosmin have extremely low odor thresholds [159], which impart undesirable tastes and odors in fish; catfish suffers in particular from problems caused by these two compounds. Although both compounds can be identified in fish using the techniques applicable to volatile aroma compounds in general (SDE [160]; solvent extraction [161]; headspace adsorption [21]; SPME [162]), procedures have been developed specifically for the accurate quantification of methylisoborneol and geosmin. Microwave distillation, a form of steam distillation, has been used as an effective means of quantitatively extracting methylisoborneol and geosmin from the fish matrix [163,164], as has vacuum distillation [165]. The aqueous extract that results can then be extracted further, using SPME [163,166], solid-phase extraction [164], or trapping on an adsorbent [165].
24.5 Separation and Identification of Aroma Components To determine the important compounds in an aroma extract, the complex mixture needs to be separated into its components. The amount of isolate is usually small, containing many compounds of diverse chemical structures, varying greatly in concentration, and important components are
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often present in extremely low amounts. The success of any aroma analysis depends mainly upon the efficiency of separation and the sensitivity of detection. GC using bonded phase fused silica capillary columns is universally used as the separation method in aroma analysis. Such columns can separate complex mixtures, and the most commonly used stationary phases are Carbowax 20 M, a polar phase, and the two nonpolar phases, 100% poly(dimethylsiloxane) and poly(5% diphenylsiloxane/95% dimethylsiloxane). The retention times of an aroma compound on two columns with different stationary phases, relative to the retention times of a series of straight-chain alkanes can be helpful in its identification; databases containing retention data for volatile compounds are available [167]. GC is a widely used technique and will not be discussed here. Structure elucidation of the chromatographically separated components is the next step in the analysis of an aroma isolate. GC–MS allows direct analysis of the separated components and provides the most efficient means of volatile identification. Compounds eluting from the GC column enter the ion source of the mass spectrometer, where they are ionized and break into fragments. The fragments are separated by their mass-to-charge ratio, resulting in a characteristic spectrum, which will provide structural information. Several types of mass spectrometer are suitable for the identification of aroma compounds, although most of the work discussed in this chapter was performed using single quadrupole mass spectrometers. Although single quadrupoles are by far the most common type of mass spectrometer for GC–MS, other types of mass spectrometer are used [168]. Ion traps and triple quadrupoles offer all of the capabilities of the single quadrupole, plus MS–MS, i.e., the trapping of fragments from the first ionization, for further fragmentation, in order to yield more structural information about unknown compounds. Double-focusing magnetic sector mass spectrometers can acquire accurate mass data, allowing the calculation of the empirical formula of an unknown compound. These machines are relatively expensive compared to quadrupoles and less robust, so are used far less often for routine flavor analysis. Time-of-flight (TOF) machines have become increasingly popular as mass spectrometric detectors, with newer models offering rapid scan speeds (up to 500 spectra per second) and accurate mass measurement. High scan speeds are necessary when using fast GC techniques, such as two-dimensional GC (GC × GC), where small time-window fractions are diverted from the first analytical column, onto a second short column with a different stationary phase to the first column [169]. Rapid elution of the peaks from the second column (6 s maximum), allow a two-dimensional trace to be obtained. TOF machines are robust but relatively expensive, although, as they have been introduced relatively recently, they may become cheaper as they become more popular [170]. The characterization of unknown compounds is greatly facilitated by comparing their mass spectra with those of known compounds in compiled libraries, which are supplied with the GC–MS data system. Confirmation of the identity of compounds should always be carried out, preferably by comparing their mass spectra and GC retention times with those of authentic samples.
24.6
Quantification of Aroma Components
Often quantitative information on aroma compounds in a food is needed, for example, when using AEDA to determine the key compounds contributing to the aroma of a food. Quantification is rarely simple, because most extraction techniques only remove a proportion of the aroma from the food, and difficulties may arise when compounds are not resolved by GC.
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The most effective means of quantification is isotope dilution assay using GC–MS. A known amount of a 13C- or 2H-labeled internal standard is added to a slurry of the food under study, in order to quantify its nonlabeled equivalent. As the labeled and unlabeled aroma compound possess similar physical properties, the proportion of each extracted from the food will be the same. The relationship between the mass spectral peaks of the labeled standard and the compound of interest can be used to calculate accurately the amount of the compound of interest in the food. If the labeled standard is homogeneously distributed within the food, then quantitative extraction of the compound under study is not necessary [171]. Other quantification methods include the addition of an internal standard, not present in the food, of a similar chemical composition to the compound of interest, e.g., 2-methylpentanal could be used to quantify hexanal. Alternatively, one added compound can be used to approximately quantify all of the compounds in an extract, by measuring their peak areas relative to that of the added compound. 2,4,6-Trimethylpyridine, p-cymene, and ethyl heptanoate are compounds, which have been used several times for this purpose, in the analysis of fish and seafood. If the extraction of the compound of interest is quantitative, then solutions of the compound of interest can be used to plot a calibration curve (an external standard), which can then be used to quantify that compound. Lee et al., using vacuum SDE, extracted standard compounds at a series of concentrations under the same conditions as the sample, in order to accurately quantify selected compounds in lobster tail meat [114]. Conversely, standards could be added to the extract. The peak area of a known concentration of a standard added to an extract can be compared with the peak areas of all the compounds in the extract, to give an approximate concentration for all of the compounds in the extract. Standards can be injected into traps containing adsorbent and, in the case of SPME, injected onto the GC column immediately before desorption of the fiber. Methanol is a useful solvent for such standards; it can be easily purged from the trap as its affinity for Tenax is very low and as its molecular weight is 32, data acquisition down to m/z 33 will provide enough mass spectral data for successful library searching, without peaks of interest being hidden by a solvent peak.
24.7
Detection of Components of Sensory Significance
A widely used technique for determining components that contribute to aroma is GC–O. The column effluent is split between a conventional GC detector and a vent to the outside of the oven, where the odors emerging can be smelled and described [172]. AEDA is a quantitative GC–O technique, which has been used many times to estimate the relative contributions of volatile components toward the total aroma quality of cooked fish and seafood. The aroma extract under study is diluted twofold and analyzed by GC–O, then diluted twofold again and again. After a certain number of dilutions of the extract, no aromas will be perceived. The flavor dilution factor for a particular compound is defined as the highest dilution at which that compound can be perceived by GC–O. For example, if the concentration of the extract was halved at each dilution and the seventh dilution was the last at which the compound could be detected, its flavor dilution factor would be 27 (128). Hence, if the aroma extract is representative of the food from which it is derived, the most important contributors to the aroma of the food are those with the highest flavor dilution factors. It should be noted that components with high flavor dilution factors might not give GC peaks of any significant size. These flavor dilution factors can be plotted against retention time, to give an aromagram for a particular extract. When combined
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with isotope dilution analysis, AEDA is a very powerful technique for the identification of the key compounds in fish flavor [1,2]. Other quantitative GC–O techniques, such as CHARM analysis, olfactory global analysis, and OSME, have also been used for the analysis of fish aroma [101,173]. GC–O has shown that, for many raw and cooked fish and shellfish, lipid-derived compounds, such as unsaturated aldehydes, alcohols, and ketones, are character-impact compounds, while methional, dimethyl disulfide, 2-acetyl-1-pyrroline, 3-methylbutanal, and alkylpyrazines are important compounds formed in cooked fish, via the Maillard reaction. As well as the use of GC–O, several papers have been published on fish aroma, which combine aroma extraction with sensory analysis. Varlet et al. [54] extracted aroma from smoked salmon, then added the extract to unsmoked salmon, to be analyzed by a trained panel, in order to examine the representativeness of the aroma extraction method used (SDE). Morita et al. [104] prepared fish broths from 16 species, and related compounds in these broths with sensory attributes. The 16 species were divided into four groups, associated with particular aroma notes, which could be correlated with certain aroma compounds.
24.8 The Electronic Nose Originally, the term “electronic nose” was used to describe an array of chemical sensors, connected to a pattern recognition system, which responded to odors passing over it. Different odors cause different responses in the sensors and these responses provide a signal pattern, characteristic of a particular aroma. The computer evaluates the signal pattern and can compare the aromas of different samples, using pattern recognition. Sensors are usually made of metal oxides or organic polymers, although more recently surface acoustic waves and piezoelectric crystals have been used. Problems may exist when samples with a high water content are analyzed, as many of the sensors respond strongly toward water, preventing any sample differences being observed. Electronic noses have been widely used in fish aroma analysis, particularly with regard to fish freshness. Olafsdottir et al. used four gas-specific sensors, for CO, NH3, H2S, and SO2, to measure quality changes in stored cod, and related results to volatile formation (particularly ethanol and 2-methyl-1-propanol), pH change, and microbial spoilage [27]. Du et al. used an electronic nose with polymer sensors and a sensory panel to examine quality changes during storage of yellowfin tuna [174]. Jonsdottir et al. showed a good correlation between sensory results, electronic nose data, and aroma compound formation in stored, ripened cod’s roe [146]. More recently, electronic noses based on MS have been developed, which are also known as mass sensors or MS-noses. Volatile compounds are introduced directly into the mass spectrometer, without any preseparation. With these instruments, each mass scanned by the mass spectrometer can be described as a sensor, which detects any ion fragment with that mass. In fact, the mass sensor is taking all of the scans that make up a GC–MS run and then combining these scans, to provide a fingerprint of the food under study. The advantages of these machines over conventional electronic noses are that they are less prone to sensor poisoning (due to excess sample), moisture effects, and nonlinearity of signals [175]. This technique has been used to measure spoilage volatiles in the headspace of whiting, cod, and mackerel [26]. A related technique involves conventional GC–MS, followed by summing of all the spectral data to give an MS fingerprint. Slurries of fresh oysters were extracted by SPME, followed by GC–MS. Mass fragments that were shown to vary between treatments were then analyzed by a chemometric method. Five mass fragments were sufficient to completely discriminate oysters from seven production areas [176].
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24.9 Future Developments Since the mid-1990s, it has been possible to measure the release of aroma volatiles from chewed food in real time, using MS. As many volatile compounds enter the mass spectrometer at the same time, a soft ionization technique is used, i.e., one that favors the formation of a protonated molecular ion, with little additional fragmentation. The two processes most commonly used to achieve this are atmospheric pressure chemical ionization MS (APCI-MS) [177] and proton-transfer reaction MS (PTR-MS) [178]. A plastic tube is inserted into one nostril and exhaled air passes directly into the mass spectrometer, giving a characteristic sigmoid trace, with troughs during inhalation and peaks during exhalation. The recent paper by Rochat et al. [179] showed the potential of two-dimensional GC, hyphenated to a TOF MS, as an unrivaled technique for the separation of complex mixtures. The technique is extremely sensitive, as a result of low background and exceptionally high peak resolution, allowing thousands of peaks to be separated in one GC–MS trace. At present, the cost of such equipment may place it beyond the reach of most analytical laboratories, but its potential is clear to see. SAFE would appear to be the most effective aroma extraction technique currently available; yields are higher than any other technique and sample degradation does not occur readily. Its potential has been shown in the analysis of numerous foods. None of the three techniques described in this section have been used to analyze fish aroma so far, although their potential is clear. Even so, many of the other techniques described in this chapter will continue to be used for the foreseeable future.
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11. Whitfield, F.B., Biological origins of off-flavors in fish and crustaceans, Water Sci. Technol., 40, 265, 1999. 12. Whitfield, F.B. et al., Distribution of bromophenols in Australian wild-harvested and cultivated prawns (shrimp), J. Agric. Food Chem., 45, 4398, 1997. 13. Serot, T. et al., Effect of dietary lipid sources on odour-active compounds in muscle of turbot (Psetta maxima), J. Sci. Food Agric., 81, 1339, 2001. 14. Serot, T., Regost, C., and Arzel, J., Identification of odour-active compounds in muscle of brown trout (Salmo trutta) as affected by dietary lipid sources, J. Sci. Food Agric., 82, 636, 2002. 15. Turchini, G.M. et al., Effects of dietary lipid sources on flavour volatile compounds of brown trout (Salmo trutta L.) fillet, J. Appl. Ichthyol., 20, 71, 2004. 16. Turchini, G.M. et al., Effects of dietary lipid source on fillet chemical composition, flavour volatile compounds and sensory characteristics in the freshwater fish tench (Tinca tinca L.), Food Chem., 102, 1144, 2007. 17. Runge, G. et al., Amounts of volatile sulfur-compounds in the edible part of carp (Cyprinus carpio L.) fed different fat and a-tocopheryl acetate dietary supplements, Agribiol. Res., 43, 183, 1990. 18. Pennarun, A.L. et al., Comparison of two microalgal diets. 2. Influence on odorant composition and organoleptic qualities of raw oysters (Crassostrea gigas), J. Agric. Food Chem., 51, 2011, 2003. 19. Kin, J.-S., Ma, W.C.J., and Chung Y.H., Enhancement of bromophenol content in cultivated green grouper (Epinephelus coloides), J. Fisheries Sci. Technol., 10, 113, 2007. 20. Hirano, T. et al., Studies on the odor of fishes.1. Identification of volatile compounds in ayu fish and its feeds, Nippon Suisan Gakkaishi, 58, 547, 1992. 21. Farmer, L.J. et al., Flavor and off-flavor in wild and farmed Atlantic salmon from locations around Northern Ireland, Water Sci. Technol., 31, 259, 1995. 22. Grigorakis, K., Taylor, K.D.A., and Alexis, M.N., Organoleptic and volatile aroma compounds comparison of wild and cultured gilthead sea bream (Sparus aurata): Sensory differences and possible chemical basis, Aquaculture, 225, 109, 2003. 23. Alasalvar, C., Taylor, K.D.A., and Shahidi, F., Comparison of volatiles of cultured and wild sea bream (Sparus aurata) during storage in ice by dynamic headspace analysis gas chromatography mass spectrometry, J. Agric. Food Chem., 53, 2616, 2005. 24. Prost, C., Serot, T., and Demaimay, M., Identification of the most potent odorants in wild and farmed cooked turbot (Scophtalamus maximus L.), J. Agric. Food Chem., 46, 3214, 1998. 25. Turchini, G.M. et al., Discrimination of origin of farmed trout by means of biometrical parameters, fillet composition and flavor volatile compounds, Ital. J. Anim. Sci., 3, 123, 2004. 26. Duflos, G. et al., Determination of volatile compounds to characterize fish spoilage using headspace/ mass spectrometry and solid-phase microextraction/gas chromatography/mass spectrometry, J. Sci. Food Agric., 86, 600–611, 2006. 27. Olafsdottir, G. et al., Characterization of volatile compounds in chilled cod (Gadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose, J. Agric. Food Chem., 53, 10140, 2005. 28. Wierda, R.L. et al., Analysis of volatile compounds as spoilage indicators in fresh king salmon (Oncorhynchus tshawytscha) during storage using SPME-GC-MS, J. Agric. Food Chem., 54, 8480, 2006. 29. Edirisinghe, R.K.B., Graham, A.J., and Taylor, S.J., Characterisation of the volatiles of yellowfin tuna (Thunnus albacares) during storage by solid phase microextraction and GC-MS and their relationship to fish quality parameters, Int. J. Food Sci. Technol., 42, 1139, 2007. 30. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Identification of volatile aroma compounds from oxidized frozen whitefish (Coregonus clupeaformis), Can. Inst. Food Sci. Technol. J., 17, 178, 1984. 31. Choi, S.H. and Kato, H., Odor of fermented small shrimp. 4. Changes in cooked odor of Antarctic krill during frozen storage, Agric. Biol. Chem., 48, 545, 1984. 32. Triqui, R., Sensory and flavor profiles as a means of assessing freshness of hake (Merluccius merluccius) during ice storage, Eur. Food Res Technol., 222, 41, 2006.
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33. Triqui, R. and Bouchriti, N., Freshness assessments of Moroccan sardine (Sardina pilchardus): Comparison of overall sensory changes to instrumentally determined volatiles, J. Agric. Food Chem., 51, 7540, 2003. 34. Ganeko, N. et al., Analysis of volatile flavor compounds of sardine (Sardinops melanostica) by solid phase microextraction, J. Food Sci., 73, S83, 2008. 35. Prost, C. et al., Effect of storage time on raw sardine (Sardina pilchardus) flavor and aroma quality, J. Food Sci., 69, S198, 2004. 36. Alasalvar, C., Quantick, P.C., and Grigor, J.M., Aroma compounds of fresh and stored mackerel (Scomber scombrus), in Flavor and Lipid Chemistry of Seafoods, Shahidi, F. and Cadwallader, K.R., Eds., American Chemical Society, Washington, DC, 1997, p. 39. 37. Frankel, E.N., Formation of headspace volatiles by thermal-decomposition of oxidized fish oils vs oxidized vegetable oils, J. Am. Oil Chem. Soc., 70, 767, 1993. 38. Girard, B. and Nakai, S., Static headspace gas chromatographic method for volatiles in canned salmon, J. Food Sci., 56, 1271, 1991. 39. Girard, B. and Nakai, S., Grade classification of canned pink salmon with static headspace volatile patterns, J. Food Sci., 59, 507, 1994. 40. Girard, B. and Durance, T., Headspace volatiles of sockeye and pink salmon as affected by retort process, J. Food Sci., 65, 34, 2000. 41. Khayat, A., Hydrogen sulfide production by heating tuna meat, J. Food Sci., 42, 601, 1977. 42. Lerke, P.A. and Huck, R.W., Objective determination of canned tuna quality—Identification of ethanol as a potentially useful index, J. Food Sci., 42, 755, 1977. 43. De Quiros, A.R.B. et al., Comparison of volatile components in fresh and canned sea urchin (Paracentrotus lividus, Lamarck) gonads by GC-MS using dynamic headspace sampling and microwave desorption, Eur. Food Res. Technol., 212, 643, 2001. 44. Oliveira, A.C.M. et al., Headspace gas chromatography-mass spectrometry and electronic nose analysis of volatile compounds in canned Alaska pink salmon having various grades of watermarking, J. Food Sci., 70, S419, 2005. 45. Przybylski, R., Eskin, N.A.M., and Malcolmson, L.J., Development of a gas chromatographic system for trapping and analyzing volatiles from canned tuna, Can. Inst. Sci. Technol. J., 24, 129, 1991. 46. Cardinal, M. et al., Effects of the smoking process on odour characteristics of smoked herring (Clupea harengus) and relationships with phenolic compound content, Food Chem., 96, 137, 2006. 47. Cardinal, M. et al., Effect of various smoking techniques on the nature of volatile compounds and on the sensory characteristics of salmon meat, Sci. Aliments, 17, 679, 1997. 48. Guillen, M.D. and Errecalde, M.C., Volatile components of raw and smoked black bream (Brama raii) and rainbow trout (Oncorhynchus mykiss) studied by means of solid phase microextraction and gas chromatography/mass spectrometry, J. Sci. Food Agric., 82, 945, 2002. 49. Guillen, M.D. et al., Headspace volatile components of smoked swordfish (Xiphias gladius) and cod (Gadus morhua) detected by means of solid phase microextraction and gas chromatography-mass spectrometry, Food Chem., 94, 151, 2006. 50. Ishiguro, K., Wakabayashi, H., and Kawaguchi, H., Changes in volatile compounds during smoking process and evaluation of major aroma constituents of dried bonito (Katuo bushi), J. Jpn. Soc. Food Sci. Technol., 48, 570, 2001. 51. Sakakibara, H. et al., Volatile flavor compounds of some kinds of dried and smoked fish, Agric. Biol. Chem., 54, 9, 1990. 52. Serot, T. et al., Effect of smoking processes on the contents of 10 major phenolic compounds in smoked fillets of herring (Cuplea harengus), Food Chem., 85, 111, 2004. 53. Varlet, V. et al., Olfactometric determination of the most potent odor-active compounds in salmon muscle (Salmo salar) smoked by using four smoke generation techniques, J. Agric. Food Chem., 55, 4518, 2007. 54. Varlet, V., Prost, C., and Serot, T., New procedure for the study of odour representativeness of aromatic extracts from smoked salmon, Food Chem., 100, 820, 2007.
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55. Varlet, V., Prost, C., and Serot, T., Volatile aldehydes in smoked fish: Analysis methods, occurrence and mechanisms of formation, Food Chem., 105, 1536, 2007. 56. Varlet, V. et al., Comparison of odor-active volatile compounds of fresh and smoked salmon, J. Agric. Food Chem., 54, 3391, 2006. 57. Chung, H.Y. et al., Static headspace analysis-olfactometry (SHA-O) of odor impact components in salted-dried white herring (Ilisha elongata), Food Chem., 104, 842, 2007. 58. Cha, Y.J. and Cadwallader, K.R., Volatile components in salt-fermented fish and shrimp pastes, J. Food Sci., 60, 19, 1995. 59. Cha, Y.J., Lee, G.H., and Cadwallader, K.R., Aroma-active compounds in salt-fermented anchovy, in Flavor and Lipid Chemistry of Seafoods, Shahidi, F. and Cadwallader, K.R., Eds., American Chemical Society, Washington, DC, 1997, p. 131. 60. Choi, S.H. and Kato, H., Odor of fermented small shrimp. 5. Volatile components of Sergia lucens and its fermented product, Agric. Biol. Chem., 48, 1479, 1984. 61. Choi, S.H. and Kobayashi, A., Odor of fermented small shrimp. 2. Cooked odor of commercial shiokara made from mysid shrimps, J. Jpn. Soc. Food Sci. Technol., 30, 404, 1983. 62. Choi, S.H., Kobayashi, A., and Yamanishi, T., Odor of fermented small shrimp. 1. Odor of cooked small shrimp, Acetes Japonicus Kishinouye—Difference between raw-material and fermented product, Agric. Biol. Chem., 47, 337, 1983. 63. Choi, S.H. and Kato, H., Odor of fermented small shrimp. 6. Flavor of fermented product of Antarctic krill prepared by modified method, J. Jpn. Soc. Food Sci. Technol., 32, 274, 1985. 64. Nordvi, B. et al., Characterization of volatile compounds in a fermented and dried fish product during cold storage, J. Food Sci., 72, S373, 2007. 65. Chung, H.Y. et al., Analysis of volatile components in frozen and dried scallops (Patinopecten yessoensis) by gas chromatography/mass spectrometry, Food Res. Int., 35, 43, 2002. 66. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Influence of processing on the volatile compounds characterizing the flavor of pickled fish, J. Food Sci., 52, 10, 1987. 67. Sharma, S.K., Basu, S., and Gholap, A.S., Effect of irradiation on the volatile compounds of shrimp (Solenocera choprii), J. Food Sci. Technol. Mysore, 44, 267, 2007. 68. Lopetcharat, K. et al., Fish sauce products and manufacturing: A review, Food Rev. Int., 17, 65, 2001. 69. Fukami, K. et al., Identification of distinctive volatile compounds in fish sauce, J. Agric. Food Chem., 50, 5412, 2002. 70. McIver, R.C., Brooks, R.I., and Reineccius, G.A., Flavor of fermented fish sauce, J. Agric. Food Chem., 30, 1017, 1982. 71. Peralta, R.R., Shimoda, M., and Osajima, Y., Further identification of volatile compounds in fish sauce, J. Agric. Food Chem., 44, 3606, 1996. 72. Shimoda, M., Peralta, R.R., and Osajima, Y., Headspace gas analysis of fish sauce, J. Agric. Food Chem., 44, 3601, 1996. 73. Michihata, T., Yano, T., and Enomoto, T., Volatile compounds of headspace gas in the Japanese fish sauce Ishiru, Biosci. Biotech. Biochem., 66, 2251, 2002. 74. Cha, Y.J., Baek, H.H., and Hsieh, T.C.Y., Volatile components in flavor concentrates from crayfish processing waste, J. Sci. Food Agric., 58, 239, 1992. 75. Cha, Y.J., Cadwallader, K.R., and Baek, H.H., Volatile flavor components in snow crab cooker effluent and effluent concentrate, J. Food Sci., 58, 525, 1993. 76. Tanchotikul, U. and Hsieh, T.C.Y., Volatile flavor components in crayfish waste, J. Food Sci., 54, 1515, 1989. 77. Elmore, J.S., Aroma, in Handbook of Muscle Foods Analysis, Nollet, L.M.L. and Toldrá, F., Eds., Taylor & Francis, Boca Raton, FL, 2008. 78. Chung, H.Y. and Cadwallader, K.R., Volatile components in blue-crab (Callinectes sapidus) meat and processing by-product, J. Food Sci., 58, 1203, 1993.
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79. Aro, T. et al., Determination of semivolatile compounds in Baltic herring (Clupea harengus membras) by supercritical fluid extraction-supercritical fluid chromatography-gas chromatography-mass spectrometry, J. Agric. Food Chem., 50, 1970, 2002. 80. Roh, H.S. et al., Isolation of off-flavors and odors from tuna fish oil using supercritical carbon dioxide, Biotech. Bioprocess Eng., 11, 496, 2006. 81. Kubota, K. et al., Odor of Antarctic krills. Part 1. Cooked odor of Antarctic krills, J. Agric. Chem. Soc. Jpn., 54, 1, 1980. 82. Shen, C., Xie, J., and Xu, X.S., The components of cuttlefish (Sepiella maindroni de Rochebruns) oil, Food Chem., 102, 210, 2007. 83. Triqui, R. and Reineccius, G.A., Flavor development in the ripening of anchovy (Engraulis encrasicholus L.), J. Agric. Food Chem., 43, 453, 1995. 84. Triqui, R. and Reineccius, G.A., Changes in flavor profiles with ripening of anchovy (Engraulis encrasicholus), J. Agric. Food Chem., 43, 1883, 1995. 85. Pennarun, A.L., Prost, C., and Demaimay, M., Identification and origin of the character-impact compounds of raw oyster Crassostrea gigas, J. Sci. Food Agric., 82, 1652, 2002. 86. Pennarun, A.L., Prost, C., and Demaimay, M., Aroma extracts from oyster Crassostrea gigas: Comparison of two extraction methods, J. Agric. Food Chem., 50, 299, 2002. 87. Selli, S. et al., Characterization of aroma-active compounds in rainbow trout (Oncorhynchus mykiss) eliciting an off-odor, J. Agric. Food Chem., 54, 9496, 2006. 88. Le Guen, S., Prost, C., and Demaimay, M., Characterization of odorant compounds of mussels (Mytilus edulis) according to their origin using gas chromatography-olfactometry and gas chromatographymass spectrometry, J. Chromatogr. A, 896, 361, 2000. 89. Le Guen, S., Prost, C., and Demaimay, M., Evaluation of the representativeness of the odor of cooked mussel extracts and the relationship between sensory descriptors and potent odorants, J. Agric. Food Chem., 49, 1321, 2001. 90. Likens, S.T. and Nickerson, G.B., Detection of certain hop oil constituents in brewing products, Proc. Am. Soc. Brew. Chem., 5, 1964. 91. Runge, G. and Steinhart, H., Determination of volatile sulfur-compounds in the edible part of carp, Agribiol. Res., 43, 155, 1990. 92. Kubota, K., Kobayashi, A., and Yamanishi, T., Cooked odor of Antarctic krills. 3. Some sulfurcontaining compounds in cooked odor concentrate from boiled Antarctic krills (Euphausia superba Dana), Agric. Biol. Chem., 44, 2677, 1980. 93. Kubota, K., Kobayashi, A., and Yamanishi, T., Cooked odor of Antarctic krills. 6. Basic and neutral compounds in the cooked odor from Antarctic krill, Agric. Biol. Chem., 46, 2835, 1982. 94. Tanchotikul, U. and Hsieh, T.C.Y., Analysis of volatile flavor components in steamed Rangia clam by dynamic headspace sampling and simultaneous distillation and extraction, J. Food Sci., 56, 327, 1991. 95. Chen, D.W. and Zhang, M., Analysis of volatile compounds in Chinese Mitten Crab (Eriocheir sinensis), J. Food Drug Anal., 14, 297, 2006. 96. Zhang, H.Z. and Lee, T.C., Gas chromatography–mass spectrometry analysis of volatile flavor compounds in mackerel for assessment of fish quality, in Flavor and Lipid Chemistry of Seafoods, Shahidi, F. and Cadwallader, K.R., Eds., American Chemical Society, Washington, DC, 1997, p. 55. 97. Chung, H.Y., Volatile components in crabmeats of Charybdis feriatus, J. Agric. Food Chem., 47, 2280, 1999. 98. Chung, H.Y., Yung, I.K.S., and Kim, J.S., Comparison of volatile components in dried scallops (Chlamys farreri and Patinopecten yessoensis) prepared by boiling and steaming methods, J. Agric. Food Chem., 49, 192, 2001. 99. Morita, K., Kubota, K., and Aishima, T., Comparing sensory and gas chromatographic profiles in aromas of boiled squid, prawn, and scallop using full factorial design, J. Food Sci., 67, 3456, 2002. 100. Morita, K., Kubota, K., and Aishima, T., Investigating sensory characteristics and volatile components in boiled scallop aroma using chemometric techniques, Food Chem., 78, 39, 2002.
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101. Le Guen, S., Prost, C., and Demaimay, M., Critical comparison of three olfactometric methods for the identification of the most potent odorants in cooked mussels (Mytilus edulis), J. Agric. Food Chem., 48, 1307, 2000. 102. Cros, S. et al., Desalination of mussel cooking juices by electrodialysis: Effect on the aroma profile, J. Food Eng., 69, 425, 2005. 103. Morita, K., Kubota, K., and Aishima, T., Sensory characteristics and volatile components in aromas of boiled prawns prepared according to experimental designs, Food Res. Int., 34, 473, 2001. 104. Morita, K., Kubota, K., and Aishima, T., Comparison of aroma characteristics of 16 fi sh species by sensory evaluation and gas chromatographic analysis, J. Sci. Food Agric., 83, 289, 2003. 105. Whitfield, F.B. et al., Distribution of bromophenols in species of marine polychaetes and bryozoans from eastern Australia and the role of such animals in the flavor of edible ocean fish and prawns (shrimp), J. Agric. Food Chem., 47, 4756, 1999. 106. Chung, H.Y., Ma, W.C.J., and Kim, T.S., Seasonal distribution of bromophenols in selected Hong Kong seafood, J. Agric. Food Chem., 51, 6752, 2003. 107. Boyle, J.L., Lindsay, R.C., and Stuiber, D.A., Bromophenol distribution in salmon and selected seafoods of fresh-water and saltwater origin, J. Food Sci., 57, 918, 1992. 108. Ma, W.C.J. et al., Enhancement of bromophenol levels in aquacultured silver seabream (Sparus sarba), J. Agric. Food Chem., 53, 2133, 2005. 109. da Silva, V.M. et al., Determination of simple bromophenols in marine fishes by reverse-phase high performance liquid chromatography (RP-HPLC), Talanta, 68, 323, 2005. 110. Chaintreau, A. Simultaneous distillation-extraction: From birth to maturity—Review, Flavour Fragrance J., 16, 136, 2001. 111. Acree, T.E., Bioassays for flavour, in Flavor Science: Sensible Principles and Techniques, Acree, T.E. and Teranishi, R., Eds., American Chemical Society, Washington, DC, 1993, p. 1. 112. Kubota, K. et al., Identification and formation of characteristic volatile compounds from cooked shrimp, in Thermal Generation of Aromas, Parliament, T.H., McGorrin, R.J., and Ho, C.-T., Eds., American Chemical Society, Washington, DC, 1989, p. 376. 113. Elmore, J.S., Mottram, D.S., and Dodson, A.T., Meat aroma analysis: Problems and solutions, in Handbook of Flavor Characterization: Sensory Analysis, Chemistry, and Physiology, Deibler, K.D. and Delwiche, J., Eds., Marcel Dekker, Inc., New York, 2004, p. 295. 114. Lee, G.H., Suriyaphan, O., and Cadwallader, K.R., Aroma components of cooked tail meat of American lobster (Homarus americanus), J. Agric. Food Chem., 49, 4324, 2001. 115. Cha, Y.J. and Cadwallader, K.R., Aroma-active compounds in skipjack tuna sauce, J. Agric. Food Chem., 46, 1123, 1998. 116. Triqui, R. and Guth H., Determination of potent odorants in ripened anchovy (Engraulis encrasicholus L.) by aroma extract dilution analysis and by gas chromatography-olfactometry of headspace samples, in Flavor and Lipid Chemistry of Seafoods, Shahidi, F. and Cadwallader, K.R., Eds., American Chemical Society, Washington, DC, 1997, p. 31. 117. Triqui, R. and Zouine, K., Sensory and instrumental assessments of the ripening process of anchovy (Engraulis encrasicholus), Food Sci. Technol., 32, 203, 1999. 118. Schlüter, S. et al., Changes in the odorants of boiled carp fillet (Cyprinus carpio L.) as affected by increasing methionine levels in feed, J. Agric. Food Chem., 47, 5146, 1999. 119. Engel, W., Bahr, W., and Schieberle, P., Solvent assisted flavour evaporation—A new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices, Eur. Food Res. Technol., 209, 237, 1999. 120. Girard, B. and Nakai, S., Nonparametric discriminant analysis of static headspace volatiles for grading fresh Pacific salmon, J. Food Qual., 17, 409, 1994. 121. Medina, I., Satué-Gracia, M.T., and Frankel, E.N., Static headspace gas chromatographic analyses to determine oxidation of fi sh muscle lipids during thermal processing, JAOCS, 76, 231, 1999.
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122. Hollingworth, T.A., Jr., Throm, H.R., and Wekell, M.M., Determination of ethanol in canned salmon, in Seafood Quality Determination, Kramer, D.E. and Liston, J., Eds., Elsevier, Amsterdam, the Netherlands, 1987, p. 153. 123. Chantarachotti, J. et al., Alaska pink salmon (Onchorynchu gorbuscha) spoilage and ethanol incidence in the canned product, J. Agric. Food Chem., 55, 2517, 2007. 124. McLachlan, D.G., Wheeler, P.D., and Sims, G.G., Automated gas chromatographic method for the determination of ethanol in canned salmon, J. Agric. Food Chem., 47, 217, 1999. 125. Lindsay, R.C., Josephson, D.B., and Olafsdottir, G., Chemical and biochemical indices for assessing the quality of fish packaged in controlled atmospheres, in Seafood Quality Determination, Kramer, D.E. and Liston, J., Eds., Elsevier, Amsterdam, the Netherlands, 1987, p. 221. 126. Yasuhara, A. and Shibamoto, T., Quantitative analysis of volatile aldehydes formed from various kinds of fish flesh during heat treatment. J. Agric. Food Chem., 43, 94, 1995. 127. Kolb, B. and Ettre, L., Static Headspace-Gas Chromatography: Theory and Practice, 2nd edn., Wiley, Chichester, U.K., 2006, p. 376. 128. Matiella, J.E. and Hsieh, T.C.Y., Analysis of crabmeat volatile compounds, J. Food Sci., 55, 962, 1990. 129. Zhang, C.H. et al., Studies on the odor of fishes. 3. Volatile compounds and their generation in smelt, Nippon Suisan Gakkaishi, 58, 773, 1992. 130. Kawai, T. et al., Flavor components of dried squid, J. Agric. Food Chem., 39, 770, 1991. 131. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Identification of compounds characterizing the aroma of fresh whitefish (Coregonus clupeaformis), J. Agric. Food Chem., 31, 326, 1983. 132. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Variations in the occurrences of enzymically derived volatile aroma compounds in saltwater and fresh-water fish, J. Agric. Food Chem., 32, 1344, 1984. 133. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Influence of maturity on the volatile aroma compounds from fresh Pacific and Great Lakes Salmon, J. Food Sci., 56, 1576, 1991. 134. Josephson, D.B., Lindsay, R.C., and Stuiber, D.A., Volatile compounds characterizing the aroma of fresh Atlantic and Pacific oysters, J. Food Sci., 50, 5, 1985. 135. Ishizaki, S. et al., Evaluation of odour-active compounds in roasted shrimp (Sergia lucens Hansen) by aroma extract dilution analysis, Flavour Fragrance J., 20, 562, 2005. 136. Hallier, A., Prost, C., and Serot, T., Influence of rearing conditions on the volatile compounds of cooked fillets of Silurus glanis (European catfish), J. Agric. Food Chem., 53, 7204, 2005. 137. Hallier, A., Serot, T., and Prost, C., Odour of cooked silurus (Silurus glanis) flesh: evaluation by sensory analysis and comparison of collection methods to assess the odour representativeness of extracts obtained by dynamic headspace, J. Sci. Food Agric., 84, 2113, 2004. 138. Hallier, A. et al., New gas chromatography-olfactometric investigative method, and its application to cooked Silurus glanis (European catfish) odor characterization, J. Chromatogr. A, 1056, 201, 2004. 139. Aro, T. et al., Volatile compounds of Baltic herring analysed by dynamic headspace sampling-gas chromatography-mass spectrometry, Eur. Food Res. Technol., 216, 483–488, 2003. 140. Piveteau, F. et al., Aroma of fresh oysters Crassostrea gigas: Composition and aroma notes, J. Agric. Food Chem., 48, 4851–4857, 2000. 141. Hsieh, T.C.Y. et al., Volatile flavor components in thermally processed Louisiana red swamp crayfish and blue crab, in Thermal Generation of Aromas, Parliament, T.H., McGorrin, R.J., and Ho, C.-T., Eds., American Chemical Society, Washington, DC, 1989, p. 386. 142. Vejaphan, W., Hsieh, T.C.Y., and Williams, S.S., Volatile flavor components from boiled crayfish (Procambarus Clarkii) tail meat, J. Food Sci., 53, 1666, 1988. 143. Jorgensen, L.V., Huss, H.H., and Dalgaard, P., Significance of volatile compounds produced by spoilage bacteria in vacuum-packed cold-smoked salmon (Salmo salar) analyzed by GC-MS and multivariate regression, J. Agric. Food Chem., 49, 2376, 2001. 144. Refsgaard, H.H.F., Haahr, A.M., and Jensen, B., Isolation and quantification of volatiles in fish by dynamic headspace sampling and mass spectrometry, J. Agric. Food Chem., 47, 1114, 1999.
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145. Methven, L. et al., Influence of sulfur amino acids on the volatile and non-volatile components of cooked salmon (Salmo salar), J. Agric. Food Chem., 55, 1427, 2007. 146. Jonsdottir, R. et al., Flavor characterization of ripened cod roe by gas chromatography, sensory analysis, and electronic nose, J. Agric. Food Chem., 52, 6250, 2004. 147. Andersen, E., Andersen, M.L., and Baron, C.P., Characterization of oxidative changes in salted herring (Clupea harengus) during ripening, J. Agric. Food Chem., 55, 9545, 2007. 148. Aidos, I. et al., Volatile oxidation products formed in crude herring oil under accelerated oxidative conditions, Eur. J. Lipid Sci. Technol., 104, 808, 2002. 149. Zhang, Z.Y. and Pawliszyn, J., Headspace solid-phase microextraction, Anal. Chem., 41, 809, 1993. 150. Kataoka, H., Recent advances in solid-phase microextraction and related techniques for pharmaceutical and biomedical analysis, Curr. Pharm. Anal., 1, 65, 2005. 151. Shirey, R.E., Optimization of extraction conditions for low-molecular-weight analytes using solidphase microextraction, J. Chromatogr. Sci., 38, 109, 2000. 152. Song, X.A. et al., Volatile compounds in the hepatic and muscular tissues of common carp, Japanese flounder, Spanish mackerel and skipjack, in More Efficient Utilization of Fish and Fish Products, Sakaguchi, M., Ed., Elsevier, Amsterdam, the Netherlands, 2004, p. 209. 153. Linder, M. and Ackman, R.G., Volatile compounds recovered by solid-phase microextraction from fresh adductor muscle and total lipids of sea scallop (Placopecten magellanicus) from Georges Bank (Nova Scotia), J. Food Sci., 67, 2032, 2002. 154. Mansur, M.A. et al., Volatile flavor compounds of some sea fish and prawn species, Fisheries Sci., 69, 864, 2003. 155. Wang, Q., O’Reilly, J., and Pawliszyn, J., Determination of low-molecular mass aldehydes by automated headspace solid-phase microextraction with in-fibre derivatisation. J. Chromatogr. A, 1071, 147, 2005. 156. Bianchi, F. et al., Fish and food safety: Determination of formaldehyde in 12 fish species by SPME extraction and GC-MS analysis, Food Chem., 100, 1049, 2007. 157. Chan, S.T. et al., Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines in fish by headspace solid-phase microextraction and gas chromatography-mass spectrometry, Eur. Food Res. Technol., 224, 67, 2006. 158. Li, X.J. et al., Novel fiber coated with amide bridged-calix[4]arene used for solid-phase microextraction of aliphatic amines, J. Chromatogr. A, 1041, 1, 2004. 159. Howgate, P., Tainting of farmed fish by geosmin and 2-methyl-iso-bomeol: A review of sensory aspects and of uptake/depuration, Aquaculture, 234, 155, 2004. 160. Heil, T.P. and Lindsay, R.C., A method for quantitative-analysis of flavor-tainting alkylphenols and aromatic thiols in fish, J. Environ. Sci. Health Part B: Pestic. Food Contam. Agric. Wastes, 23, 475, 1988. 161. Robin, J. et al., Off flavor characterization and origin in French trout farming, Aquaculture, 260, 128, 2006. 162. Schrader, K.K. et al., Geosmin and 2-methylisoborneol cause off-flavors in cultured largemouth bass and white sturgeon reared in recirculating-water systems, North Am. J. Aquaculture, 67, 177, 2005. 163. Grimm, C.C. et al., Using microwave distillation-solid-phase microextraction-gas chromatographymass spectrometry for analyzing fish tissue, J. Chromatogr. Sci., 38, 289, 2000. 164. Conte, E.D. et al., Determination of geosmin and methylisoborneol in catfish tissue (Ictalurus punctatus) by microwave-assisted distillation-solid phase adsorbent trapping, J. Agric. Food Chem., 44, 829, 1996. 165. Johnsen, P.B. and Lloyd, S.W., Influence of fat-content on uptake and depuration of the off-flavor 2-methylisoborneol by channel catfish (Ictalurus punctatus), Can. J. Fisheries Aquatic Sci., 49, 2406, 1992. 166. Lloyd, S.W. and Grimm, C.C., Analysis of 2-methylisoborneol and geosmin in catfish by microwave distillation-solid phase microextraction, J. Agric. Food Chem., 47, 164, 1999.
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167. Kondjoyan, N. and Berdagué, J.-L., A Compilation of Relative Retention Indices for the Analysis of Aromatic Compounds, INRA de Theix, Saint Genes Champanelle, France, 1996. 168. Mukhopadhyay, R., Old reliable benchtop GC/MS, Anal. Chem., 76, 213A, 2004. 169. Adahchour, M., Beens, J., and Brinkman, U.A., Recent developments in the application of comprehensive two-dimensional gas chromatography, J. Chromatogr. A, 1186, 67, 2008. 170. Čajka, T. and Hajslová, J., Gas chromatography–time-of-flight mass spectrometry in food analysis, LC-GC Eur., 2007, 25, 2007. 171. Milo, C. and Blank, I., Quantification of impact odorants in food by isotope dilution assay: Strengths and limitations, in Flavor Analysis: Developments in Isolation and Characterization, Mussinan C.J. and Morello M.J., Eds., American Chemical Society, Washington, DC, 1998, p. 69. 172. d’Acampora Zellner, B. et al., Gas chromatography-olfactometry in food flavour analysis, J. Chromatogr. A, 1186, 123, 2008. 173. Senger-Emonnot, P. et al., Odour active aroma compounds of sea fig (Microcosmus sulcatus), Food Chem., 97, 465, 2006. 174. Du, W.X. et al., Microbiological, sensory, and electronic nose evaluation of yellowfi n tuna under various storage conditions, J. Food Prot., 64, 2027, 2001. 175. Pavón, J.L.P. et al., Strategies for qualitative and quantitative analyses with mass-spectrometry-based electronic noses, Trends Anal. Chem., 25, 257, 2006. 176. Ratel, J. et al., Mass spectrometry based sensor strategies for the authentication of oysters according to geographical origin, J. Agric. Food Chem., 56, 321, 2008. 177. Taylor, A.J. and Linforth, R.S.T., Atmospheric pressure ionisation mass spectrometry for in vivo analysis of volatile flavour release, Food Chem., 71, 327, 2000. 178. Blake, R.S. et al., Demonstration of proton-transfer reaction time-of-flight mass spectrometry for real-time analysis of trace volatile organic compounds, Anal. Chem., 76, 3841, 2004. 179. Rochat, S., de Saint Laumer, J.-Y., and Chaintreau, A., Analysis of sulfur compounds from the inoven roast beef aroma by comprehensive two-dimensional gas chromatography, J. Chromatogr. A, 1147, 85, 2007.
Chapter 25
Quality Index Methods Grethe Hyldig, Emilía Martinsdóttir, Kolbrún Sveinsdóttir, Rian Schelvis, and Allan Bremner Contents 25.1 Introduction ................................................................................................................. 463 25.2 QIM ............................................................................................................................. 464 25.2.1 How to Use QIM ............................................................................................ 468 25.2.1.1 QIM Assessors ................................................................................ 469 25.2.1.2 QIM Sessions .................................................................................. 469 25.2.1.3 QIM Results ....................................................................................471 25.2.2 Development of New QIM Schemes ................................................................472 25.2.2.1 The Raw Material ............................................................................472 25.2.2.2 Setting Up the QIM Scheme and Testing the Scheme .....................473 25.2.2.3 Validation of the QIM Scheme ........................................................475 25.2.3 QIM in Relation to the EU-Scheme.................................................................476 25.3 Conclusion ................................................................................................................... 477 References ............................................................................................................................... 477
25.1
Introduction
The Quality Index Method (QIM) is a sensory analysis using a category scale, where the scheme measures the degree and rate of change in important criteria and in the sum total of these changes, which can be interpreted into equivalent days of storage and remaining shelf life. The development of the first QIM schemes were based on the work by Bremner [1] and are for whole fish stored in ice. There are now also QIM schemes developed for cod and plaice fillet [2,3] and for 463
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frozen cod [4–6]. The QIM scheme for frozen cod measures the storage history [5]. The QIM schemes have been developed from the viewpoint of the industry and from technical research, but there have also been studies on developing a version for the consumer QIM (C-QIM; C for consumer) [7,8]. C-QIM is not an acceptance test, but a tool for decision making for the consumer buying fish in a market or at the fishmonger [9]. The principle in the QIM scheme is based on the proposition that assessors cannot judge degrees of perfection but can very readily detect deviations or changes from it. A simple illustration of this would be a crack in an otherwise perfect wall. Thus defects in the product were allotted demerit points, which were summed to a total to provide an overall evaluation. The higher the number of demerit points, the more defects the product had. This approach was derived from the understanding that during storage of fish, changes occur that are readily detectable and often measurable. Th is is also in keeping with the fact that the vast majority of chemical, biochemical, and microbiological tests on fish products start from either zero or a low value and increase with both temperature and period of storage. The parameter for the QIM scheme is based on significant, well-defined characteristic changes of outer appearance attributes (eyes, skin, gills, and smell) for raw fish. In addition, the scoring allotted to each criterion is such that no single criterion could dominate and that the score values are easy to judge. The QIM scheme can be seen as a list of attributes each of which is scored on a restricted scale (0–3) and the scores are then added to provide a total—a quality index (QI). The QIM schemes are developed in such a way that there is a straight-line relationship with period of storage of the fish [1,10–16]. Further trials and theoretical investigations underpinned the validity of the scheme and demonstrated that it was capable of integrating the effects of time and temperature during storage [12,13,17]. The slope of the line is the rate of demerit point accumulation per day of storage and a simple calculation can indicate the equivalent of the number of days at 0°C that the product has been stored. If the decision had been made at which value the product should no longer be sold, or where it crosses some arbitrary set boundary between product grades, then the remaining shelf life can be calculated for the appropriate end use. Some of the problems among earlier schemes, such as the EU-scheme [18] are that they do not take into account the difference between species. To do that it is necessary to develop one scheme for each species. QIM does take the inherent differences between fish species into account and therefore it is necessary to develop QIM schemes for each fish species. This can be illustrated in the parameters for the eyes in the QIM scheme for cod (Gadus morhua) and salmon (Salmo salar) (Tables 25.1 and 25.2). There is a three-quality parameter concerning the eyes in the scheme for cod (corona, form of the eyes, and the pupil) and only two in the scheme for salmon (pupil and form of the eyes). Another example is that texture is not a parameter in the QIM scheme for plaice (Pleuronectes platessa), because here texture is not easy to measure and it does not change much during storage. In the following sections the QIMs for whole fish storage in ice are described in detail.
25.2
QIM
Today, several QIM schemes have been developed and the QIM-Eurofish Foundation (www. qim-eurofish.com) have published a QIM reference manual for the fish industry in 11 European languages (Danish, Dutch, English, French, German, Greek, Icelandic, Italian, Norwegian, Portuguese, and Spanish) covering 13 QIM schemes for commercially important species [19]. QIM schemes for the following fish species are published: brill (Rhombus laevis), cod (G. morhua), deep
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Table 25.1 QIM Scheme for Whole Farmed Salmon (S. salar) Containing Description for Each Parameter and the Given Scores in Succession from 0 to 3 Quality Parameters Skin
Description Color/appearance
Mucus
Odor
Texture
Eyes
Pupils
Form
Gills
Color/appearance
Mucus
Odor
Score
Pearl-shiny all over the skin
0
The skin is less pearl-shiny
1
The fish is yellowish, mainly near the abdomen
2
Clear, not clotted
0
Milky, clotted
1
Yellow and clotted
2
Fresh sea weedy, neutral
0
Cucumber, metal, hay
1
Sour, dish cloth
2
Rotten
3
In rigor
0
Finger mark disappears rapidly
1
Finger leaves mark over 3 s
2
Clear and black, metal shiny
0
Dark gray
1
Matt, gray
2
Convex
0
Flat
1
Sunken
2
Red/dark brown
0
Light red, pink/hazel
1
Gray-brown, brown, gray, green
2
Transparent
0
Milky, clotted
1
Brown, clotted
2
Fresh, seaweed
0
Metal, cucumber
1
Sour, moldy
2
Rotten
3 (continued)
466 ◾ Handbook of Seafood and Seafood Products Analysis Table 25.1 (continued) QIM Scheme for Whole Farmed Salmon (S. salar) Containing Description for Each Parameter and the Given Scores in Succession from 0 to 3 Quality Parameters Abdomen
Description Blood in abdomen
Odor
Score
Blood red/not present
0
Blood more brown, yellowish
1
Neutral
0
Cucumber, melon
1
Sour, reminds of fermentation
2
Rotten/rotten kale
3
QI (0–24)
Table 25.2 QIM Scheme for Whole Farmed Cod (G. morhua) Containing Description for Each Parameter and the Given Scores in Succession from 0 to 3 Quality Parameter Appearance
Description Skin
Stiffness
Eyes
Cornea
Form
Color of pupil
Score
Bright, iridescent pigmentation
0
Rather dull, becoming discolored
1
Dull
2
In rigor
0
Firm, elastic
1
Soft
2
Very soft
3
Clear
0
Opalescent
1
Milky
2
Convex
0
Flat, slightly sunken
1
Sunken, concave
2
Black
0
Opaque
1
Gray
2
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Table 25.2 (continued) QIM Scheme for Whole Farmed Cod (G. morhua) Containing Description for Each Parameter and the Given Scores in Succession from 0 to 3 Quality Parameter Gills
Description Color
Smell
Mucus
Blood
Fillets
Color
Color
Score
Bright
0
Less colored, becoming discolored
1
Discolored, brown spots
2
Brown, discolored
3
Fresh, sea weedy, metallic
0
Neutral, grassy, musty
1
Yeast, bread, beer, sour milk
2
Acetic acid, sulfuric, very sour
3
Clear
0
Milky
1
Milky, dark, opaque
2
Red
0
Dark red
1
Brown
2
Translucent, bluish
0
Waxy, milky
1
Opaque, yellow, brown spots
2
QI (0–23)
water shrimp (Pandalus borealis), farmed salmon (Salmo salar), whole fjord shrimp (P. borealis), haddock (Melanogrammus aeglefinus), herring (Clupea harengus), peeled shrimp (P. borealis), plaice (P. platessa), pollock (Pollachius virens), redfish (Sebastes mentella/marinus), sole (Solea vulgaris), and turbot (Scophthalmus maximus). Moreover Andrade et al. [20] have published schemes for Atlantic mackerel (Scomber scombrus), horse mackerel (Trachurus trachurus), and European sardine (Sardina pilchardus), Barbosa and Vaz-Pirez [21] a QIM scheme for common octopus (Octopus vulgaris), Huidobro et al. [11] for raw gilthead Sea bream (Sparus aurata), Baixas-Nogueras et al. [22] for Mediterranean Hake (Merluccius merluccius), Pons-Sánchez-Cascado et al. [23] for Mediterranean anchovies (Engraulis encrasicholus), and Herrero et al. [6] for frozen Hake (M. capensis and M. paradoxus). Furthermore QIM schemes have been developed for cod fillets (G. morhua) [3], flounder (Paralichthys patagonicus) [24], air and MA-packed maatjes herring (C. harengus) [25], cuttlefish (Sepia officinalis), and broadtail shortfin
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squid (Illex coindetii) [26], farmed Atlantic halibut (Hippoglossus hippoglossus L.) [27], and tub gunard (Chelidonichthys lucernus) [28]. Collaborative work has resulted in development of the Australian Quality Index Manual [29] that in its first version contains schemes for six species: Atlantic salmon (S. salar) in the head-on, gilled, and gutted form, whole Goldband snapper (Pristipomoides multidens), whole Sea mullet (Mugil cephalus), whole Snapper (Pagrus auratus), whole Tiger flathead (Neoplatycephalus richardsonii), and cooked whole Black tiger prawns (Penaeus monodon). A second version with schemes for another eight species will be released at the end of 2008. Tables 25.1 and 25.2 show the schemes for salmon and cod.
25.2.1
How to Use QIM
QIM is an objective method and, is easy to work with, since it includes instructions and easily understood illustrational material. Training QIM assessors for the industry implies both training for being a sensory assessor, including the standard sensory procedures, as well as training for implementation of QIM in practice. The sampling system, methods, and procedures for sensory evaluation must be very well defined to serve its purpose in quality management. QIM is well suited to train assessors and monitor performance of the panel. The QIM sessions must take place without any disturbance among the assessors. Assessors should know the nature and limits of the sense organs and learn how to recognize and evaluate appearance, taste, odor, and texture of fish after different periods of storage. Sensory evaluation of whole fish is generally carried out by trained assessors in the reception or processing halls of fish factories or at auction sites. In quality control procedures, special facilities or rooms are preferred for sensory evaluation, but it is not always possible. In industry and auctions the testing area should be located giving consideration to what is practical. The demands for the testing areas are 1. The noise level shall be kept to a minimum; during a sensory session the assessors must be able to work without any interruption. 2. Lighting is very important. It is preferable that the light is either real daylight according to ISO standard [30] but as a minimum be an intensity of 600–1500 lx/m2. 3. It must be free of any foreign odors. As a minimum there must not be any waste or other matter, or operation with a strong smell nearby. 4. There must be no eating, drinking, or smoking allowed in the testing area. 5. Testing area must be easy to clean and disinfect. Regular cleaning and disinfecting shall take place. It must be ensured that the cleaning agents used do not leave odors in the testing area. 6. The temperature should be kept low and constant. The aim of nonbiased sampling is to obtain a representative random sample from a lot. It is vital that the sample is selected randomly to ensure that it is representative. The number of fish to be sampled is determined by the accepted uncertainty, the characteristic of the lot, and economy [31,32]. From a defined homogeneous lot, preferably three to five fish (10 for small fish species) should be assessed according to QIM schemes. An homogeneous lot of fish should be assessed, i.e., from the same catching day. Number the boxes in a standard way, for example always from
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left to right and from top to bottom and generate 3–10 random numbers. Take one fish out of each of the 3–10 numbered boxes as decided. Make sure the fishes are taken from different places in the boxes (not always from the top layer). Evaluate all 3–10 fish using the QIM schemes as provided.
25.2.1.1
QIM Assessors
QIM assessors must be selected on their ability to evaluate appearance, color, odor, and texture. Assessors must also be healthy and possess normally sensitive taste and odor senses [33,34]. Personal characteristics are also very important such as conscientiousness and accuracy and they must be able to work in a group without disturbing the other assessors with noise, talk, and making faces. Depending on the regular duties of the individual, he or she must be readily available. For a company it is necessary to have a panel leader and a group of tested and trained assessors. The assessors for the QIM evaluation are then picked out from the group depending on availability, but it must be emphasized that all assessors in the sensory group are used frequently. The training of the sensory panel should begin by describing the procedures of the sensory evaluation, what is expected of the assessors, etc. The nature and limits of the sense organs are described, such as the importance of breathing deeply and resting between samples during odor evaluation. The schemes intended for use must be carefully explained. The general descriptions of the parameters are shown in Table 25.3. It should be emphasized to the assessors that they must not let their hedonic personal judgment interfere with the evaluation. For training, three to four samples of fish of different known storage periods in ice and treatment are used. The storage time of the fish is introduced to the assessors before they evaluate the fish and they are asked if they can agree on the scores that should be given for each sample. The samples are number coded. All assessors should become very familiar with fish of all freshness stages, i.e., not only raw material that is on the borderline of production. Training results should be evaluated. Average and standard deviation of each sample is calculated and a comparison is made between the assessors, i.e., by performing statistical analysis (analysis of variance, for example). The ability of the assessors can be examined during repeated evaluation of the same samples. Repetition of the training will show the capabilities of the assessors. Regular training of the sensory panel should be done and performance of the assessors monitored. It is also important to keep the assessors motivated. Finally the assessors must be able to perform QIM in a fast and accurate way and be in agreement with other QIM panels in proficiency tests.
25.2.1.2
QIM Sessions
The panel leader prepares the evaluation by giving the fish samples three-digit codes, and places the fish in random order on the table. They are kept cool either by placing them on a cooling plate or on ice. To avoid bias samples should always be coded with two- to three-digit numbers that provide no information about the samples. The samples must be kept cool under evaluation and the assessors should not see the samples being placed. The boxes where the samples have been taken from must also be removed from the testing area, because this might enhance expectation error. Order of presentation should be random and the order should be balanced. The assessors must be told in which order they should evaluate the samples. Hunger or satiation can influence the performance of the assessors. The assessors must not eat or smoke for an hour before the sensory evaluation. The assessors must be quiet and concentrating
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Table 25.3 General Description of Parameters Appearance It is important that the fish do not lie for such a long time that the skin dries out. Skin
The whole fish is inspected for the appearance of the skin and fins.
Mucus
The appearance of mucus on the skin is assessed. Mucus can be difficult to find on fish such as salmon, but it is often located around the dorsal fin.
Odor
The odor of the skin is assessed by smelling the spine. If the fish has been lying more than 15 min on the table, it should be turned over and smelled on the other side.
Texture Texture/firmness: The texture is assessed by pressing a finger (firmly, but not too hard) on the spine muscle and observing if/how fast the flesh recovers. Only fish in rigor is given a score of 0. Prerigor fish is soft/very soft and therefore given a high score, but if it is known that it is a prerigor fish, the texture should be 0. Belly
The consistency of the belly is assessed by pinching it between fingers or by stroking it with the fingertips.
Eyes Avoid touching the eyes with your fingers. If one eye is damaged, assess the other one. Eyes where the cornea is swollen are often difficult to assess, but the membrane may be stung or cut for easier assessment of the eye. Cornea
Color and clearness of the cornea is assessed.
Form
The form of the eyes is assessed by looking at the eye directly or from the side.
Gills The gills are assessed by lifting the opercula. If the gills have been cut on one side of the fish, assess the gills that have not been cut. Avoid touching the gills since the appearance and mucus of gills can easily be destroyed. Gill color
The color of the gills is assessed.
Gill odor
Odor of the gills is assessed by, lifting the opercula and smelling by the gill bow.
Mucus in gills
Color and appearance of the mucus is assessed.
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471
General Description of Parameters
Viscera Fish kept in ice with the viscera (ungutted) must be opened. The appearance of the viscera is assessed.
Color of Blood in Abdomen Usually, remains of blood in abdomen are visible in gutted fish. Blood may also be assessed in the cut wound (near the gills), if no remaining blood is left in the abdomen.
Odor in Abdomen Odor in the abdomen is assessed by smelling inside the abdomen.
Fillets/Cut Surface Color of fillets is assessed by the cut surface at the flaps or by assessing the fillets. Some fish such as redfish must be filleted from one side to be able to see the fillets and viscera.
during the evaluation. Trained assessors can evaluate 40 fish with QIM in 20 min, and the method is nondestructive. During continuous assessment of odor, assessors become insensitive to odors after some time. People become desensitized to odors as the receptors in the olfactory senses become saturated. Therefore, it is necessary to rest and breathe fresh air between samples when evaluating odor. Also, by taking a deep breath, the airflow through the olfactory senses increases and the odor becomes easier to detect. When applying the QIM schemes, the outer appearance of the fish, eyes, gills, and texture are evaluated. The odor of gills is evaluated, and for some species the odor and mucus of the skin is also evaluated. The color of blood, and fillets (or the cut surface at the flaps) is evaluated in gutted fish. All attributes are to be assessed in the same order for each fish. For some fish species that are not gutted, such as redfish, dissolution of viscera is evaluated as well. The assessor must evaluate all the parameters involved in the scheme (he or she cannot determine which parameters are most important). The assessors write down the scores given. For (quality) control purposes it is important to write down the information about the batch, date of assessment, and name of the assessors prior to assessing the fish. To make the scheme uniform and easy to use and to ensure all criteria were scored it was programmed into a handheld computer [35–37] and a prototype dedicated handheld device was developed [17]. An Icelandic company has developed software which can be connected to a handheld computer. The software includes both QIM schemes and photographs of fish at different spoilage stage, but this instrument is not commercially available [38].
25.2.1.3 QIM Results The scores for all the characteristics are summarized to give an overall sensory score, the so-called QI. If a score for one of the parameters is missing, it is not possible to calculate the total sum and thereby the QI for the assessed fish. If this situation is an incident (for example damaged eyes
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makes it impossible to assess this attribute) the best way to deal with it is to leave that particular fish out and take another sample. If it occurs on a regular basis (for example, due to washing procedures at a company the mucus is always removed) the scheme should be adapted and a new calibration curve constructed in which the particular attribute (e.g., mucus) is removed. Having the QI on an electronic basis means the data can be rapidly communicated from a boat, the quayside, or an auction, and that it can be used in management systems to plan supply and production and to allocate product to different grades or to end uses according to production or market requirements. The full assessment data can be stored in databases. This ability to transmit a meaningful QI along with identity and traceability information over the Internet represents a major advantage and a progressive step in electronic marketing of fish products. It further enhances opportunities for quality chain management to ensure product of known properties is handled correctly along the supply chain. As the QI increases linearly with storage time in ice, the information may be used in production management [19]. From the QI results, an estimate can be calculated for the remaining shelf life (equals total shelf life minus predicted storage time). In the following the calibration curves for cod, salmon, and plaice are shown: (R 2 = 0.966)
Cod: QI = 1.20 × days in ice − 0.04
Salmon: QI = 0.692 × days in ice + 1.57 (R 2 = 0.953) Plaice: QI = 1.28 × days in ice
(R 2 = 0.89)
It is emphasized that remaining shelf life should be used with some precaution due to the uncertainty in the estimation. Various factors can affect the remaining shelf life. It depends on the handling of the fish. Rapid cooling after the catch and an uninterrupted cold storage, different fishing gear, bleeding, and gutting methods are important, and the season and catching ground can also have an effect. In the literature several storage studies are reported and the estimated shelf life of different species is recorded [12,13,39–43]. These are summarized in Table 25.4.
25.2.2
Development of New QIM Schemes
To develop a new QIM scheme there are several considerations to take into account. It is necessary to have some specific knowledge about the fish species, to have on hand two tested and trained sensory panels, a facility to conduct storage experiments under standardized conditions, and to be able to make a statistical validation of the developed QIM scheme. In the following paragraphs this standardized development is described in detail.
25.2.2.1
The Raw Material
The selection of fish species is based on practical considerations and economic value. The fishing gear used to catch them and the fishing grounds where they are caught correspond with this selection and are fi xed for QIM schemes. The handling of the fish should be according to Good Manufacturing Practice (GMP) [32]. GMP can mean various standards or technical specifications, such as that the fish is gutted at sea (if gutting is the normal procedure) and washed. The fish is directly cooled down to 0°C in melting ice or equivalent cooling media. The fish is stored
Quality Index Methods Table 25.4
473
The Estimated Shelf Life for Some Fish Species
Species
Estimated Shelf Life in Ice
Brill (R. laevis)
14 days
Cod (G. morrhua)
15 days
Deep water shrimp (P. borealis)
a
◾
6 days
Farmed salmon (S. salar)
20 days
Fjord shrimp (P. borealis)
6 days
Haddock (M. aeglefinus)
15 days
Herring (C. harengus)
8 days
Peeled shrimp (P. borealis)
6 daysa
Plaice (P. platessa)
13 days
Pollock (P. virens)
18 days
Redfish (S. mentella/marinus)
18 days
Sole (S. vulgaris)
15 days
Turbot (S. maximus)
15 days
The storage life before peeling.
in fish boxes, with sufficient ice, which may be replenished, during the whole storage period. The storage trials begin with homogeneous batches of fish, preferably from one haul, with known history such as date of catch, storage condition, etc. For the complete development of a new QIM scheme at least three storage trials are needed and the experiment must begin at the time of catch and continue till after the end of shelf life. The QIM schemes developed with these batches are valid for these conditions.
25.2.2.2 Setting Up the QIM Scheme and Testing the Scheme For the development of a draft QIM scheme, the first storage experiment is needed. In this stage the fish is described in detail during the complete ice storage until the end of an expected shelf life. This is done at fi xed time intervals of 12–48 h, depending on the expected shelf life of the species. The shorter the expected shelf life is, the shorter the time intervals required. A group of maximum five experienced assessors, including a panel leader, is put together. This group is selected, besides the usual criteria for sensory assessors, for their good use of vocabulary and knowledge of the fish species. The panel leader is experienced in control of the discussion and enables everyone to express their meaning. Approximately five fishes are used at the time and they are discarded after each session. During the sessions the fishes are placed on chill plates or ice. Further sessions are continued until the fish is completely spoiled. All attributes are listed and described in detail. The attributes to be assessed varies per species: for example for flat fish species the appearance of both sides might be relevant which is, obviously, not the case for round fish species. The appearance for shrimp will
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have a completely different description than for cod. All descriptions are written down, preferably in terms the whole group agrees on. A description is written on how the assessment is done, for example the assessment of the texture attribute for cod is done by pressing the finger (firmly but not too hard) on the spine muscle and observing how fast the flesh recovers. After this first storage trial, the panel leader selects those attributes that change over the storage period, the descriptions are grouped together per attribute and major changes are scored with “demerit” points from 0 to 3. If, for example, the maximum of three demerit points are scored within the first 5 days, but it is generally known that then the shelf life is, in total, about 14 days, the description per demerit point needs to be changed in such a way that the scoring covers more of the complete shelf life. This results in the draft QIM scheme that will be used in the next storage trial. The testing of the draft QIM scheme is done with a second batch of fish during a new storage trial. For this test a new panel of approximately 10 assessors (experts in QIM) is organized, the assessors who participated in the development of the scheme are included. During the storage trial at fi xed time intervals, again depending on the shelf life but less frequently than in the first storage trial, five fishes are assessed individually by using the draft QIM scheme. Each expert scores every attribute and comments are written down. After each session the scores, comments, and questions on how to assess the attributes are discussed among the assessors. The results are analyzed and the attributes are selected for being discriminative and for ease of assessment. Statistical analyses used to assist these decisions are Principal Component Analyses (PCA) or calculation of linear regression lines per attribute [12,13]. These decisions on attributes and scores are of major importance for meeting several of the above-mentioned considerations like the balance between the strength of the scheme, reliability of the results, and practical use of the scheme, and cannot be made without knowledge on the fish species, spoilage pattern, practical importance of the sensory attributes, and QIM principles in general. At the same time the draft scheme is tested the end of shelf life needs to be determined. The shelf life is defined as the number of days that whole, fresh (gutted) fish can be stored in ice until it becomes unfit for human consumption. It needs to be emphasized that the estimated shelf life is based upon optimal catching and storage conditions [12,13,19]. Spoilage due to microbial activity is the main limitation of the shelf life. Another cause of spoilage may be rancidity, especially in fatty fish species. The flesh of newly caught fish is free of bacteria. However, considerable amounts of bacteria may be in the viscera, the gills, and on the skin, which can give contamination during processing. When the fish is stored whole in ice, the deterioration caused by bacteria is minimal for the first days of storage and then it will increase. In general, when fish is stored in ice the flavor and odor compounds that characterize newly caught fish decrease and disappear in the first few days during storage, and the fish flesh becomes almost flavorless and odorless for a while. Then an increase in bad-smelling sulfur and nitrogenous volatiles will result in rejection of the fish for human consumption. This can be measured by descriptive sensory profiling or by using the Torry-scale. A tested and trained sensory panel evaluates cooked samples from the storage experiment. The sensory panel must be a second panel of different composition in order to overcome bias. The descriptive sensory profiling and the Torryscale are described in the previous chapter. From the results of the descriptive sensory analysis the shelf life is defined. For future use of the QIM scheme, i.e., training of QIM inspectors and illustration of the descriptions of the different attributes, pictures are of utmost importance. During the development of QIM, pictures need to be taken of all the changes of the different attributes. These pictures
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are preferably taken by a professional photographer in order to be clear, and with the right use of flashlights. In practice the importance of the quality of the pictures and the difficulties in taking them are highly underestimated. This can result in low-quality pictures which are not suitable for printing in reference manuals. The next step in the development of a QIM scheme is the so-called calibration curve. As mentioned in the introduction of this chapter, the QIM resulted in a scoring system that gave a linear relationship with storage period and the slope of the line is the rate of demerit point accumulation per day of storage. A simple calculation can indicate the equivalent of the number of days at 0°C that the product has been stored (i.e., predicted storage time). To calculate the calibration curve, a simple linear regression analysis is performed. The data for this calculation is obtained from a third more complex storage trial. Before this third trial the QIM panel is trained for the new developed QIM scheme. The QIM panel will assess according to good sensory practice. They will evaluate unknown coded samples in random order. This implies that, during the storage trial several new batches, which have been stored for different equivalent days in ice, are randomly allocated so that fish of different QIM are assessed in one session. From each batch five fishes are assessed within each trial and a maximum of five assessments are done per fish. If more than five assessors assess the fish a double batch is needed. The storage trial is finished when the fish is spoiled, meaning some days after the determined end of shelf life, in order to get a complete calibration curve for the QIM scheme. The average results are used to calculate the linear regression. The equation is calculated as well as the variation around the curve. The intercept at the y-axis is of major importance and should be close to 0. This follows the QIM principle of “deviations from a perfect product.” The correlation coefficient between the QI and the days in ice should be close to 1.0. With this result the calibration curves give reliable prediction of the storage time. The calculated Standard Error of Prediction (SEP) results in the reliability of the QIM assessments. Sveinsdóttir et al. [12,13] published results that with the assessment of three salmon per batch the reliability was 2 days, and with assessment of five salmon per batch the reliability was 1.5 day.
25.2.2.3
Validation of the QIM Scheme
To finalize the QIM scheme the foregoing steps need to be combined and will result in a QIM scheme that is more than only a table with attributes, descriptions, and scores. The scheme is developed under standardized conditions that need to be described in order to know the validity of the scheme under different circumstances. If a scheme becomes too specific then it may not suit all the circumstances of catch and handling for that species. A fish caught by handline/longline will generally show fewer signs of deterioration than one caught by trawling, and its starting characteristics when stored in ice will be different. A trawled fish stored in refrigerated seawater will have a different appearance to those stored in ice [44]. In both these instances the QIM scores at the start of storage will be different, but later during the storage period any differences may be insignificant. Either a general scheme that does not differentiate on the basis of catch or storage medium should be used, or particular schemes should be developed that allow for this difference in properties. It depends on circumstances, anticipated storage life, and the use of the scheme. Huidobro et al. [45] reported that the QIM of washed sea bream developed similarly to that of unwashed sea bream for up till 10 days storage in ice. After that, less slime and lower trimethylamine (TMA) formation in the washed fish retarded the normal increase in score, in comparison to unwashed fish. This illustrates another
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instance where the process may influence the score. But anticipated shelf life should also be taken into account since these were aquacultured sea bream destined for sale as fresh (i.e., unfrozen) fish. Clearly there would be a failure in the market and distribution system if such a prime product was not sold in much less than 10 days, and a scheme should not necessarily be required to cover all treatment options beyond this time. To a large extent all changes in stored fish are governed by time and temperature with the proviso, as we have seen above, that species and catch and handling practices, and maybe seasonal factors, are similar. If the commercial fish handling system maintains fish at a steady temperature of 0°C then any lack of ability to integrate is not of concern. However, it is a matter of common and recorded observation that temperature in the fish chain is not always well controlled. It may be that some schemes provide an incorrect assessment depending on how long the fish has been at elevated temperatures. However, most other instruments and schemes have not been tested in this regard either. The Torrymeter for example cannot integrate time and temperature at the correct rate [17]. The original scheme, on which QIM is based, was shown to be capable of integrating the effects of time and temperature [46]. Experiments at the DTU AQUA (former Danish Institute for Fisheries Research) in 2003 were set up to check whether the developed QIM schemes could integrate the time/temperature effects. The results from experiments with cod and plaice showed that if the fish have been stored at elevated temperatures (between 0°C and 5°C) and then stored at 0°C, the development in QI would follow the calibration curve for the fish species. Some tropical reef fish have inordinately long shelf-lives of more than 3 weeks when they are iced soon after catch and kept cold during storage [46]. Th is seems to be due to the absence of spoilage psychrotrophic bacteria, unless they are introduced, and to a stable level of IMP in the flesh. Consequently, the flesh remains acceptable in flavor for considerable periods of time when virtually all the external indicators that would be used in a QIM scheme, are at, or near, their maxima. However, it is still obvious that the fish have been stored for a considerable period and their remaining commercial life must be extremely limited. The use of a QIM approach for these species is still valid, but care must be taken in formulating a scheme in which the maximum QIM score is not reached while the fish is still quite marketable with some remaining shelf life. To validate the new developed QIM scheme a storage experiment at another location, season, or catching ground can be conducted [12,13].
25.2.3
QIM in Relation to the EU-Scheme
Specific for seafood the EU regulation: “Council Regulation (EC) No. 2406/96 of 26 November 1996 [18], laying down common marketing standards for certain fishery products” has only one sensory method—the EU-scheme for fresh fish. Th is method is to be used at the first point of sale and implies freshness and other quality items (parasites, pressure marks, injuries, blemishes, and bad discoloration). There are different schemes for Whitefish, Bluefish, Selachii, Cephalopods, and Crustaceans. This method is to be used by experts or by the competent authority (inspection body). As already mentioned the EU-scheme does not take into account the differences between species as it only uses general parameters, and there are also problems with mixing subjective and objective sensory in the scheme. Several studies have shown that the QIM proved to be more reliable in assessing sensory changes of different fish species as compared to the EU grading scheme [47].
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Table 25.5 The Relation between the EU Classification and the QI Score for Four Fish Species EU Classification
QI Score Plaice (P. platessa) Cod (G. morhua) Sole (S. vulgaris) Turbot (S. maximus)
E
0–5
0–4
0–5
0–5
A
6–16
5–13
6–19
6–19
B
17–21
14–16
20–27
20–26
>22
>17
>28
>27
Rejected
If the industry using the QIM needs to refer the scores to the current EU classification E, A, and B, QIM-Eurofish advises the following relation between the QI scores and EU classification: In case the average score of the assessed fish samples (3–5) per batch is between the mentioned QIM ranges, the results should be rounded off to the nearby QIM range. Table 25.5 clearly shows that A, after the EU classification, is too broad and not detailed enough to use in electronic auctions and in production managements.
25.3
Conclusion
The QIM has been proven in the marketplace to be an easy to use, practical tool. It is widely used in research as a reference method and can be applied to a wide variety of species including crustacea and cephalopods. It is versatile and can integrate time and temperature effects since it is designed to mimic the kinetics of microbial, enzymic action, and the changes in bulk properties. Consequently the range of schemes becoming available is quite extensive. Further development of handheld devices containing these schemes and the ability to link the results with photos, traceability, authenticity, catch, and market information is required to make fuller use of the available information and to enhance electronic marketing, safety, and quality assurance procedures.
References 1. Bremner A. A Convenient easy-to-use system for estimating the quality of chilled seafood. In Scott DN and Summers C (eds.); Proceedings of the Fish Processing Conference, Nelson, New Zealand, 23–25 April 1985; Fish Process. Bull., 7, 59, 1985. 2. Larsen EP, Hyldig G, and og Frederiksen M. Fersk fi sk—Kvalitet i detailhandlen. DFU Rapport udgivet af Danmarks Fiskeriundersøgelser, Afdeling for Fiskeindustriel Forskning, Lyngby, Denmark, 1998–1999. 3. Bonilla AC, Sveinsdottir K, and Martinsdottir E. Development of quality index method (QIM) scheme for fresh cod (Gadus morhua) fillets and application in shelf life study. Food Control, 18, 352, 2007.
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4. Hyldig G and Nielsen J. A rapid sensory method for quality management. In Ólafsdóttir G and others (eds.). Methods to Determine the Freshness of Fish in Research and Industry; Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3CT942283, Nantes Conference, Nov. 12–14, International Institute of Refrigeration, Paris, France, p. 297, 1997. 5. Warm K, Bøknæs N, and Nielsen J. Development of quality index methods for evaluation of frozen cod (Gadus morhua) and cod fillets. J. Aquat. Food Prod. Technol., 7, 45, 1998. 6. Herrero AM, Huidobro A, and Careche M. Development of quality index method for frozen hake (M. capensis and M. paradoxus). J. Food Sci., 68, 1086, 2003. 7. Warm K. Sensory Quality Criteria for New and Traditional Fish Species of Relevance to Consumer Needs. Danish Institute for Fisheries Research, Lyngby, The Royal Veterinary and Agricultural University, Copenhagen, Denmark, p. 1, 2000. 8. Larsen E, Hyldig G, Dalgaard P, Bech AC, and Østerberg C. Consumers and experts responses to fresh cod fillets. In Luten JB, Oehlenschlager J, and Ólafsdóttir G (eds.). Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen Academic Publ., Wageningen, the Netherlands, p. 345, 2003. 9. Nielsen J, Hyldig G, and Larsen E. Eating quality of fish—A review. J. Aquat. Food Prod. Technol., 11, 125, 2002. 10. Larsen EP, Heldbo J, Jespersen CM, and Nielsen J. Development of a standard for quality assessment on fish for human consumption. In Huss HH, Jacobsen M, and Liston J (eds.). Quality Assurance in the Fish Industry. Elsevier, Amsterdam, the Netherlands, p. 351, 1992. 11. Huidobro A, Pastor A, and Tejada M. Quality index method developed for raw gilthead seabream (Sparus aurata). J. Food Sci., 65, 1202, 2000. 12. Sveinsdóttir K, Martinsdóttir E, Hyldig G, Jørgensen B, and Kristbergsson K. Application of quality index method (QIM) scheme in shelf life study of farmed Atlantic Salmon (Salmo salar). J. Food Sci., 67(4), 1570, 2002. 13. Sveinsdóttir K, Hyldig G, Martinsdóttir E, Jørgensen B, and Kristbergsson K. Development of quality index method (QIM) scheme for farmed Atlantic Salmon (Salmo salar). Food Qual. Prefer., 14, 237, 2003. 14. Nielsen D, Green D, Bolton G, Hodson R, and Nielsen J. Hybrid Striped Bass—A QIM Scheme for a Major U.S. Aquaculture Species; Poster at First joint trans Atlantic fisheries technology conference (TAFT), 10–14 June 2003 Reykjavik, Iceland: 33rd WEFTA Meeting, 2003. 15. Hyldig G and Nielsen J. QIM—A tool for determination of fish freshness. In Shahidi F and Simpson BK (eds.). Seafood Quality and Safety. Advances in the New Millennium. ScienceTech Publishing Company, St. John’s, NL, p. 81, 2004. 16. Hyldig G and Green-Petersen DMB. Quality index Method—An objective tool for determination of sensory quality. J. Aquat. Food Prod. Technol., 13(4), 71, 2005. 17. Bremner HA, Olley J, and Vail AMA. Estimating time-temperature effects by a rapid systematic sensory method. In Advances in Food Research: Seafood Quality Determination. Elsevier Press, Amsterdam, the Netherlands, p. 413, 1987. 18. Anonymous, Council regulation (EC) No. 2406/96 of November 26, 1996, Laying Down Common Marketing Standards for Certain Fishery Products. Official J. Eur. Commun., No. L334, 1–14, 1996. 19. Martinsdóttir E, Sveinsdóttir K, Luten J, Schelvis-Smit R, and Hyldig G. Sensory Evaluation of Fish Freshness. A Reference Manual for the Fish Industry. QIM-Eurofish, the Netherlands, 2001, 2004. Available from http://www.qim-eurofish.com. 20. Andrade A, Nunes ML, and Batista I. Freshness quality grading of small pelagic species by sensory analysis. In Ólafsdóttir G and others (eds.). Methods to Determine the Freshness of Fish in Research and Industry; Proceedings of the Final Meeting of the Concerted Action “Evaluation of Fish Freshness” AIR3CT942283, Nantes Conference, Nov. 12–14, International Institute of Refrigeration, Paris, France, p. 333, 1997.
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21. Barbosa A and Vaz-Pires P. Quality index method (QIM): Development of a sensorial scheme for common octopus (Octopus vulgaris). Food Control, 15, 161, 2004. 22. Baixas-Nogueras S, Bover-Cid S, Veciana-Nogues T, Nunes ML, and Vidal-Carou MC. Development of a quality index method (QIM) to evaluate freshness in Mediterranean Hake (Merluccius merluccius). J. Food Sci., 68(3), 1067, 2003. 23. Pons-Sánchez-Cascado S, Vidal-Carou MC, Nunes ML, and Veciana-Nogués, MT. Sensory analysis to assess the freshness of Mediterranean anchovies (Engraulis encrasicholus) stored in ice. Food Control, 17, 564, 2006. 24. Massa AE, Palacios DL, Paredi ME, and Crupkin M. Postmortem changes in quality indices of icestored flounder (Paralichthys patagonicus). J. Food Biochem., 29, 570, 2005. 25. Lyhs U and Schelvis-Smit R. Development of a quality index method (QIM) for Maatjes Herring stored in air and under modified atmosphere. J. Aquat. Food Prod. Technol., 14(2), 63, 2005. 26. Vaz-Pires P and Seixas P. Development of new quality index method (QIM) schemes for cuttlefish (Sepia officinalis) and broadtail shortfin squid (Illex coindetii). Food Control, 17(12), 942, 2006. 27. Guillerm-Regost C, Haugen T, Nortvedt R, Carlehög M, Lunestad BT, Kiessling A, and Rora AMB. Quality characterization of farmed Atlantic Halibut during ice storage. J. Food Sci., 71(2), 83, 2006. 28. Bekaert K. Development of Quality Index Method scheme to evaluate freshness of tub gunard (Chelidonichthys lucernus). In Luten JB, Jacobsem C, Bekaert K, Sæbö A, and Oehlenschläger J (eds.). Seafood from Fish to Dish. Wageningen Academic Publishers, the Netherlands, p. 289, 2006. 29. Boulter M, Poole S, and Bremner A. Australian Quality Index Manual. Fisheries Research and Development Corporation, Canberra ACT, 2006. 30. ISO 8589. Sensory Analysis—General Guidance for the Design of Test Rooms, 1st edn., International Standard, Geneva, Switzerland, 1988. 31. NMKL procedure No. 12. Guiding on Sampling. Nordic Committee on Food Analysis, Copenhagen, Denmark, 2002. Available at http://www.nmkl.org. 32. Codex: Recommended International code of Practice for Fresh Fish CAC/RCP 9–1976Codex standards for methods of analysis and sampling, “Sampling Plans for Prepackaged Foods (AQL 6.5),” XOT 13–1969, Rome, FAO/WHO Codex. Alimentarius. Codex XOT 13–1969, 1976. 33. ISO 8586-1. Sensory Analysis—General Guidance for the Selection, Training and Monitoring of Assessors. Part 1: Selected Assessors, 1st edn. International Standard, Switzerland, 1993. 34. ISO 11035. Sensory Analysis—Identification and Selection of Descriptors for Establishing a Sensory Profile by a Multi-Dimensional Approach, 1st edn., International Standard, Geneva, Switzerland, 1994. 35. Branch AC and Vail AMA. Bringing fish into the computer age. Food Technol. Aust., 37, 352, 1985. 36. Heldbo, J. Information Technology and Production Management in the White Fish Industry. Industrial PhD thesis, Danish Institute for Fisheries Research and The Royal Veterinary and Agricultural University, Copenhagen, Denmark, 1990. 37. Jónsdóttir SM, Hyldig G, Nielsen J, Bleechmore T, and Silberg S. Rapid PC based sensory methods. Infofish Int., 2, 54, 1999. 38. Luten JB. Development and implementation of a computerised sensory system (QIM) for evaluating fish freshness. CRAFT FAIR CT97 9063. Final Report for the period from 01–01–98 to 31–03–00, Wageningen, the Netherlands, RIVO The Netherlands Institute for Fisheries Research, p. 18, 2000. 39. Howgate P. Approaches to the Definition and Measurement of Storage Life of Chilled and Frozen Fish. Torry Research Station, Aberdeen, U.K., 1985. 40. Martinsdóttir E and Blomsterberg F. Sjálfvirk ferskleikamæling með RT-gæðaflokkara 12. RIT Icelandic Fisheries Laboratories, 1987. 41. Magnússon H, Martinsdóttir E, and Steinþórsson P. Áhrif frystingar og frystigeymslu þorsks eftir þíðingu. 26. Icelandic Fisheries Laboratories REPORT no. 26, 1990.
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42. Rehbein H, Martinsdóttir E, Blomsterberg F, Valdimarsson G, and Öehlenschläger J. Shelf life of ice-stored redfish, Sebastes marinus and S. mentella. Int. J. Food Sci. Technol., 29, 303, 1994. 43. Martinsdóttir E, Sveinsdóttir K, and Ólafsdóttir G. Development and Implementation of a Computerised Sensory System (QIM) for Fish Freshness. Icelandic Fisheries Laboratories Project Report 11, 2000. 44. Nielsen D and Hyldig G. Influence of handling procedures and biological factors on the QIM evaluation of whole herring (Clupea harengus L.). Food Res. Int., 37(10), 975, 2004. 45. Huidobro A, Pastor A, Lopez-Caballero ME, and Tejada M. Washing effect on the quality index method (QIM) developed for raw gilthead seabream (Sparus auratus). Eur. Food Res. Technol., 212, 408, 2001. 46. Bremner HA, Statham JA, and Sykes SJ. Tropical species from the North-West Shelf of Australia. Sensory assessment and acceptability of fi sh stored on ice. Proceedings of the Sixth Session IPFC Working Party on Fish Technology and Marketing, Melbourne 1984 FAO, Fish Rep., 317(Suppl.), 41, 1984. 47. Triqui R and Bouchriti N. Freshness assessment of Moroccan Sardine (Scardina pilchardus): Comparison of overall sensory changes to instrumentally determined volatiles. J. Agric. Food Chem., 51, 7540, 2003.
Chapter 26
Sensory Descriptors Grethe Hyldig Contents 26.1 Introduction ..................................................................................................................481 26.1.1 Sensory Analysis ............................................................................................. 482 26.1.2 Objective and Subjective Sensory Analysis ..................................................... 482 26.2 The Human Senses ....................................................................................................... 482 26.2.1 The Sense of Vision......................................................................................... 482 26.2.2 The Olfactory Sense........................................................................................ 483 26.2.3 The Sense of Touch ......................................................................................... 483 26.2.4 The Gustatory Sense ....................................................................................... 483 26.2.5 The Chemical/Trigeminal Sense ..................................................................... 484 26.2.6 The Sense of Hearing ...................................................................................... 484 26.3 The Use of Sensory Descriptors .................................................................................... 484 26.4 Sensory Descriptors in Seafood .................................................................................... 487 26.4.1 Appearance ..................................................................................................... 487 26.4.2 Odor ............................................................................................................... 488 26.4.3 Taste/Flavor .................................................................................................... 488 26.4.4 Texture ............................................................................................................492 References ................................................................................................................................495
26.1
Introduction
The sensory quality of seafood is influenced by the treatment and processing from harvest, through transportation, storage, and processing. Sensory analysis of seafood has therefore played a natural part of the seafood chain for years. 481
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26.1.1
Sensory Analysis
In sensory analysis appearance, odor, flavor, and texture are evaluated using the human senses of vision, smell, taste, touch, and hearing. Scientifically, the process can be divided into three steps: (1) detection of a stimulus by the human sense organs; (2) evaluation and interpretation by a mental process; and (3) then the response of the assessor to the stimuli.
26.1.2
Objective and Subjective Sensory Analysis
A special aspect of sensory analysis is that it can be both objective and subjective. The objective tests include discriminative (triangle test, forced choice) and descriptive (profiling, structured scaling) sensory tests. Both groups of tests are analytical measurements of the intrinsic quality of the product. The subjective tests are used for consumer testing and to measure the attitude and emotional response of the consumer toward the product. The subjective tests can be applied in the fields such as market research and product development where the consumer reaction is required. In the objective tests, a trained panel objectively describes the attributes of products using defined sensory descriptors [1–3], whereas the subjective tests use untrained human beings (consumers) who answer subjectively, for example, how much they like/dislike a product. The objective sensory analytical methods do not depend on whether assessors like or dislike a certain item. Instead, it operates with the determining of intensities of sensory descriptors such as sweetness, amine, sourness, softness, and the like. In the following text, the focus will be on sensory descriptors that are used in objective sensory tests.
26.2
The Human Senses
In a sensory test, the first sense that the assessor uses is the vision. The vision is used to measure the appearance such as color and texture properties. The next sense is the olfactory that are used for detecting odors. Then the assessor touches the sample, with the fingers, directly or indirectly by using a tool (e.g., spoon or fork) and in the mouth where also the tongue and lips are used to measure the texture properties. In the mouth, the sense of gestation and the trigeminal sense are used to measure the taste, the pain, and the cooling effects. During the whole assessment, the sense of hearing is used to measure the sound both outside and inside the mouth, like when a cracker is broken or during chewing.
26.2.1
The Sense of Vision
The visual sense is often equated only with color but provides also input on many appearance attributes such as size, shape, and surface structure. In particular, the visual senses can provide an early and strong expectation of the flavor and textural properties of food. The visual receptors, the rods and cones, are located in the retina of the eye. These receptors contain light-sensitive pigments, which change shape when stimulated by light energy, leading to the generation of electrical nerve impulses that travel along the optic nerves to the brain. There are ∼120 million rods in the retina. The maximum rod concentration is ∼20° from the foveal area; this area is the parafovea. The 6 million cones operate at higher light intensities (levels of illumination) and provide chromatic information (color), allowing photopic vision.
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The light reflected from an object, or the light passing through an object, falls on the corona, travels through the lens, and from there to the retina, where most of the light falls on a small hallow in the retina, the fovea, a small depression located in a yellow-colored spot (macula lute) on the retina. The rods are capable of operating at extremely low light intensities (<1 lx). The rods yield only achromatic (black/white) information, and under low light conditions, these rods give a scotopic vision with no color perception. The cones are concentrated on the fovea, where the highest color resolution occurs. When viewing an object, the unconscious movement of the eyes serves to bring the image of the object onto the foveal areas. The cones contain three color-sensitive pigments, each responding to red (two polymorphic variations 552 and 557 nm), green (at 530 nm), or blue light (at 426 nm) most sensitively [4,5]. People lacking one of these pigments are color-blind categories, and depending on which pigments they are missing can be classified into different groups. The genes for the more common forms of color-blindness are recessive and carried on the X chromosome. Thus, the trait is seen much more frequently with men than with women.
26.2.2 The Olfactory Sense The odor response is much more complex. The olfactory receptors are located very high in the nasal cavity. There are several million receptors on each side of the nose, and they are highly ciliated. The function of the cilia is to greatly increase the surface area exploring the receptors to chemical stimuli. Odors are detected as volatiles entering the nasal passage, either directly via the nose or indirectly through the retronasal path via the mouth. Some 150–200 odor qualities have been recognized [6]. The odor receptors are easily saturated, and specific anosmia (blindness to specific odors) is common. It is thought that the wide range of possible odor responses contributes to variety in flavor perception.
26.2.3
The Sense of Touch
Texture is perceived by the sense of touch and comprises two components: somesthesis, a tactile, surface response from skin; and kinesthesis (or proprioception), a deep response from muscles and tendons. The touch stimuli themselves can arise from tactile manipulation of the food with hands and fingers, either directly or through the intermediary of utensils such as a knife or a spoon. Oral contact with food can occur through the lips, the tongue, the palate, and the teeth, all of which provide textural information. For many foods, visually stimuli will generate an expectation of textural properties. As food enters the mouth and is either bitten or manipulated between tongue and palate, changes occur to the structure of the food that strongly influence the way in which tastants and odorants are released from the food. Temperature increase (cold foods) or decrease (hot foods) and dilution by saliva are of particular importance.
26.2.4
The Gustatory Sense
Taste is defined as the response by the sense organs on the tongue and soft palate contains the taste receptors. These have been defined as five primary basic taste sensations—salt, sweet, sour, bitter, and umami.
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The taste receptors are organized in groups of cells, known as taste buds, located within specialized structures called papillae. These are located mainly on the tip, sides, and rear upper surface of the tongue. Taste buds are onion-like structures and each taste bud contains approximately 50–100 taste cells. A single taste cell may be multimodal, with several types of receptors; one type may be more active than others on that cell. Each taste receptor cell is connected through a synapse to a sensory nerve ending, which is a peripheral afferent fiber, sending the taste-coding information to the brain. The taste buds are located on the surface of the tongue within taste papillae. On the front and edges of the tongue, there are slightly larger mushroom-shaped fungiform papillae (reddish spots). There are over 100 on each side. These papillae contain from two to four taste buds each [7,8]. Along the sides of the tongue, about two-thirds of the way back from the tip to the root several parallel grooves. These are the foliate papillae and each contains several hundred taste buds. At the back of the tongue there are the large button-shaped bumps arranged in an inverted-V. These are circumvallate papillae, which also contain several hundred taste buds. Frequency counts of taste buds show that people with higher taste sensitivity tend to possess more taste buds [9]. Saliva plays an important part in taste function, both as a carrier of sapid molecules to the receptors and because it contains substances capable of modulating taste receptors. Saliva contains sodium and other cations, bicarbonate capable of buffering acids, and range of proteins and mucopolysaccharides that give it its slippery and coating properties.
26.2.5 The Chemical/Trigeminal Sense The chemical sense corresponds to a pain response through stimulation of the trigeminal nerve. This effect is produced by chemical irritants such as ginger and capsaicin (from chili), of which both give a heat response, and chemicals such as menthol and sorbitol, which give a cooling response. Astringency is a chemically induced complex of tactile sensations.
26.2.6
The Sense of Hearing
Sound emission from crisp and crunchy foods has been shown to be of great importance in the perception of their texture (e.g., [10]) and to form a basis for discrimination of crisp and crunchy foods.
26.3 The Use of Sensory Descriptors In analytical sensory studies, a trained panel objectively describes the attributes of products [11,12]. The criteria for specific sensory descriptors, the key aspects of the words, which should be considered are: (1) that the descriptors should be orthogonal; (2) that they should be based on underlying structure if it is known; (3) that the descriptors should be based on a broad reference set; and (4) that they should be precisely defined, and finally, that they should be “primary” rather than “integrated.” When developing a set of sensory descriptors that can be used for sensory characterization of a product, the process is split up in a qualitative and a quantitative part. The qualitative part consisted of collecting descriptive words for appearance, odor, taste, and texture. First of all assessors suggested words and then words from literature are added to the vocabulary. The main point of the qualitative part is to group similar words in a mental map by the assessors with help of the panel leader. A verbal explanation of the descriptors is developed in cooperation with both the panel leaders and the panel. References of the different attributes are found. If it is necessary to reduce the vocabulary further multivariate data analysis can be used. In the quantitative part, the panel is trained in use of the scale that is chosen for the sensory analysis.
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In a study of Green-Petersen et al. [13] they performed a sensory profile of the most common chilled and frozen salmon products available to consumers on the Danish market. The sensory profiling was made on 12 salmon products, varying in salmon species, origin, storage method, and time. Samples stored in ice between 7 and 16 days, frozen for 1 month or stored in modified atmosphere for 5 days all had sensory profiles dominated by sea/seaweed odor, juicy and oily texture, fresh fish oil, sweet, and mushroom flavor. The list of words from this study in the qualitative part of developing the set of sensory descriptors is shown in Table 26.1.
Table 26.1 Words from the First Session in the Qualitative Part in Developing a Set of Sensory Descriptors Used for Sensory Profiling of Salmon Odor Green grass
Cucumber
Sea
Seaweed
Amine/farmyard
Sour/sour socks
Rancid
Sourish
Sickly sweet
Warm milk
Sweet
Wet dog
Caramel
Burnt
Hay/dry grass
Pleasant farmyard
Sweetness/ fermented grass
Bear and bread
Mushroom
Forest floor/earthy
Bright/dull
Slimy surface
Shiny
Pearl-shiny
Discoloration
Coagulated protein
Moisture in the bowl
Orange oil droplets
Oily
Salmon
Orange
Reddish
Warm milk
Sweetness
Fish oil/fresh nuts
Nutty
Mushroom
Sea
Sourish
Sickly sweet
Hey
Amine/farmyard
Rancid
Cooked potato
Green
Sour
Bitter
Chemical
Metal
Salty
Mealy taste
Cooked egg white
Cream
Fermented green grass
Brown soap
Cabbage/broccoli
Fiber
Watery
Soft/firm
Dry/juicy
Fatty
Oily
Loos flakes
Flaky (firm)
Sticky
Fibrous
Grainy
Mealy
Appearance
Taste/Flavor
Texture
Source: Green-Petersen, D.M.B. et al., J. Sens. Stud., 21, 415, 2006.
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As it can be seen the first list of words consisted of 20 words for odor, 12 for appearance, 24 for taste/flavor, and 12 for texture. Based on the criteria that the words should be relevant and discriminate clearly between the salmon samples, they should be nonredundant, and be cognitively clear to the assessors, the words were reduced and the selected descriptors for the sensory profiling are shown in Table 26.2.
Table 26.2
Sensory Descriptors Used for Sensory Profiling of Salmon
Descriptor
Description
Odor Sea/seaweed
Fresh seaweed, fresh sea
Sourish
Acidic, fresh citric acid
Sweet
Sweet
Rancid
Rancid fish, paint, varnish
Sour
Sour dishcloth/sour sock
Appearance Discolored
Brown or yellow spots, dark areas
Salmon color
Evaluated with a SalmoFan ruler (Roche)a
Texture Juicy
The sample’s ability to hold water after 2–3 chews
Firm
Force required to compress the sample between the molars
Oily
Amount of fat coating in the mouth
Flavor Fresh fish oil
Fresh oil, fresh green hazelnut
Sweet
Sweet, warm milk
Sourish
Acidic, fresh citric acid
Cooked potatoes
Cooked peel potatoes
Mushroom
Mushroom flavor
Rancid
Rancid fish, paint, and varnish
Salt
Salt
Source: Green-Petersen, D.M.B. et al., J. Sens. Stud., 21, 415, 2006. a
Evaluated with a SalmoFan ruler (Roche) on a interval scale from 20 to 34, where 20 is more light salmon color (pink) than 34.
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◾ 487
26.4 Sensory Descriptors in Seafood Criteria for selection of attributes, which can discriminate between the samples, have to be relevant for the specific seafood, must discriminate clearly between the samples, must be nonredundant, and be cognitively clear to the assessors. To see if these demands are fulfilled, the sensory data can be analyzed concerning the signal to noise relation for each assessor and attribute. The signal to noise analysis can be evaluated with multivariate data analysis [14]. To be cognitively clear and to make reference samples it is good to have a definition of the sensory attribute. Reference samples for the different attributes can be found not only in seafood samples, but also in other commodities such as vegetables, e.g., cucumber and boiled potato. Another example is the sensory attribute warm milk, where only heated and not boiled milk can be used, because when boiling milk a sulfurous odor is developed.
26.4.1 Appearance Appearance can be color/discolor and texture properties such as flaky or gaping. Flaky is assessed by pressing on the sample with a fork; if the muscle separates in flakes, the sample has a high intensity of flakiness. The fish is gaping when the muscle splits up in factions or chaps. Gaping can be seen both in raw and heat-treated fish muscle. For assessing color, it is helpful to use standards, such as the natural color system (NCS®) or for salmon the SalmoFan™ lineal from Roche. A description of the attributes is given in Table 26.3.
Table 26.3
Sensory Descriptors for Appearance
Attribute
Definition
References
Beige
Intensity of beige color
[15]
Brownish
Light brown
[16]
Brown
The brownness of the meat near the head (cross-section at cut end) of cooked shrimp
Coagulated protein
Whitish form on top of and beside sample
[16]
Amount of coagulation that appears on the surface of the salmon steak
[15]
Color intensity and shade
1 = white/yellow, 7 = strong red
[17]
Crumbly
Crumbliness of steak when cut with a knife
[15]
Discoloration
Uneven color distribution
[18]
Discolored
Brown or yellow spots, dark areas
[13]
Discolored
Not the natural color
[19]
Fat droplets in water
Quantity and size of fat droplets in the liquid
[18] (continued)
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Table 26.3 (continued)
Sensory Descriptors for Appearance
Attribute Flacky
Definition
References
Visible flakiness of steak when cut with a knife
[15]
Tissue parts into flakes by pressing with fork
[16]
Tissue parts into flakes by pressing with fork
[19]
Grayish
Light gray
[16]
Glossiness
The amount of light reflected from the meat, from dull to glossy
[20]
Hue
From red = 1 to yellow = 9
[21,22]
Yellow = 1, red = 9
[23]
Juicy appearance
Amount of juice that has seeped onto the plate from the salmon
[15]
Moist appearance
Visible moistness of steak when cut with a knife
[15]
Orange
Intensity of orange color in the uncut steak
[15]
Paleness
Intensity of paleness in the uncut steak
[15]
Peach
Intensity of peach color in the uncut steak
[15]
Pink
Intensity of pink color in the uncut steak
[15]
Red/orange
The redness of the surface
[20]
Separation
Oiliness of the juice seeping out of the salmon
[15]
Shiny
Gloss of tissue caused by oil
[16]
Whiteness
Intensity of white color in the uncut steak
[15]
Whitish
Not totally white
[16]
Yellow water
Degree of yellow water liquid present
[18]
Note: Only references where the evaluation of the property is described are included in the table.
26.4.2 Odor The odor has often a higher intensity than flavor in seafood. A description of the odor attributes is given in Table 26.4.
26.4.3 Taste/Flavor A description of the attributes for taste/flavor and aftertaste is given in Table 26.5.
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◾ 489
Table 26.4 Sensory Descriptors for Odor Attribute
Definition
References
Acid
Vinegar
[24]
Amine
A solution of trimethylamine
[25]
Urine
[24]
Fishy/trimethylamine
[19]
Bacon
Odor corresponding to the product
[25]
Blue cheese
Musty
[24]
Boiled corn
Sweet aroma in boiled corn
[26]
Boiled egg
Smell of boiled egg
[26]
Boiled prawn
Typical aroma in boiled prawn
[26]
Butter
Caramel
[24]
Cabbage
Gas/garlic
[24]
Canned tuna
Aroma of canned tuna
[27]
Cardboardy
Marine fish off-odor notes
[28]
Cheese
Sour feet
[24]
Cold ash
Odor of ash once the fire is out
[25]
Cooked fish
Aroma of cooked fish
[26]
Typical aroma of cooked fish
[27]
Cooked potato
As newly cooked potato
[19]
Cucumber-like
Grated cucumber
[28]
Earthy
Intensity of any earthy/peaty odor
[15]
Farmyard
Intensity of manure/cow-dung odor
[15]
Fat
Fresh fish oil, unripe hazelnut
[29]
Fish oil
Smell of canned mackerel or sardine
[27]
Fish oil
“Fatty” odor, herring oil, and typical smell of fresh herring
[30,31]
Fish oil
Intensity of oily (fish oil) odor
[15]
Fish/herring oil
“Fatty” odor, herring oil, and typical smell of fresh herring
[30,31]
Fishy
Smell of raw not fresh fish
[27] (continued)
490
◾ Handbook of Seafood and Seafood Products Analysis Table 26.4 (continued)
Sensory Descriptors for Odor
Attribute
Definition
References
Fishy
Trimethylamine crystals (marine fish off-odor notes)
[28]
Fresh odor
The typical odor of rainbow trout, an element of fresh, and slightly acidulous odor intensifies the freshness
[32]
Fried chicken
Aroma of fried chicken
[27]
Green
Fresh green odor of soybean milk
[27]
Green aroma
Freshly cut grass
[24]
Grilled fish
Aroma of grilled fish, roasted fish oil
[27]
Ham
Cooked meat
[24]
Herring
Odor corresponding to the product
[25]
Hydrogen sulfide
Egg
[24]
Irritate
Ammonia-like
[26]
Marine/ seaweed
Fresh marine
[19]
Metallic
Warm metal, blood
[30,31]
Rancid
Rancid fish, paint, and varnish
[13,30,31]
Rotten seaweed
Putrefied seaweed
[19]
Sea/seaweed
Fresh seaweed, fresh sea
[13]
Sickly sweet
Sickly sweetness
[19]
Smokiness
Intensity of smoky odor
[29]
Sour
Sour caused by putrefaction
[19]
Sour dishcloth/sour sock
[13]
Sourish
Acidic, acetic acid, and citric acid
[13,29–31]
Sweet
Sucrose-like
[29]
Sweet
[13,30,31]
As a wet dog
[19]
Wet dog
Note: Only references where the evaluation of the property is described are included in the table.
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◾ 491
Table 26.5 Sensory Descriptors for Flavor and Aftertaste Attribute
Definition
References
Acidulous
Fruit-acid-like flavor
[33]
Amine
Fishy/trimethylamine
[19]
Bitter
Quinine-/or caffeine-like
[16,19,20,29]
Chicken-like aftertaste
Intensity of chicken-like aftertaste
[15]
Chemical
Aromatic associated with lotion and cleanser
[34]
Cooked potato
As newly cooked peeled potatoes
[13,19]
Cooked shrimp
The flavor associated with cooked shrimp
[20]
Dried sea mussel
Aromatics associated with dried sea mussel
[34]
Dried shrimp
Aromatics associated with crustacean such as dried shrimp
[34]
Earthy aftertaste
Intensity of earthy aftertaste
[15]
Earthy flavor
Intensity of any earthy/peaty flavor
[15]
Farmyard flavor
Intensity of manure/cow-dung flavor
[15]
Fatty flavor
Impression of fatness perceived as aromatic notes and mouth coating film
[35]
Fishy flavor
Intensity of any other fish-like flavors
[15]
Fresh fish oil
Fresh oil, fresh green hazelnut
[13]
Fresh taste
The typical taste of rainbow trout. In this investigation, the typical taste was scored after the sample had been masticated five times. As element of fresh, slightly acidulous taste intensifies the freshness
[32]
Herring
Typical taste of fresh herring
[30,31]
Iodine aftertaste
Aftertaste associated with the chemical iodine
[20]
Marine
Fresh marine
[16]
Metallic
Flavor associated with metal
[18]
Warm metal, blood
[19,29–31]
Metallic aftertaste
Intensity of metallic aftertaste
[15]
Monosodium glutamate (MSG)
Fundamental taste sensation of which monosodium glutamate or other nucleotides are typical
[34]
Mushroom
Raw mushroom flavor
[13,19] (continued)
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Table 26.5 (continued)
Sensory Descriptors for Flavor and Aftertaste
Attribute
Definition
References
Musty
Flavor associated with damp earth
[18]
Oily
Intensity of fish oil flavor
[15,18]
Fresh fish oil, unripe hazelnut
[29]
Oily aftertaste
Intensity of fish oil aftertaste
[15]
Rancid
Paint, varnish
[30,31]
The atypical taste associated with oxidized fat
[32]
Rancid fish, paint, and varnish
[13,30,31]
Salmon-like flavor
Intensity of distinctive salmon-like flavor
[15]
Salty
Intensity of salt-like flavor
[15,20]
Typical taste of salt, seawater
[29–31]
The taste on the tongue associated with sodium ions
[34,35]
Smokiness
Intensity of smoky flavor
[15,29]
Sourish
Acidic, acetic acid, and citric acid
[13,30,31]
Sourish like in fruit or as fresh fruit acids
[19,29]
Sweet as in warm milk
[13,30,31]
Sucrose-like
[16,19,20,29,34]
Sweet/fresh
Characteristic flavor of cooked Arctic char fillets
[18]
Time
Time when aftertaste starts
[15]
Sweet
Note: Only references where the evaluation of the property is described are included in the table.
26.4.4 Texture Texture is by definition a sensory parameter and only a human being can perceive, describe, and quantify texture adequately. However, texture is very difficult to evaluate due to its complexity. In many cases, several terms are used to describe the same characteristics. In other cases, the same term is used to describe several characteristics. Moreover, the same word may have different meanings to different people [36]. Therefore, the use of sensory texture profiling requires highly trained assessors [6,37]. Sensory perception of texture has been thoroughly reviewed by Jack et al. [38]. The best way to get a “full picture” of the texture is by the Texture Profile Method [39–42]. A texture profile is defined as the sensory analysis of the texture complex of a food in terms of its mechanical, geometrical, and compositional (fat and moisture) characteristics, the degree of each trait present, and the order in which they appear from the first bite through complete mastication [43]. In Table 26.6 there is a long list of words used in the literature for describing texture properties.
Sensory Descriptors Table 26.6
◾ 493
Sensory Descriptors for Texture
Attribute Adhesiveness
Definition
References
Degree to which the sample sticks to the mouth surface
[44]
Force required to remove the material that adheres to the mouth during the normal eating process
[45]
Astringency
The feeling which shrivels the tongue-associated with tannin or alum
[34]
Chewiness
The time required to masticate the sample to a consistency acceptable for swallowing
[20]
Cohesiveness
The extent to which a material can be deformed before it ruptures
[46]
Degree to which the sample deforms before it ruptures during a bite between the molar teeth
[44]
Degree to which a substance is compressed between the teeth before it breaks
[45]
Crispness
The amount of force exerted during first incisor bite that generates a high pitched sound
[20]
Elasticity
The ability of the material to return to its original shape after deformation. Judged by compressing the substance slightly between the molars, or between the tongue and the palate, and noting to what extent the material returns to its original shape
[46]
Elasticity
The ability of the sample to regain its form after the first compression by the molars
[30,31]
Fatty mouth feel
Amount of fat coating the mouth surfaces after three chews
[30,31]
Fibrous
The presence of individual muscle fibers in the shrimp meat
[20]
Firm
The force required to compress between the tongue and the palate (first bite/first chew)
[19]
Force required to compress the sample between the molars
[13]
The effort to bite through the fish sample with the front teeth
[47,48]
The force required to compress the sample between the molar teeth
[30,31,44]
The force required to compress the material between the molars, or between the tongue and the palate
[46]
The amount of force needed to deform the head-end of the shrimp meat by first biting through skin with incisors, then chewing with molars (skin toward molars)
[20]
Firmness
(continued)
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Table 26.6 (continued)
Sensory Descriptors for Texture
Attribute
Definition
References
Grainy
The amount of small, rounded particles
[19]
Gritty
Heterogeneity. Amount of particles in the sample after three chews
[30,31]
Hardness
Resistance to breakdown on chewing to a state suitable for swallowing
[46]
Force required to compress a substance between molar teeth (in the case of solids) or between the tongue and the palate (semisolids)
[45]
The perceived force required to compress the sample using the molar teeth
[49]
Chewing with molars
[48]
Force required to bite the shrimp between second and third segment with incisors
[50]
Compress the sample between the molar teeth and the release pressure
[51]
Amount of wetness
[19]
The sample’s ability to hold water after two to three chews
[13,30,31]
The amount of moisture to masticate the sample to a consistency acceptable for swallowing
[20]
Water release during chewing
[35]
Oiliness
Degree to which oil is perceived after chewing
[29]
Oily
Amount of fat coating in the mouth
[13]
Sliminess
The feeling of a slimy film in the mouth
[20]
Soft
Resistance to a very slight opening and shutting of the jaws
[52]
Force required to compress the samples
[16]
Degree to which a product returns to its original shape once it has been compressed between the teeth
[45]
Degree to which the sample rapidly returns to its original shape after a partial deformation between the molar teeth
[44]
Resistance to breakdown in substructures when compressing between the tongue and the palate
[52]
Juicy
Juiciness
Springiness
Tenderness
Note: Only references where the evaluation of the property is described are included in the table.
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◾ 495
References 1. ISO 3972. Sensory analysis—Methodology—Method of investigating sensitivity of taste. Reference number ISO 3972: 1991 (E). 2. ISO 8586-1. Sensory Analysis—General Guidance for the Selection, Training and Monitoring of Assessors—Part 1: Selected Assessors. International Organization for Standardization, Geneva, Switzerland, 1993. 3. ISO/DIS 13299.2. Sensory analysis—Methodology—Guidance for establishing a sensory profile. ISO, Denmark, 1998. 4. Mebs SL and Nathans J. Absorption spectra of human cone pigments. Nature, 356, 433, 1992. 5. Mebs SL and Nathans J. Role of hydroxyl-bearing amino acids in differently tuning the absorption spectra of the human red and green cone pigments. Photochem. Photobiol., 58, 706, 1993. 6. Meilgaard M, Civille GV, and Carr BT. Sensory Evaluation Techniques, 4th edn., CRC Press Ltd., London, U.K., 2006. 7. Arvidson K. Location and variation in number of taste buds in human fungiform papillae. Scand. J. Dental Res., 87, 435, 1979. 8. Miller IJ and Bartoshuk LM. Taste perception, taste bud distribution and spatial relationships. In T.V. Getchell, R.L. Doty, L.M. Bartoshuk, and J.B. Snow, eds. Smell and Taste in Health and Disease. Raven, New York, p. 205, 1991. 9. Bartoshuk LM, Duff y VB, and Miller IJ. PTC/PROP tasting: Anatomy, psychophysics and sex effects. Physiol. Behav., 56, 1165, 1994. 10. Vickers ZM. Sound perceptions and food quality. J. Food Qual., 14(1), 87, 1991. 11. ISO 11035. Sensory Analysis—Identification and Selection of Descriptors for Establishing a Sensory Profile by a Multidimensional Approach. International Organization for Standardization, Geneva, Switzerland, 1994. 12. ISO 5492. Sensory Analysis—Vocabulary. International Organization for Standardization, Geneva, Switzerland, 1992. 13. Green-Petersen DMB, Nielsen J, and Hyldig G. Sensory profiling of the most common salmon products on the Danish market. J. Sens. Stud., 21, 415, 2006. 14. Thybo AK and Martens M. Analysis of sensory assessors in texture profiling of potatoes by multivariate modelling. Food Qual. Prefer., 11, 283, 2000. 15. Farmer LJ, McConnell JM, and Kilpatrick DJ. Sensory characteristic of farmed and wild Atlantic salmon. Aquaculture, 187, 105, 2000. 16. Warm K, Nielsen J, and Hyldig G. Sensory quality criteria for five fish species. J. Food Qual., 23, 583, 2000. 17. Sivertsvik M, Rosnes JT, Vorre A, Randell K, Ahvenainen R, and Bergslien H. Quality of whole gutted salmon in various bulk packages. J. Food Qual., 22, 387, 1999. 18. Ginés R, Valdimarsdottir T, Sveinsdottir K, and Thorarensen H. Effect of rearing temperature and strain on sensory characteristics, texture, colour and fat of Arctic charr (Salvelinus alpinus). Food Qual. Prefer., 15, 177, 2004. 19. Larsen E, Hyldig G, Dalgaard P, Bech AC, and Østerberg C. Consumers and experts responses to fresh cod fillets. In Luten, J.B., Oehlenschlager, J., and Ólafsdóttir, G. (eds.) Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen Academic Publ., Wageningen, The Netherlands, 2003, p. 345. 20. Erikson MC, Bulgarelli MA, Resurreccion AVA, Vendetti RA, and Gates KA. Sensory differentiation of shrimp using a trained descriptive analysis panel. LWT, 40, 1774, 2007. 21. Einen O and Skrede G. Quality characteristics in raw and smoked fillets of Atlantic salmon, Salmo salar, fed high-energy diets. Aquacul. Nutr., 4, 99, 1998.
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22. Hemre G-I, Karlsen Ø, Eckhoff K, Tveit K, Mangor-Jensen A, and Rosenlund G. Effect of season, light and diet on muscle composition and selected quality parameters in farmed Atlantic cod, Gadus morrhua L. Aquacul. Res., 35, 683, 2004. 23. Rørå AMB, Kvåle A, Mørkøre T, Rørvik KA, Stein SS, and Thomassen MS. Process yield, colour and sensory quality of smoked Atlantic salmon (Salmo salar) in relation to raw material characteristics. Food Res. Int., 31, 601, 1998. 24. Stohr V, Joffraud JJ, Cardinal M, and Leroi F. Spoilage potential and sensory profile associated with bacteria isolated from cold-smoked salmon. Food Res. Int., 34, 797, 2001. 25. Cardinal M, Gunnlaugsdottir H, Bjoernevik M, Ouisse A, Vallet JL, and Leroi F. Sensory characteristic of cold-smoked Atlantic salmon (Salmo salar) from European market and relationships with chemical, physical and microbiological measurements. Food Res. Int., 37, 181, 2004. 26. Morita K, Kubota K, and Aishima T. Sensory characteristics and volatile components in aroma of boiled prawns prepared according to experimental designs. Food Res. Int., 34, 473, 2001. 27. Morita K, Kubota K, and Aishima T. Comparison of aroma characteristics of 16 fi sh species by sensory evaluation and gas chromatographic analysis. J. Sci. Food Agricul., 83, 289, 2003. 28. Hong LC, Leblanc EL, Hawrysh ZJ, and Hardin RT. Quality of Atlantic mackerel (Scomber scombrus L.) fillets during modified atmosphere storage. J. Food Sci., 61, 646, 1996. 29. Vogel BF, Ng YY, Hyldig G, Mohr M, and Gram L. Potassium lactate combined with sodium diacetate can inhibit growth of Listeria monocytogenes in vacuum-packed cold-smoked salmon and has no adverse sensory effects. J. Food Prot., 69, 2134, 2006. 30. Nielsen D, Hyldig G, Nielsen HH, and Nielsen J. Sensory properties of marinated herring (Clupea harengus)—Influence of fishing ground and season. J. Aquat. Food Prod. Technol., 13, 3, 2004. 31. Nielsen D, Hyldig G, Nielsen J, and Nielsen HH. Sensory properties of marinated herring (Clupea harengus) processed from raw material from commercial landings. J. Sci. Food Agricul., 85(1), 127, 2005. 32. Johansson L, Kiessling A, Kiessling K-H, and Berglund L. Effects of altered ration levels on sensory characteristics, lipid content and fatty acid composition of rainbow trout (Oncorhynchus mykiss). Food Qual. Prefer., 11, 247, 2000. 33. Einen O and Thomassen MS. Starvation prior to slaughter in Atlantic salmon (Salmo salar). II. Muscle composition and evaluation of freshness, texture and colour characteristics in raw and cooked fillets. Aquaculture, 169, 37, 1998. 34. Kang M-W, Chung S-J, Lee H-S, Kim Y, and Kim K-O. The sensory interactions of organic acids and various flavors in Ramen soup systems. J. Food Sci., 72(9), 639, 2007. 35. Turchini GM, Moretti VM, Mentasi T, Orban E, and Valfré F. Effects of dietary lipid source on fillet chemical composition, flavour volatile compounds and sensory characteristics in the freshwater fish tench (Tinca tinca L.). Food Chem., 102, 1144, 2007. 36. Szczesniak AS. Classification of textural characteristics. J. Food Sci., 28, 385, 1963. 37. Barroso M, Careche M, and Borderías AJ. Quality control of frozen fish using rheological techniques. Trends Food Sci. Technol., 9, 223, 1998. 38. Jack FR, Paterson A, and Piggott JR. Perceived texture: Direct and indirect methods for use in product development. Int. J. Food Sci. Technol., 30, 1, 1995. 39. Johnson EA, Peleg M, Sawyer FM, Segars RA, and Cardello A. Mechanical methods of measuring textural characteristics of fish flesh. Refrigeration Sci. Technol., 4, 93, 1981. 40. Bourne MC. Texture profile analysis. Food Technol., 22, 62, 1978. 41. Breene WM. Application of texture profile Analysis to instrumental food texture evaluation. J. Texture Stud., 6, 53, 1975. 42. Friedman HH, Whitney JE, and Szczesniak AS. The texturometer—A new instrument for objective texture measurement. J. Food Sci., 28, 390, 1963. 43. Brandt MA, Skinner EZ, and Coleman JA. Texture profile method. J. Food Sci., 28, 404, 1963. 44. Hamann DD and Webb NB. Sensory and instrumental evaluation of material properties of fi sh gels. J. Texture Stud., 10, 117, 1979.
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45. Sánchez MT. Food texture: Concept and measurement. Alimentaria, 272, 29, 1996. 46. Borderias AJ, Lamua M, and Tejada M. Texture analysis of fish fillets and minced fish by both sensory and instrumental methods. J. Food Technol., 18, 85, 1983. 47. Hurling R, Rodell JB, and Hunt HD. Research note: Fiber diameter and fish texture. J. Texture Stud., 27, 679, 1996. 48. Iseya Z, Sugiura S, and Sarki H. Procedure for mechanical assessment of texture change in dried fish meat. Fisheries Sci., 62, 772, 1996. 49. Cardello AV, Sawyer FM, Maller O, and Digman L. Sensory evaluation of the texture and appearance of 17 species of North Atlantic fish. J. Food Sci., 47, 1818, 1982. 50. Gundavarapu S, Hung Y-C, and Reynolds AE. Consumer acceptance and quality of microwavecooked shrimp. J. Food Qual., 21, 71, 1998. 51. Leblanc RJ and Leblanc EL. Effect of retort process time on the physical and sensory quality of canned lobster (Homarus americanus) meat. J. Food Process. Preserv., 14, 345, 1990. 52. Schubring R and Oehlenschläger J. Comparison of the ripening process in salted Baltic and North Sea herring as measured by instrumental and sensory methods. Z. Lebensm. U. Forsch. A, 205, 89, 1997.
Chapter 27
Sensory Aspects of Heat-Treated Seafood Grethe Hyldig Contents 27.1 Introduction ................................................................................................................. 500 27.2 Seafood ........................................................................................................................ 500 27.2.1 The Anatomy of Shellfish ................................................................................ 500 27.2.2 The Anatomy of Fish ...................................................................................... 500 27.2.3 Difference between Fish Species and between Individuals ...............................501 27.2.4 Handling of Seafood ...................................................................................... 502 27.3 Sensory Analysis of Seafood ......................................................................................... 502 27.3.1 Assessors ......................................................................................................... 503 27.3.2 Sessions........................................................................................................... 503 27.3.2.1 Test Room ...................................................................................... 503 27.3.2.2 Scales .............................................................................................. 504 27.3.2.3 Between Samples ............................................................................ 504 27.3.3 Preparation of Heat-Treated Seafood Samples................................................. 505 27.3.3.1 Fish Samples Heat Treated in Oven ................................................ 505 27.3.3.2 Fish Samples Cooked in Water Bath ............................................... 506 27.3.3.3 Fish Broth ....................................................................................... 506 27.3.3.4 Prawns and Shrimps ....................................................................... 506 27.3.3.5 Prawns Broth .................................................................................. 506 27.3.4 Sensory Attributes .......................................................................................... 506 27.4 Examples of Sensory Characterization of Seafood ........................................................ 507 References ................................................................................................................................510 499
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27.1 Introduction Sensory analysis plays an important role in quality control and quality assurance in the fish sector. To a certain extent sensory analysis is also used in product development and optimization. Both objective and subjective sensory testing are used. The objective tests include discriminative (triangle test, forced choice) and descriptive (profiling, structured scaling) sensory tests. Both groups of test are analytical measurements of the intrinsic quality of the product, whereas the affective (subjective test) methods are used for consumer testing and to measure the attitude and emotional response of the consumer toward the product. All these methods are used in the seafood chain. Descriptive sensory analysis such as profiling can be used to characterizing “postmortem changes” of all kind of seafood. Descriptive testing provides quantitative data and can both be very simple and used for assessment of a single attribute, or more complex and give a total characterization of sensory quality. Several conditions are unique for objective sensory profile of seafood and will be described in the following text.
27.2 Seafood 27.2.1
The Anatomy of Shellfish
Shellfish is a broad term for all aquatic animals that have a shell of some kind. Both saltwater and freshwater invertebrates are considered shellfish. “Shellfish” is a misnomer, because these invertebrates are definitely not fishes. The term “finfish” is sometimes used to distinguish ordinary (vertebrate) fish from shellfish. Shellfish is separated into two basic categories—crustaceans and mollusks. Crustaceans have elongated bodies and jointed soft (crustlike) shells. The crustacean family includes crabs, crayfish, barnacles, lobster, prawns, and shrimps. Mollusks are invertebrates with soft bodies covered by a shell of one or more pieces. Mollusks are further divided into gastropods, bivalves, and cephalopods. Gastropod is often referred to as a “univalve,” a gastropod can be any of several mollusks with a single (univalve) shell and single muscle. Among the more common gastropods are the abalone, limpet, periwinkle, snail, and whelk. Bivalve is any soft-bodied mollusk, such as a clam, scallop, oyster, or mussel, which has two shells hinged together by a strong muscle. Cephalopod is a class of mollusk that includes the octopus, squid, and cuttlefish. It is the most biologically advanced of the mollusks. All cephalopods share two common characteristics—tentacles attached to the head, and ink sacs, which they use to evade their predators. The sensory quality of shellfish can vary considerably according to season, fishing ground, farming conditions, stress, and the harvesting conditions.
27.2.2 The Anatomy of Fish Fish (vertebrae) have a backbone composed of segments (vertebrae)—and a cranium covering the brain. The backbone runs from the head to the tail. The vertebrae are extended dorsally to form neural spines, and in the trunk region, they have lateral processes that bear ribs. The ribs are cartilaginous or bony structures in the connective tissue between the muscle segments. Usually, there are also a corresponding number of false ribs or “pin bones” extending more or less horizontally into the muscle tissue. These bones have to be removed if a bone free fillet is the goal.
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Fish has muscle cells running in parallel and connected to sheaths of connective tissue (myocommata) anchored to the skeleton and the skin. The bundles of parallel muscle cells are called myotomes. The myocommata run in an oblique pattern perpendicular to the long axis of the fish, from the skin to the spine. This anatomy is ideally suited for the flexing muscle movements necessary for swimming through the water. All muscle cells extend the full length between two myocommata, and run parallel with the longitudinal direction of the fish. The muscle mass on each side of the fish makes up the fillet, of which the upper part is termed the dorsal muscle and the lower part the ventral muscle.
27.2.3 Difference between Fish Species and between Individuals The variation in the chemical composition of fish is closely related to feed intake, migratory swimming, and sexual changes in connection with spawning. Fish will have starvation periods for natural or physiological reasons (such as migration and spawning) or due to external factors such as shortage of food. Fish raised in aquaculture may also show variation in chemical composition, but in this case, several factors can be controlled, and the chemical composition may be predicted. To a certain extent, the fish farmer is able to design the fish by selecting the farming conditions. It has been reported that factors such as feed composition, environment, fish size, and genetic traits all have an impact on the composition and quality [1–3]. There is not only difference between species, but also a considerable variation between individuals. These variations must be taken into account when setting up experiments using sensory analysis to characterize fish and fish products. In farmed salmon (Salmo salar), e.g., the lipid content can differ from 10% to 19% lipid in salmon from the same farm. Moreover, for herring there is a wide variation in lipid content within catches. A single catch can contain herring (Clupea harengus) with lipid content ranging from 1% to 25%. This variation is due to heterogeneity caused by mixing between stocks [4]. The lipid fraction is the component showing the greatest variation. Often, the variation within a certain species will display a characteristic seasonal curve with a minimum around the time of spawning. Although the protein fraction is rather constant in most species, variations have been observed such as protein reduction occurring in salmon during long spawning migrations [5,6] and in Baltic cod during the spawning season [7]. Fish is often divided into lean and fatty fish. A possible method for discriminating between lean and fatty fish species is to term fish that store lipids only in the liver as lean, and fish storing lipids in fat cells distributed in other body tissues as fatty fish. Typical lean species are the bottom-dwelling ground fish like cod fish and flat fish species. The lipid content of fillets from lean fish is low and stable whereas the lipid content in fillets from fatty species varies considerably. In many species most of the muscle is white or has a light color, but many fish species have a certain amount of dark tissue of a brown or reddish color. The dark muscle is located just under the skin along the body side. A typical pelagic fish as herring will contain ∼25% of dark muscle required for prolonged aerobic muscle activity. The proportion of dark muscle varies with the activity of the fish. The larger dark muscle is found in fish that are more active. There are many differences in the chemical composition of the two muscle type, e.g., higher levels of lipids and myoglobin in the dark muscle. From a technological point of view, the high lipid content of dark muscle is important and can give high intensities of desirable sensory notes, but also a shorter shelf life due to lipid oxidation resulting in rancid and sour notes among others. Demersal species such as cod
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has a white flaky and tender muscle; textural changes such as toughness and chewiness pose the most noticeable problem during storage. The white muscle is used for quick attacks or escapes— not for long destinations. Whether a fish is lean or fatty the actual fat content is important for the technological characteristics postmortem. The changes taking place in fresh lean fish may be predicted from the knowledge of biochemical reactions in the protein fraction, whereas in fatty species the changes in the lipid fractions have to be taken into account. The implication may be that the storage time is reduced due to lipid oxidation, or special precautions have to be taken to avoid this complication. The reddish meat color found in salmon and sea trout does not originate from myoglobin but is due to the red carotenoid, astaxanthin. The function of this pigment has not been clearly established, but it has been proposed that the carotenoid may play a role as an antioxidant.
27.2.4
Handling of Seafood
“Rigor mortis” begins immediately or shortly after death if the fish is starved and the glycogen reserves are depleted, or if the fish is stressed. The technological significance of rigor mortis is of major importance, when the fish is filleted before or in “rigor.” In rigor, the fish body will be completely stiff; the filleting yield will drop significantly, and rough handling can cause gaping in the fillets. If the fillets are removed from the bone “prerigor” the muscle can contract freely and the fillets will shorten following the onset of rigor. Dark muscle may shrink up to 52% and white muscle up to 15% of the original length [8]. If the fish is cooked prerigor the texture will be very soft and pasty. In contrast, the texture is tough but not dry when the fish is cooked in rigor. “Postrigor” the flesh will become firm, succulent, and elastic. In some fisheries, bleeding of the fish is very important as a uniform white fillet is desirable. Discoloration of the fillet may also be a result of rough handling during catch and catch handling while the fish is still alive. Physical rough handling in the net (long trawling time, very large catches) or on the deck (stepping on the fish or throwing boxes, containers, and other items on top of the fish) may cause bruises, rupture of blood vessels, and blood oozing into the muscle tissue (hematoma). Shellfish is harvested and stored alive or shortly after harvesting processed and frozen. Shellfish acts as a filter and therefore contain mud and bacteria; hence, they should be washed in clean seawater as soon as they are harvested. After being washed, shellfish should be packed and kept cool under storage and transport.
27.3
Sensory Analysis of Seafood
Objective descriptive sensory requires a tested and trained panel together with a test room [9,10]. In the sensory method, profiling, a series of attributes will be developed to describe the sensory characteristics describing appearance, smell, taste, and texture (see the previous chapter). The panel must be trained in identification of the attributes and furthermore the panel must be trained in using a scale for describing the intensity of the attributes. In a session, the samples will be served in random order, and between the samples, there will be served something that can be used to clean the mouth.
Sensory Aspects of Heat-Treated Seafood
27.3.1
◾
503
Assessors
When selecting and training assessors for sensory analysis of seafood, it is very important to be remembered that, some people cannot taste rancid flavor, iodine, geosmin, and some have a very low response to cold-storage flavor and rancidity. Moreover, some people are allergic or hypersensitive to different fish proteins, shellfish, or histamine. Special care must be taken to a number of relations. The assessors must not be afraid of fish bones. Some flavors are very special such as iodine (from bromophenols) and muddy (from geosmin or 2-methyl-iso-borneol) must be known to the assessors. Further, there might be considerable differences between the individual seafood, and it can be a challenge to have the homogeneous samples and it is even more complicated to get replicates for a panel of 12. In all cases, the assessors require intensive training and a detailed briefing before each session. Interpretation of the stimulus and response must be trained very carefully in order to receive objective responses. It is very easy—as an example—to give an objective answer to the question: is the fish in rigor (completely stiff ), but more training is needed if the assessor has to decide whether the fish is post- or prerigor. Training is essential as it provides all assessors with a common vocabulary, thus decreasing the risk of different quantitative and qualitative interpretation of descriptors [9,11]. Furthermore, continuous training during long-term projects, preferably using reference material, can reduce the risk of drifting. If the training is interrupted, the assessors might forget descriptor meanings and/ or rating levels with time. The performance of a sensory panel has to be evaluated and calibrated just as any other instrument in the laboratory in order to give reliable results. If this is not the case, the risk of drawing wrong conclusions from the experiment is high. By serving the same product as a reference before each session, assessors can recapitulate the descriptors easily and can recalibrate their evaluations to the same scale. The additional use of coded reference samples served together with the actual samples makes it possible to monitor the performance of the panel. Multivariate data analysis allows for quick calculations and results are easily interpreted with the visual layout [12].
27.3.2 Sessions It is important to serve the heated samples hot and in containers with lids. In this way, the assessors get the full impression of the odor just after opening the lid. When the samples are cooked in plastic bags odor attributes are assessed immediately after opening the vacuum bags, while flavor and texture are evaluated after removing the samples from the bag [3,13]. Each assessor evaluated the samples in replicate and all the samples must be served to each assessor in a randomized order to minimize possible carry-over effects between the samples. The number of samples for each session depends on the number of attributes and the type of the sample. For the cooked samples without any spices added, the assessors can evaluate ∼10 attributes in six samples within a session. If there are many attributes, it is necessary to cut down the number of samples in each session. It is important to give the assessors a short break, 1–3 min, between each sample.
27.3.2.1 Test Room There are international and national standards and guidelines for the design and construction of sensory assessment rooms [9–11,14,15].
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In quality control procedures, special facilities or rooms are preferred for sensory evaluation, but it is not always possible. In industry and auctions, the testing area should adhere to the ISO 8589 standard as closely as possible. ◾ The test room must be a separate room and it must be easy accessible to the assessors. There must be a booth for each assessor. ◾ Temperature must be constantly around 20°C–24°C. ◾ Noise level shall be kept to a minimum; during a session, the assessors must be able to work without any interruption. ◾ Free of any foreign odor. As a minimum, there must not be any waste or other matter, or operation with a strong smell nearby. It must be possible to change the air in the test room. ◾ Testing area must be easy to clean and disinfect. Regular cleaning and disinfecting shall take place. It must be ensured that the cleaning agents used do not leave odors in the testing area. The colors in the test room must be light gray such as NCS-S1002Y-S3002Y. ◾ Lighting is very important. It is preferable to have real daylight according to ISO standard [10] but as a minimum, there should be an intensity of 600–1500 lx/m2. ◾ No eating, drinking, or smoking shall be allowed in the testing area.
27.3.2.2
Scales
Different type of scales can be used, but the most common is the line scale and the nine-point scale. For assessing the color it is helpful if standards are used, such as the natural color system (NCS®). The most commonly used scale (Figure 27.1A) is the 15 cm unstructured line scale, with two anchor points: “little” and “much” of attribute intensity, e.g., placed 1.5 cm and 13.5 cm from the endpoint of the scale [16–23]. An intensity scale from 0 to 100 (Figure 27.1B) is also suggested [24–26]. Here is 0 defined as minimum mark or nothing of the attribute and 100 as the maximum mark of the attribute. Others are using a nonstructured continuous line scale of different length: 8 cm [27], 10 cm [28–30], or 15 cm [31–34]. The nine-point scale (Figure 27.1C) can be either an continuous intensity scales going from 0 (low intensity) to 9 (high intensity) [35,36] or a line scale where the responses are transformed into numbers after assessment, where 1 equals no intensity and 9 equals high intensity [2–3,13,32,37].
27.3.2.3
Between Samples
Different cleaning items can be used to dissipate residual flavors and particles between evaluations depending on the samples. Tap water can be used if it is not too bitter and it should be served at room temperature together with unsalted crackers or crisp bread. The tap water can be filtered through a domestic water filter to remove any extraneous flavors [38], but also distilled water combined with unsalted crackers can be used [39]. If the samples have off-flavor or taint, some acidic solution can be used in combination with a longer break between the samples. Bett et al. [40] used unsalted soda crackers and citric acid (0.03%) solution as “palate cleansers.” Peeled cucumber in slices can be used together or instead of crackers.
Sensory Aspects of Heat-Treated Seafood Little
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505
Much
(A)
(B)
(C)
Figure 27.1 Intensity scales. (A) Unstructured line scale with two anchor points; (B) scale from 0 to 100; (C) nine points going from 0 to 9.
27.3.3 Preparation of Heat-Treated Seafood Samples Sampling for sensory analysis must be as representative as possible and therefore consideration must be given not only to how and where the sample is cut, but also to the effect of the chosen method for heat treatment. The preparation must have a minimal sensory impact of the “innate” characteristics of the samples. Different types of seafood products demand different sample preparation. The optimal amount of seafood prepared for each assessor is a portion size of 30–100 g. All the samples must be marked individually with three-digit or four-digit codes. The samples must be placed in individual containers such as porcelain bowls and covered with porcelain lids. For fat fish species, it is important to consider whether the samples should be with or without the skin. As the dark muscle is right under the skin and if the fillet are skinned some of the dark muscle will be removed. This process can exercise an influence on rancid flavor due to the high lipid content in the dark muscle. If the samples for replicates for each assessor are taken from the same position of each fillet, it is possible to have real replicate and thereby identify if there is any differences between assessors or between fillets from different parts of the fish. If the texture is of no importance, it is possible to make minced fish samples to eliminate the differences between individuals. For shellfish, there can be some special conditions due to sample size. For example, shrimp can be very different in size; some are large enough to be assessed in one “mouthful,” but others are so small that several shrimps are needed for one “mouthful.” Concerning the variation between individuals, it can be an advance to ask the assessors to take three or four shrimps, depending of the size, at a time.
27.3.3.1 Fish Samples Heat Treated in Oven The samples from fish species such as cod (Gadus morhua), salmon (S. salar), and saithe (Pollachius virens) are cut from the loin part in 8 × 4 × 2 cm pieces corresponding to approximately 75 g. Smaller fish species such as rainbow trout (Oncorhynchus mykiss), herring (C. harengus), and plaice (Pleuronectes platessa) require a whole fillet, which gives the samples of ∼40–50 g for plaice and herring and approximately 75–100 g for rainbow trout. The samples are heated, e.g., in a convection oven in their own juice at 100°C to an internal temperature of 70°C.
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Fish Samples Cooked in Water Bath
Fish can also be cooked in a water bath as suggested by Bjerkeng et al. [13], Nortvedt and Tuene [3], Sivertsvik et al. [41], and Hemre et al. [42]; 1.5 cm thick cutlets of salmon (S. salar) and cod (G. morhua) or loin part of Atlantic halibut (Hippoglossus hippoglossus L.) fillets were packaged in diffusion tight plastic bags under vacuum and cooked in water bath at 85°C for 15 min or at 70–72°C for 1 h without salt or other spices.
27.3.3.3
Fish Broth
For very different fish species, it can be an advantage to have a sample preparation where the fish are cut into small pieces and boiled to a broth. This was carried out in the study of Ref. [34]. All the species except loach were gutted, and tuna and swordfish were also skinned. Small fish species such as banded blue-sprat, sardine, loach, pond smelt, and goby were boiled completely, while the others were cut into 3–4 cm lengths before boiling as in ordinary domestic cooking. Each sample (200 g) was placed in a three-necked separable 31 round flask along with 200 mL of boiling water, than heated with refluxing for 30 min by a mantle heater. The fish broth (350 mL) obtained after filtering the boiled mixture through three layers of cotton gauze was divided into 35 mL aliquots in 50 mL glass vials. Fish broth (35 mL) prepared from 20 g fish was placed in a 260 mL disposable plastic cup, covered with a plastic Petri dish, and served to the panelists at 40°C.
27.3.3.4
Prawns and Shrimps
Frozen prawns and shrimps must either be thawed for 1 h at room temperature before cooking or added directly to the boiling water, depending on the size. The cooking time in boiling fresh water is 3 min. After cooking the prawns/shrimps are drained, cooled with cold water, peeled, and held at 7°C–10°C until evaluation [16,31,37].
27.3.3.5 Prawns Broth If a sensory profiling of whole prawn is required, it is possible to make a broth where both shell and the meat are assessed at the same time. Morita et al. [28] thawed frozen prawns (kuruma prawns (Penaeus monodon) and black tiger prawns (P. monodon)) with running water, 190 g meat and/or 30 g shell were cut into 1 cm2 pieces and after adding 400 mL of deionized distilled water, the mixture boiled for 30 min. Broth was obtained by filtering the boiled mixture through three layers of cotton gauze. The broth was divided into 35 mL each and preserved in 50 mL glass vials at −50°C until performing sensory evaluation.
27.3.4 Sensory Attributes The criteria for selection of sensory attributes are that they can discriminate between the samples, are relevant to the specific seafood, they must be nonredundant, and be cognitively clear to the assessors. In the previous chapter, there is a long list of attributes used in the literature.
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27.4 Examples of Sensory Characterization of Seafood Green-Petersen et al. [23] gave an overview of both the sensory properties and differences between the most common salmonids products available on the Danish market. Twelve salmon samples, which differed in storage condition, packaging, and species were used in the experiment. All the samples were obtained from local shops or companies and bought as consumer products. There were nine samples of S. salar, two were modified atmosphere packed (MAP), four were stored in ice, and three were frozen. There were also two frozen samples of Oncohynchus kisutch and one frozen sample of Oncorhynchus keta. The four samples stored in ice had very similar sensory profile described by the attributes sea/seaweed odor, the flavors fresh fish oil, sweet and mushroom, and the texture juicy and oily. Even though the ice storage samples represented different fish farms, different batches, and different storage times in ice (7 or 16 days), they did not observe any clear sensory differences which were in agreement with Sveinsdóttir et al. [20]. The MAP samples stored for 5 days and the ice storage samples had a relatively similar sensory profile. Whereas the MAP packed that was stored for 7 days was rather different, it was rancid and sour. For the frozen samples, the results show that a longer freezing time gave a more soft texture, an increase in discolor and a decrease in sea/seaweed odor. However, they found that freezing only had little influence on flavor. The results indicate that in general, the sensory properties of O. keta and S. salar are fairly similar. Furthermore, the results indicate that the sensory properties of O. kisutch are very different from the sensory properties of O. keta and S. salar. The two samples of O. kisutch had a high intensity of firm texture, rancid flavor, and were discolored, combined with a low intensity of juicy and oily texture, sea/seaweed odor and the flavors sweet, fresh fish oil, and mushroom. The length of starvation before slaughtering can have considerable influence on the sensory attributes. In farmed Atlantic salmon (S. salar), fresh flavor was significantly reduced in groups starved from 30 to 86 days, whereas no significant differences were found among groups starved for 0–30 days. The sensory test was performed on day 13–16 postmortem fish [35]. The content of the feed can influence not only the growth rate, but also the sensory characteristics of the fish. For Atlantic halibut (H. hippoglossus) fed with a diet containing 20% or 39% fat, the sensory test showed that larger fish (2.1–2.7 kg) were characterized by a fresher and more acidic flavor and a more juicy consistency, compared to smaller fish (1.4 kg). The growth of the 20 halibut used in the sensory test showed that high growth was associated with small fish, which achieved higher scores for off-flavor and rancid flavor than the larger halibut with lower growth rate. This does not imply that a high growth rate is a contributing factor to the off-flavor or the rancid flavor of the small fish, but shows that these characteristics are typical for the small fish [3]. The odor for turbot (Psetta maxima) fed with three experimental diets (with 9% (w/w) added lipid–fish oil, soybean oil, or linseed oil) were described as having a fatty fish note, even though the lipid content of turbot muscle was very low. Grass and hay notes were also found to characterize the odor of flesh [43]. There can be large variation in the sensory characteristic of different fish species, but also similarities. Cardello et al. [39] characterized 17 fish species by sensory profiling and by using cluster analyzing of the data they found that the 17 species could be grouped in three major groups. The first group is characterized by primarily low fat, low flavor intensity, white-fleshed fish, and consists of tilefish (Lopholatilus chamaeleonticeps), pollock (P. virens), haddock (Malanogrammus aeglefinus), wolfish (Anarhichas lups), Atlantic cod (G. morhua), cusk (Brosme brosme), white hake (Urophycis tenuis), whiting (Merluccius bilinearis), blackback flounder (Pseudopleuronectes americanus), Atlantic halibut (H. hippoglossus), monkfish (Lophius americanus), and grouper (Mycteroperca microlepis). The second major group consists of the high fat, high flavor intensity, dark-fleshed
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fish, including bluefish (Pomotomus saltatrix), Atlantic mackerel (Scomber scombrus), weakfish (Cynoscion regalis), and striped bass (Morone saxatilis). The third group consists solely of swordfish (Xiphias gladius), but none of the sensory parameters described the swordfish very good. The swordfish was included in another study by Ref. [34]. In this study different saltwater fish, migratory coastal fish, coastal bottom fish, pelagic fish, deep-sea fish, freshwater fish, anadromous fish, and brackish water fish, in total 16 species was sensory characterized. By multivariate data analysis, four groups were observed in a biplot using PCA (principal component analysis). Red sea bream snapper (Pagrus major), common Japanese conger (Conger myiaster), Japanese eel (Anguilla japonica), and freshwater species, i.e., loach (Misgurmus anguillicaudatus), pond smelt (Hypomesus nipponensis), and carp (Cyprinus carpio) were grouped around the flavor “green.” Migratory coastal fish species, i.e., sardine (Sardinops melanosticta), banded blue-sprat (Spratelloides gracilis), and chub mackerel (Scomber japonicus), were located close to “fish oil,” “grilled fish,” “sea breeze,” and “fishy.” Swordfish (Xiphas gladius), sablefish (Anoplopoma fimbria), and chum salmon (O. keta) were located close to “fried chicken.” Slime flounder (Microstmus achne), pacific cod (Gadus macrocephalus), blue-fin tune (Thunnus thynnus), and yellowfin goby (Acanthogobius flavimanus) were grouped around “cooked fish,” “sweet,” “canned tuna,” and “roasted soy sauce.” During ice storage of fish the intensity of the sensory attributes will change. Sensory profiling of ice stored farmed salmon showed that sensory attributes characterizing the salmon (S. salar) on day 1 were seaweed, cucumber, and sourish odor; sweetish, sourish, fish oil; and mushroom flavor and after 22–24 days of storage the salmon were characterized by rancid, sour, amine odor, and rancid flavor. The sensory attributes used in another storage experiment with farmed salmon stored in ice were grouped into “positive sensory parameters” and “negative sensory parameters.” The samples from every second storage day were analyzed. The changes in the sensory attributes indicate that the salmon was approaching the end of acceptable flavor after 20–21 days, when the salmon was characterized by increasing the intensity of sour, amine, and rancid odor and flavor. All positive attributes had a high intensity and were very characteristic for the salmon at the beginning of the storage time, but after 21–22 days of storage they were hardly detectable. It was concluded from the result of the sensory profiling that the shelf life for salmon, where the fish is no longer fit for human consumption, was 20 days in ice [20]. In storage studies with MAP, the development of sensory attributes are different than for ice storage of the same fish species. Hong et al. [17] found that the odor of Atlantic mackerel (S. scombrus L.) during MAP storage changed from day 0 (seaweed, fishy, and rancid) to 7 days of storage (seaweed, cucumber-like, sour, fishy, painty, and rancid) and again from 14 days of storage (seaweed, sour, fishy, and rancid) to 21 days of storage (seaweed, sour, fishy, metallic, and rancid). The rapid development of rancidity in stored muscle from fish such as mackerel, herring, capelin, and bluefish is often attributed to the high level of lipid that these species contain. However, studies by Richards and Hultin [29] indicate that blood-mediated oxidation of washed cod (G. morhua) lipids required ≤0.1% phospholipid to cause rancidity. They also found with longterm, frozen storage of mackerel (S. scombrus) muscle, sensory quality deterioration occurred in spite of no measurable change in fatty acid composition. Manipulation of the environmental salinity affected the flavor of frozen white shrimp (Penaeus vannamei). Since free amino acids are major osmo-effectors in shrimp and also primary flavor producers in marine products, more flavorful shrimp can be produced by acclimation to high environmental salinities [16]. Bak et al. [44] investigated the effect of packaging atmosphere, temperature fluctuation, and light exposure on frost formation, lipid oxidation, discoloration, and meat toughness of
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shell-on-cold-water shrimps (Pandalus borealis) during 12 months of frozen storage. They found that the single most important handling factor affecting shrimp quality was the exposure to oxygen during storage. Rancid flavor was significantly higher for the samples packed in atmospheric air compared to the samples packed in modified air. The effect of light on the rancid flavor of atmospheric air-packed shrimps was significant. The sensory score for rancid flavor for modified air-packed samples did not increase significantly between 9 and 12 months of frozen storage. The sensory evaluation of shrimp meat toughness revealed a clear effect of the packaging method and storage conditions. They found a significantly higher score for toughness for the samples packed in atmospheric air compared to the samples packed in modified air. In addition, storage in light resulted in significantly tougher shrimp meat. The sensory score for toughness was almost constant for the samples packed in modified air for the first 9 months of storage, whereas a significant increase was observed between 9 and 12 months of storage. Erikson et al. [45] investigated different frozen shrimp, which was obtained from commercial distributors. The samples were identified as Gulf brown shrimp (Penaeus aztecus), Gulf white shrimp (Penaeus setiferus), Gulf pink shrimp (Penaeus duorarum), Georgia brown shrimp (P. aztecus), Georgia white shrimp (P. setiferus), Burma black tiger shrimp (P. monodon), Belise white shrimp (P. vannamei or Penaeus stylirostris), Colombia white shrimp (P. vannamei or P. stylirostris), Honduras white shrimp (P. vannamei or P. stylirostris), and Mexican white shrimp (P. vannamei or P. stylirostris). Sensory attributes that distinguish the frozen shrimp was associated with appearance. In the case of Burma black tiger shrimp, distinguishing features were shell darkness and stripe darkness of the raw products and red/orange color of the cooked meat product. Belise white shrimp, were found to have a muted shell color and significantly lower shell and stripe darkness than the other shrimp samples. While the high intensity of brown meat color differentiated the Colombia white shrimp from other shrimp, Georgia white shrimps were unique from the other samples in the high iridescence displayed in the tail of the raw product. Erikson et al. [45] found that iridescence of Georgia white shrimp was nearly twice as much than the other shrimp samples including Gulf white shrimp (the same species of shrimp, but harvested from a different location). Commercial claims stated that Georgian white shrimps are sweet, but Erikson et al. [45] found the highest sweetness intensity in Burma black tiger shrimp. Georgia white shrimps were characterized as having the highest cooked shrimp flavor, but the intensity was not significantly different from Gulf brown, Georgia brown, Gulf pink, or Mexican white shrimp. Erikson et al. [45] also found that notable differences in aroma or basic taste attributes occurred, which could be ascribed to variations in processing applied to the commercially available shrimp samples. For example, old shrimp aroma in the raw product was significantly higher in Mexican white shrimp than other shrimp samples. At the same time, blotchiness ratings were also significantly higher in Mexican white raw and cooked shrimps and they concluded that the shrimp could have been held for some period of time prior to freezing. Another variation in processing that was also perceived by the trained panel was the addition of the additives, sodium tripolyphosphate and sodium bisulfite, in Gulf shrimp. In fact salty intensity in Gulf pink shrimps were nearly twice than those of other shrimp while their sweetness scores were 20% lower than other shrimp. In the investigation, the trained panel found significant differences between fresh and commercially available frozen shrimp (Georgia white shrimp (P. setiferus) and Gulf pink shrimp (P. duorarum)) for 18 of the 30 sensory attributes. In terms of appearance, fresh shrimps were glossier than frozen shrimp for both the raw and cooked products. The most notable change in appearance for the raw product, however, was a loss in tail iridescence. While for cooked products the most notable changes in appearance were loss of red/orange color on the shrimp surface. Frozen shrimp had a higher intensity of cooked shrimp flavor, cooked shrimp aroma, and
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ocean/seawater aroma than fresh shrimp whereas no difference in the old shrimp aroma was found. They also found changes in texture, shrimp that had been frozen were firmer and less juicy than fresh shrimp. Because the frozen shrimp were commercially obtained in this study, the period of time they had been in frozen storage is unknown.
References 1. Reinitz GL, Orme LE, and Hitzel FN. 1979. Variations of body composition and growth among strains of rainbow trout (Slamo gairdneri). Transactions of the American Fisheries Society 108:204–207. 2. Einen O and Skrede G. 1998. Quality characteristics in raw and smoked fillets of Atlantic salmon, Salmo salar, fed high-energy diets. Aquaculture Nutrition 4:99–108. 3. Nortvedt R and Tuene S. 1998. Body composition and sensory assessment of three weight groups of Atlantic halibut (Hippoglossus hippoglossus) fed three pellet sizes and three dietary fat levels. Aquaculture 161:295–313. 4. Nielsen D, Hyldig G, Nielsen HH, and Nielsen J. 2005b. Lipid content in herring (Clupea harengus L.)—Influence of biological factors and comparison of different methods of analyses: Solvent extraction, Fatmeter, NIR and NMR. Lebensmittel-Wissenschaft und-Technologie 38:537–548. 5. Ando S, Hatano M, and Zama K. 1985. Deterioration of chum salmon (Oncorhynchus keta) muscle during spawning migration—1. Changes in proximate composition of chum salmon muscle during spawning migration. Comparative Physiology and Biochemistry 80B:303–307. 6. Ando S and Hatano M. 1986. Biochemical characteristics of chum salmon muscle during spawning migration. Bulletin of the Japanese Society of Fisheries 52:1229–1235. 7. Borresen T. 1992. Quality aspects of wild and reared fish. In Huss HH, Jacobsen M, and Liston J (eds.), Quality Assurance in the Fish Industry; Proceedings of an International Conference, Copenhagen, Denmark August 1991. Elsevier, Amsterdam, the Netherlands, pp. 1–17. 8. Buttkus HJ. 1963. Red and white muscle of fish in relation to rigor mortis. Journal of the Fisheries Board of Canada 20:45–58. 9. ISO 8586-1. 1993. Sensory Analysis—General Guidance for the Selection, Training and Monitoring of Assessors—Part 1: Selected Assessors. International Organization for Standardization, Switzerland. 10. ISO 8589. 1988. Sensory Analysis—General Guidance for the Design of Test Rooms. Reference number ISO 8589:1988(E). 11. ISO 11035. 1994. Sensory Analysis—Identification and Selection of Descriptors for Establishing a Sensory Profile by a Multidimensional Approach. International Organization for Standardization, Switzerland. 12. Nielsen D, Hyldig G, and Sørensen R. 2005c. An effective way to minimize drifting and monitor the performance of a sensory panel during long-term projects—A case study from a project on herring quality. Journal of Sensory Studies 20:35–47. 13. Bjerkeng B, Refstie S, Fjalestad KT, Storebakken T, Røbotten M, and Roem AJ. 1997. Quality parameters of the flesh of Atlantic salmon (Salmo salar) as affected by dietary fat content and full-fat soybean meal as a partial substitute for fish meal in the diet. Aquaculture 157:297–309. 14. NMKL Procedure NR. 6. 1998. Generelle retningslinier for kvalitetssikring af sensoriske laboratorier. Nordisk Metodikkomité for Levnedsmidler. http://www.nmkl.org/Engelsk/index.htm 15. Meilgaard M, Civille GV, and Carr BT. 2006. Sensory Evaluation Techniques, 4th ed., CRC Press, Boca Raton, FL. 16. Papadopoulos LS and Finne G. 1986. Effect of environmental salinity on sensory characteristics of penaeid shrimp. Journal of Food Science 51:812–814. 17. Hong LC, Leblanc EL, Hawrysh ZJ, and Hardin RT. 1996. Quality of Atlantic mackerel (Scomber scombrus L.) fillets during modified atmosphere storage. Journal of Food Science 61:646–651. 18. Warm K, Nielsen J, and Hyldig G. 2000. Sensory quality criteria for five fish species. Journal of Food Quality 23:583–601.
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19. Bøkness N, Jensen KN, Guldager HS, Østerberg C, Nielsen J, and Dalgaard P. 2002. Thawed chilled Barents Sea cod fillets in modified atmosphere packaging-appæication of multivariate data analysis to select key parameters in good manufacturing practice. Lebensmittel-Wissenschaft und-Technologie 35:436–443. 20. Sveinsdóttir K, Hyldig G, Martinsdóttir E, Jørgensen B, and Kristbergsson K. 2003. Development of quality index method (QIM) scheme for farmed Atlantic salmon (Salmo salar). Food Quality and Preferences 14:237–245. 21. Nielsen D, Hyldig G, Nielsen HH, and Nielsen J. 2004. Sensory properties of marinated herring (Clupea harengus)—Influence of fishing ground and season. Journal of Aquatic Food Product Technology 13:3–24. 22. Nielsen D, Hyldig G, Nielsen J, and Nielsen HH. 2005a. Sensory properties of marinated herring (Clupea harengus) processed from raw material from commercial landings. Journal of the Science of Food and Agriculture 85(1):127–134. 23. Green-Petersen DMB, Nielsen J, and Hyldig G. 2006. Sensory profiling of the most common salmon products on the Danish market. Journal of Sensory Studies 21:415–427. 24. Schubring R and Oehlenschläger J. 1997. Comparison of the ripening process in salted Baltic and North Sea herring as measured by instrumental and sensory methods. Zournal von Lebensmittel Untersuchung Forschung A 205:89–92. 25. Rasmussen RS, Ostenfeld TH, Rønsholdt B, and McLean E. 2000. Manipulation of end-product quality of rainbow trout with finishing diets. Aquaculture Nutrition 6:17–23. 26. Ginés R, Valdimarsdottir T, Sveinsdottir K, and Thorarensen H. 2004. Effect of rearing temperature and strain on sensory characteristics, texture, colour and fat of Arctic charr (Salvelinus alpinus). Food Quality and Preferences 15:177–185. 27. Stohr V, Joffraud JJ, Cardinal M, and Leroi F. 2001. Spoilage potential and sensory profile associated with bacteria isolated from cold-smoked salmon. Food Research International 34:797–806. 28. Morita K, Kubota K, and Aishima T. 2001. Sensory characteristics and volatile components in aroma of boiled prawns prepared according to experimental designs. Food Research International 34:473–481. 29. Richards M and Hultin HO. 2001. Rancidity development in a fish model system as affected by phospholipids. Journal of Food Lipids 8:215–230. 30. Cardinal M, Gunnlaugsdottir H, Bjoernevik M, Ouisse A, Vallet JL, and Leroi F. 2004. Sensory characteristic of cold-smoked Atlantic salmon (Salmo salar) from European market and relationships with chemical, physical and microbiological measurements. Food Research International 37:181–193. 31. Whitfield FB, Helidoniotis F, Shaw KJ, and Svoronos D. 1997. Distribution of bromophenols in Australian wild-harvested and cultivated prawns (Shrimp). Journal of Agriculture and Food Chemistry 45:4398–4405. 32. Rørå AMB, Kvåle A, Mørkøre T, Rørvik KA, Stein SS, and Thomassen MS. 1998. Process yield, colour and sensory quality of smoked Atlantic salmon (Salmo salar) in relation to raw material characteristics. Food Research International 31:601–609. 33. Whitfield FB, Helidoniotis F, and Smith D. 2002. Role of feed ingredients in the bromophenol content of cultured prawns. Food Chemistry 79:355–365. 34. Morita K, Kubota K, and Aishima T. 2003. Comparison of aroma characteristics of 16 fish species by sensory evaluation and gas chromatographic analysis. Journal of the Science of Food and Agriculture 83:289–297. 35. Einen O and Thomassen MS. 1998. Starvation prior to slaughter in Atlantic salmon (Salmo salar) II. White muscle composition and evaluation of freshness, texture and colour characteristics in raw and cooked fillets. Aquaculture 169:37–53. 36. Johansson L, Kiessling A, Kiessling K-H, and Berglund L. 2000. Effects of altered ration levels on sensory characteristics, lipid content and fatty acid composition of rainbow trout (oncorhynchus mykiss). Food Quality and Preference 11:247–254.
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37. Edmunds WJ and Lillard DA. 1979. Sensory characteristics of oysters, clams and cultured and wild shrimp. Journal of Food Science 44:368–373. 38. Farmer LJ, McConnell JM, and Kilpatrick DJ. 2000. Sensory characteristic of farmed and wild Atlantic salmon. Aquaculture 187:105–125. 39. Cardello AV, Sawyer MF, Prell P, Maller O, and Kapsalis J. 1983. Sensory methodology for the classification of fish according to “Edibility Characteristics”. Lebensmittel-Wissenschaft und-Technologie 16:190–194. 40. Bett KL, Ingram DA, Grimm CC, Vinyard BT, Boyette KDC, and Dionigi CP. 2000. Alteration of the sensory perception of the muddy/earthy odorant 2-methylisoborneol in channel catfish Ictalurus punctatus fillet tissues by addition of seasonings. Journal of Sensory Studies 15:459–472. 41. Sivertsvik M, Rosnes JT, Vorre A, Randell K, Ahvenainen R, and Bergslien H. 1999. Quality of whole gutted salmon in various bulk packages. Journal of Food Quality 22:387–402. 42. Hemre G-I, Karlsen Ø, Eckhoff K, Tveit K, Mangor-Jensen A, and Rosenlund G. 2004. Effect of season, light and diet on muscle composition and selected quality parameters in farmed Atlantic cod, Gadus morhua L. Aquaculture Research 35:683–697. 43. Sérot T, Regot C, Prost C, Robin J, and Arzel J. 2001. Effect of dietary lipid sources on odouractive compounds in muscle of turbot (Psetta maxima). Journal of the Science of Food and Agriculture 81:1339–1346. 44. Bak LS, Andersen AB, Andersen EM, and Bertelsen G. 1999. Effect of modified atmosphere packaging on oxidative changes in frozen stored cold water shrimp (Pandalus borealis). Food Chemistry 64:169–175. 45. Erikson MC, Bulgarelli MA, Resurreccion AVA, Vendetti RA, and Gates KA. 2007. Sensory differentiation of shrimp using a trained descriptive analysis panel. Food Science and Technology 40:1774–1783.
SAFETY
V
Chapter 28
Assessment of Seafood Spoilage and the Microorganisms Involved Robert E. Levin Contents 28.1 Introduction ..................................................................................................................516 28.2 Chemical Causes of Seafood Spoilage ...........................................................................517 28.3 Assays for Assessing the Quality of Seafood ..................................................................517 28.3.1 Assay for TMA.................................................................................................517 28.3.2 Assessment of Fish Quality Based on the Refractive Index of Eye Fluid ...........518 28.3.3 Vacuum Distillation Procedure for Determination of Volatile Acids and Volatile Bases in Fish Tissue .............................................................................518 28.4 Taxonomy of Psychrotrophic Intense Spoilage and Nonspoilage Bacteria on Seafood ....................................................................................................................518 28.4.1 The Genus Pseudomonas ...................................................................................518 28.4.1.1 Pseudomonas fragi .............................................................................521 28.4.1.2 Pseudomonas perolens ........................................................................521 28.4.2 The Genus Alteromonas ....................................................................................521 28.4.2.1 Alteromonas nigrifaciens ....................................................................521 28.4.3 The Genus Shewanella ......................................................................................521 28.4.3.1 Shewanella putrefaciens .....................................................................522 28.4.4 The Genera Moraxella and Acinetobacter ..........................................................523 28.4.5 The Genera Flavobacterium and Cytophaga .......................................................524 28.4.6 The Genus Brochothrix .....................................................................................524 515
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28.4.7 The Genus Photobacterium ...............................................................................524 28.4.8 The Genus Lactobacillus ...................................................................................525 28.4.9 The Genus Vibrio .............................................................................................525 28.4.10 The Genus Aeromonas.......................................................................................525 28.5 The Microbiology of Modified Atmosphere Stored Seafood ..........................................525 28.6 The Microbiology of Gamma-Irradiated Seafood ..........................................................526 28.7 Determination of Varius Bacterial Counts from Seafood ..............................................527 28.7.1 Determination of Total Aerobic Plate Counts ..................................................527 28.7.1.1 Selective Enumeration of Members of the Genus Pseudomonas ........527 28.7.1.2 Enumeration of Fluorescent Pseudomonads .....................................527 28.8 Useful Tests for Confirming the Identity of Seafood Spoilage Bacteria .........................528 28.8.1 Genetic Transformation Assay for Confirming the Identity of Psychrobacter immobilis Isolates..............................................................................................528 28.8.1.1 Motility ........................................................................................... 529 28.8.1.2 The Oxidase Test .............................................................................530 28.8.2 Litmus Milk .....................................................................................................530 28.8.3 Proteolysis ........................................................................................................530 28.8.4 Detection of H2S Production ...........................................................................531 28.8.5 DNase Activity.................................................................................................531 28.8.5.1 Molecular Techniques for Detection and Enumeration of Seafood Spoilage Bacteria ................................................................531 References ................................................................................................................................532
28.1
Introduction
Seafood can be expected to harbor a wide variety of bacterial species and genera. However, among the psychrotrophic seafood spoilage bacteria one finds relatively few genera and species that can be considered intense spoilage organisms. An early study undertaken to identify the major intense fish spoilage bacterial genera and species was by Castell and Anderson [1]. Their study involved the use of an autoclaved fish medium composed of equal weights of macerated fresh cod muscle and water in addition 0.05% agar that was poured into petri dishes. Known numbers of organisms from pure bacterial cultures were inoculated into this fish tissue medium followed by incubation at 3°C with daily assessment of spoilage odors. Three categories of pure cultures were described. The first group of organisms represented by enteric bacteria, Bacilli, and micrococci yielded no off odors at 3°C because they were unable to grow at this low temperature. The second group produced musty, sour, or sweetish odors at 3°C and consisted of flavobacteria, Achromobacter, and micrococci. The third group consisted of organisms that produced offensive odors rapidly at 3°C and consisted of Pseudomonas spp. Achromobacter sp., Serratia marcescens, and Proteus vulgaris. P. vulgaris is never found on seafood and the isolate used was from the American type culture collection (ATCC) (C. Castell, personal communication). This reduces the intense spoilage organisms to members of the genera Pseudomonas and Achromobacter as recognized in 1948. A later study, undertaken to identify the major psychrotrophic bacterial genera on freshly caught cod was by Georgala [2]. Among a total of 727 isolates, the following were identified: 51.5% Pseudomonas, 41.8% Achromobacter, 3.3% Vibrio, 1.5% Flavobacterium, 0.7% Micrococcus, and 0.7% miscellaneous. Since these early studies, various attempts have been made to further elucidate the major intense fish spoilage bacterial genera and species. This has
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resulted in a certain expansion and taxonomic alterations of the two originally recognized intense spoilage genera, Pseudomonas and Achromobacter, which this chapter elucidates.
28.2
Chemical Causes of Seafood Spoilage
A number of chemical agents that are products of microbial metabolism have been found to be associated with seafood spoilage. Included most notably among these is trimethylamine (TMA), the cause of the characteristic odor associated with spoiled seafood. Trimethylamine oxide (TMAO) is considered a compatible osmolyte in the muscle tissue of marine fish and is reduced by the bacterial enzyme TMAO-reductase to TMA. TMAO is present primarily in pelagic fish. In addition, other marines are encountered, such as putrescine and cadaverine derived from the bacterial decarboxylation of the amino acids arginine and lysine, respectively. Ammonia is usually derived from the oxidative deamination of amino acids by spoilage bacteria and is most readily sensed in the later stages of spoilage. Mercaptans are noxious sulfur-containing compounds derived from the activity of bacteria on cysteine in addition to the bacterial release of hydrogen sulfide from sulfur-containing amino acids. Volatile spoilage compounds fall into several categories: volatile amines, volatile acids (formic and acetic), and volatile reducing substances. All volatile compounds can readily be quantified presently by gas chromatography. However, the original description of a vacuum distillation procedure for the determination of volatile acids and bases in fish tissue by Tomiyama et al. [3] may be of value to some in that it requires no extraction and low cost equipment (see the following text).
28.3 Assays for Assessing the Quality of Seafood 28.3.1 Assay for TMA The quantitative presence of TMA in pelagic fish still remains a major chemical criterion of quality since most of the TMA is produced by bacterial reduction of the TMAO and therefore reflects the numbers of spoilage bacteria on fish tissue and the days of refrigeration or iced time. The chemical assay for quantitation of TMA in fish as described by Dyer [4] is presented. Fish tissue (100 g) is blended with 200 mL of 7.5% trichloroacetic acid (TCA) and filter clarified. One mL is then transferred to a tube and 3.0 mL of water added. One mL of 4.0% formaldehyde is added in addition to 10 mL of toluene and 3.0 mL of 50% potassium carbonate. The tube is capped and vortexed for 30 s. Five mL of the toluene (top) layer is transferred to a tube containing about 0.3 g of anhydrous sodium sulfate and is shaken for 10 s to remove trace amounts of water. The 5.0 mL of toluene is decanted into a dry tube and mixed with 5.0 mL of 0.02% picric acid–toluene solution. The intensity of the resulting yellow color is determined at 410 nm. For a standard curve use 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of a stock TMA solution (0.682% TMA·HCl in water). Results are reported in mg of TMA nitrogen (TMA-N) per 100 g of muscle tissue. Fresh cod and haddock have been found to contain no more than 0.2 mg of TMA nitrogen per 100 g of tissue. In contrast, at the time of spoilage the TMA content has been found to be 6–8 mg of TMA nitrogen per 100 g of tissue [5]. Castell et al. [6] found that cod and haddock could be graded into three categories based on sensory and TMA analysis. Group I consisted of fresh fish having TMA values (mg of TMA nitrogen per 100 g of tissue) of 0.52–0.89 with an average of 0.59. Group II consisted of stale fish having TMA values of 1.3–4.5 with an average of 3.1. Group II consisted of
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spoiled fish with TMA values of 2.6–8.3 with an average of 6.8. However, Hillig et al. [7] found a lack of correlation between sensory evaluation and TMA values for pollock and whiting.
28.3.2 Assessment of Fish Quality Based on the Refractive Index of Eye Fluid Proctor et al. [8] found a linear relationship between the refractive index of eye fluid from haddock and organoleptic score. They placed the quality of haddock into four refractive index categories: very good (1.3347–1.3366), fair to good (1.3367–1.3380), poor (1.3381–1.3393), and not marketable (1.3394 or higher). The procedure involves removal of the eyes from the head, making a slit in the posterior portion of the eyes, and allowing the fluids from each eye to drain into the same beaker. The fluids are then centrifuged (speed not specified) and then they are passed through glass wool. An Abbé refractometer or similar refractive index monitor is then used to determine the refractive index to the fourth decimal point using two or three drops of the eye fluid.
28.3.3 Vacuum Distillation Procedure for Determination of Volatile Acids and Volatile Bases in Fish Tissue The procedure of Tomiyama et al. [3] involves the blending of 85 g of fish tissue with 200 mL of water. Four hundred mL of a MgSO4 solution (600 g made to 1 L, followed by the addition of 20 mL of 6 N H2SO4) is then added, and thoroughly agitated, and then filtered through a filter paper. The filtrate should be adjusted to pH 2.0. Fifty mL of the filtrate (equivalent to 5.0 g of tissue) are then added to a 500 mL round bottom three-neck flask in a temperature controlled water bath (75°C). If the volatile acid number is to be determined 10 of 0.01 N NaOH are placed in the receiving vessel; if the volatile base content is to be determined 10 mL of N/28 H2SO4 are placed in the receiving vessel. For volatile acid determination, vacuum distillation is then initiated. If volatile bases are to be determined, 10 mL of 10% NaOH are added to the sample flask. In the determination of volatile acid number neutral red is used as the indicator and titration is with 0.01 N NaOH; in the determination of the volatile bases content methyl red is used as the indicator and titration is with 0.005 N H2SO4. An illustration of the vacuum distillation apparatus and a detailed description of its use is presented by Ref. [55].
28.4 Taxonomy of Psychrotrophic Intense Spoilage and Nonspoilage Bacteria on Seafood 28.4.1
The Genus Pseudomonas
The genus Pseudomonas is characterized as consisting of obligately aerobic gram-negative rods with polar flagella. The molar G + C (guanine + cytosine) content for members of this genus is recognized as being from 58% to 70%. Any organism outside this range is not considered a member of the genus Pseudomonas. The intense fish spoilage psychrotrophic species of the genus Pseudomonas can be divided into two convenient major groups consisting of fluorescent and nonfluorescent isolates. Among the fluorescent pseudomonads, we find that isolates of P. fluorescens are protease positive while isolates of P. putida are protease negative which constitute the major distinction between these two intense fish spoilage fluorescent species. Stanier et al. [9] established seven
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biotypes for isolates of Pseudomonas fluorescence (A–G) and two biotypes (A and B) for isolates of P. putida based on metabolic characteristics which Gennari and Fragotto [10] made use of for distinguishing fluorescent isolates from seafood and other food products. However, when it comes to the nonfluorescent fish spoilage pseudomonads, little is known about their species designations. Hugh and Leifson [11] developed a convenient culture method for distinguishing between oxidative and fermentative Gram-negative bacteria. It is known as the “Hugh–Leifson test” or the “O/F” test. The medium involved consists of: peptone, 0.2%; NaCl, 0.5%; KH2PO4, 0.03%; glucose, 1.0%; bromthymol blue, 0.03%; and agar, 0.3% pH 7.1. The low-level phosphate is used to slightly stabilize the pH and to promote fermentation. The low level of agar is to prevent convection currents. Glucose is added after autoclaving from a sterile 10% solution. Two tubes are inoculated with each culture and one is sealed with sterile mineral oil to a depth of about 1 cm to exclude oxygen. The initial pH of 7.1 results in a green color. An oxidative organism will produce a yellow acid reaction in the open tube starting at the top and with time proceeding downward and no reaction in the sealed tube. Some nonoxidizers and nonfermentors produce no change in the covered tube and only an alkaline reaction in the open tube. Other nonoxidizers and nonfermentors produce no reaction in either tube. Fermentative organisms will produce an acid reaction throughout both tubes. Shewan et al. [12] established a broad grouping of Gram-negative organisms found in fish and in other habitats that was based on 10 phenotypic characteristics. They then applied the Hugh– Leifson test to distinguish the various Gram-negative organisms prevailing on fish, which yielded four distinguishable metabolic groups of Pseudomonas from fish (Figure 28.1). This grouping of
Behavior in the test of Hugh and Leifson [12]
Oxidative Green fluorescent diffusible pigment
No diffusible pigment
Pseudomonas, Pseudomonas, group I group II
Alkaline
No action
Fermentative
No diffusible pigment
No diffusible pigment
No diffusible pigment
Pseudomonas, group III
Pseudomonas, group IV
Acid, no gas in glucose (some strains or traces of gas)
Acid, much gas in glucose at 20°C
Sensitive to the pteridine compound (O/129)
Insensitive to the pteridine compound (O/129)
Vibrio
Aeromonas
Figure 28.1 A grouping of the Gram-negative asporogenous rods, polar-flagellate, oxidase positive, and not sensitive to 2.5 i.u. of penicillin, on the results of four other tests. (Redrawn from Shewan, J. et al., J. Appl. Bacteriol., 23, 379, 1960. With permission.)
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Pseudomonas isolates from seafood is still used because many such isolates do not adhere to recognized species of Pseudomonas. Shewan et al. [13] presented a dichotomous key for the screening of cultures from seafood involving all of the major genera. This diagrammatic outline made use of the gram stain, pigmentation, flagellation, the cytochrome oxidase test, and the medium of Hugh and Leifson for the determination of oxidative versus fermentative metabolism and is presented in Figure 28.2. Isolated culture Gram-reaction
Negative
Positive
Rods, no spores Coryneforms
Cocci Micrococcus
Kovacs' oxidase test
Positive polar flagella
No pigment
Yellow or orange pigment
Achromobactera
Flavobacterium, Cytophaga
Negative peritrichous flagella no pigment fermentative in Hugh and Leifson's medium acid + gas from glucose at 25°C
Reaction in Hugh and Leifson's medium
Oxidative acid, but no gas from glucose
Nonmotile
Motile
Enterobacteriaceae
None, or alkali formed
Green fluorescent Pseudomonas spp. Some nongreen Pseudomonas spp. No acid from glucose Nongreen Pseudomonas spp.
Fermentative
Acid, but no gas from glucose
Acid, much gas from glucose
Sensitive to compound O/129
Aeromonas
Vibrio
Figure 28.2 Outline of the sequence of tests used in the screening of bacterial cultures from seafood. aMembers of the genus Achromobacter presently allocated to the genera Moraxella and Acinetobacter. (Redrawn from Shewan, J. et al., J. Appl. Bacteriol., 23, 463, 1960. With permission.)
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28.4.1.1 Pseudomonas fragi Among the off-odors that frequently develop during the early stages in the spoilage of refrigerated fillets are those that have been described as “sweet” and “fruity.” The responsible organism was found to be nonproteolytic and identified as Pseudomonas fragi which has been characterized as producing a “sweet, ester-like odor resembling that of the flower of the May apple” [14]. Isolates of P. fragi from seafood are characterized as being nonfluorescent, nonproteolytic, do not produce TMA, or H2S, but are capable of producing ammonia from amino acids, are lipolytic and are isolated from both fresh and spoiled fillets [14].
28.4.1.2 Pseudomonas perolens During the early stages of fish spoilage, a “musty” odor is sometimes noted. When the implicated organisms are in pure culture, they give rise to a “stored potato” odor. The responsible organism has been found to be P. perolens. Isolates of this organism are neither proteolytic nor lipolytic, do not produce TMA, and produce little or no change in milk, but do produce ammonia from amino acids, and H2S [15].
28.4.2
The Genus Alteromonas
The genus Alteromonas was originally created to accommodate organisms having typical phenotypic characteristics of the genus Pseudomonas but which have a molar G + C content of less than 58%, thereby excluding them from the genus Pseudomonas.
28.4.2.1 Alteromonas nigrifaciens A. nigrifaciens was originally known as Pseudomonas nigrifaciens in the older literature. This is an extremely intense fish spoilage organism characterized by producing an intense black melanin type of pigment. The organism is often overlooked in that maximum pigment production occurs with 1.5%–2.5% NaCl added to the culture medium with incubation from 4°C to 15°C. The presence of tyrosine (0.1%) has been found to be essential for pigment production [16]. In the absence of pigment production, this organism appears as a typical pseudomonad. The cells of this organism are motile by means of a single polar flagellum. Cultures are obligately aerobic, cytochrome oxidase positive, gelatinase positive, lipase positive, amylase positive, and produce putrescine, cadaverine, and spermidine. Sodium ions are required for growth. The molar G + C content is 39%–41%.
28.4.3
The Genus Shewanella
Members of the genus Shewanella were formerly considered pseudomonads. Venkateswaran [17] has reviewed the taxonomy of this genus at length. All isolates are Gram-negative, nonsporeforming rods, motile by means of a single polar flagellum, and are 2–3 mm in length. There are presently 12 recognized species, some of which produce salmon or pink-colored colonies. All species are cytochrome oxidase and catalase positive and negative for the production of amylase. Most species are gelatinase positive and lipase has been reported to be produced by several species. All species reduce TMAO to TMA and reduce nitrate to nitrite and the majority produce H2S from thiosulfate. Several species reduce elemental sulfur.
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28.4.3.1 Shewanella putrefaciens The organism presently known as Shewanella putrefaciens was first isolated from tainted butter and classified as a member of the genus Achromobacter by Derby and Hammer [18]. It was transferred to the genus Pseudomonas in 1941 by Long and Hammer [19]. In 1972, it was allocated to the genus Alteromonas by Lee et al. [20] on the basis of its much lower mol% G + C DNA content than the acceptable range of 58–70 mol% G + C for members of the genus Pseudomonas [21]. In 1985, it was transferred to the newly established genus Shewanella under the family Vibrionaceae due to its perceived closer relationship with the genus Vibrio [22]. The type species of S. putrefaciens, ATCC strain 8071 has a molar G + C content of 46% and strains in this species vary from 43% to 48% G + C [23–25]. Isolates of S. putrefaciens are intense psychrotrophic fish spoilage organisms as the species designation implies. They are visually characterized as producing salmon pigmented colonies, particularly on the surface of Peptone Iron Agar (PIA) where crowded surface colonies produce uniformly black colonies while well-isolated colonies usually produce salmon pigmented colonies with intense black centers (Figure 28.3). With pour plates of PIA, intensely black pinpoint size subsurface colonies develop. All such isolates produce an extracellular DNAse [26] in addition to an extracellular protease and lipase. Isolates of S. putrefaciens have been found on occasion to dominate at the time of intense fish spoilage [27]. Mg ions are a critical requirement for maintaining the integrity of the cell membrane [28]. If one prepares decimal dilutions of fish tissue in saline for plate counts the organism will rupture unless at least 0.001 M Mg++ ions are added to the saline
Figure 28.3
Typical colonies with black centers of S. putrefaciens on PIA.
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by way of MgCl2. This requirement is not widely recognized. Phosphate buffer enhances the lytic phenomenon by presumably pulling Mg++ ions out of the membrane or sacculus.
28.4.4
The Genera Moraxella and Acinetobacter
The genera Moraxella and Acinetobacter were originally the Gram-negative nonpigmented, nonflagellated, obligately aerobic coccobacilli that were allocated to the former genus Achromobacter in the older literature. The genus Achromobacter was eventually eliminated so that now all such Gram-negative coccobacilli from seafood are placed into the genera Moraxella or Acinetobacter. Without species differentiation, such bacterial isolates have often been placed into the “Moraxella– Acinetobacter” group. The molar G + C value for isolates of Moraxella varies from 40% to 46% and for Acinetobacter varies from 40% to 47%. These two genera are distinguished primarily on the basis that the Moraxella are sensitive to penicillin (1 i.u. disc) and are cytochrome oxidase positive while members of the genus Acinetobacter are resistant to penicillin and are cytochrome oxidase negative. Juni and Hyme [29] however found that fishery isolates of both Acinetobacter and Moraxella were cytochrome oxidase positive. A major metabolic distinction between these two genera results from the ability of Moraxella isolates to produce significant amounts of phenylethanol from the amino acid phenylalanine [30] while Acinetobacter isolates produce little or no phenylethanol. Members of both genera however appear to be closely related genetically. Juni and Hyme [29] developed a genetic transformation assay whereby the DNA from members of both of these psychrotrophic genera isolated from fish and meat products is able to transform a mutant recipient unable to synthesize hypoxanthine-to-hypoxanthine synthesis. In a subsequent report by Juni and Hyme [31] the designation Psychrobacter immobilis was proposed for all Gramnegative, aerobic, and cytochrome oxidase positive coccobacilli on the basis of genetic compatibility (genetic transformation). Such a designation eliminates the use of the genera Moraxella and Acinetobacter for such psychrotrophic food isolates and to that extent may be more convenient for individuals working in the area of seafood microbiology. In a later study, Rossau et al. [32] found that on the basis of DNA–rRNA hybridization, members of the genera Moraxella, Psychrobacter, and Acinetobacter constitute a separate genotype cluster and proposed the family Moraxellaceae to accommodate these organisms. González et al. [33] isolated 979 Gram-negative, nonmobile, aerobic coccobacilli from fresh water fish stored in ice. A total of 106 randomly selected isolates were found to consist of Psychrobacter (64 strains), Acinetobacter (24 strains), Moraxella (6 strains), Chryseobacterium (5 strains), Myroides odoratus (2 strains), Flavobacterium (1 strain), Empedobacter (1 strain), and three unidentified strains. Among the 64 Psychrobacter, 14 isolates were P. phenylpyruvica. These authors considered Acinetobacter to be the only oxidase negative genus within the family Moraxellaceae. These authors concluded that identification at the genomic species level is complex, time consuming, and in some cases impossible. In contrast to earlier studies, these authors concluded that there is considerable confusion of the role of Moraxellaceae in the spoilage of presumably raw refrigerated proteinaceous foods. In their study, this genus contributed only 9.0% of the total flora, and the majority failed to produce typical spoilage compounds (TMA and H2S). However, among the 106 randomly selected isolates, 50 were found to be mesophiles, and 56 psychrotrophs indicating that a disproportionate number of isolates were derived from fish prior to prolonged iced storage. Their observation that the Moraxellaceae decreased as spoilage progressed contradicts earlier studies on the spoilage of marine fish. A key factor in this discrepancy may be that the predominant species at the time of spoilage of fresh water fish may differ notably from that of marine fish.
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28.4.5 The Genera Flavobacterium and Cytophaga Members of both these genera are characterized as producing yellow, orange, or red carotenoid pigments. The Flavobacteria may be motile by peritrichous flagella or nonmotile. The Cytophaga, if motile exhibit gliding motility and lack flagella. Both genera are characterized as being obligately aerobic and weakly active on carbohydrates. Not all isolates of both genera are capable of utilizing glucose. Many isolates of both genera have little or no effect on litmus milk. Some isolates have been found to be proteolytic and some are able to produce H2S, moreover none are lipolytic. McMeekin [34] subjected 59 yellow-pigmented bacterial isolates from various foods to 78 phenotypic properties and with the aid of adansonian taxonomy distinguished six groups. One of the problems encountered in attempting to allocate individual strains to one or the other of these pigmented genera involves atypical strains. In an earlier study, Castell and Maplebeck [35] examined 245 isolates of Flavobacterium (132 yellow and 113 orange) from fish for the ability to exhibit fish spoilage activities. Seventy-eight percent of the yellow isolates and 92% of the orange isolates grew at 2°C–3°C. Thirty-six percent of the yellow isolates and only 4% of the orange isolates produced TMA. Forty percent of the yellow isolates and 84% of the orange isolates were proteolytic. When isolates were inoculated onto sterile fish tissue incubated at 3°C the orange cultures of Flavobacterium began to develop disagreeable odors after 5–8 days and many became quite putrid by the 10th or 11th day. Sterile fish tissue inoculated with the yellow cultures yielded no perceptible spoilage odors, even after 15 days but did discolor the fish tissue yellow, indicative of growth. In contrast, fish tissue inoculated with Pseudomonas isolates became offensive after 48 and 72 h. It is a widely recognized observation, that members of the brightly pigmented genera Flavobacterium and Cytophaga are frequently encountered on fresh fish where they may constitute 10%–30% of the initial flora and are rarely among the dominant flora of stale fish. Although isolates of these pigmented organism have been found under noncompetitive conditions to eventually spoil fish tissue, on a practical basis, under commercial conditions they are generally outgrown by the more intense spoilage pseudomonads that grow more rapidly under refrigerated conditions than members of the these two pigmented genera. As a result, many workers group such isolates into the “flavobacterium–cytophaga” group rather than attempting to determine clearly and arduously which pigmented genus they belong.
28.4.6 The Genus Brochothrix These are Gram-positive nonsporeforming rods closely related to the genus Lactobacillus and are considered heterofermentative with regard to lactic acid production. Log-phase cells are typically rods, while older cells are coccoids, a feature common to coryneforms. Only two species are recognized: B. thermosphacta and B. campestris. These organisms are important in the spoilage of modified atmosphere (MA) stored seafood [36]. In contrast to B. thermosphacta, B. campestris is rhamnose and hippurate positive. Both species have a molar G + C content of 36%.
28.4.7
The Genus Photobacterium
These are Gram-positive nonsporeforming, peritrichously flagellated rods possessing fermentative metabolism with sugars and are therefore facultative anaerobes. Isolates are luminescent (glow in the dark). The molar G + C for the genus is 39%–42%. Because of their facultatively anaerobic metabolism, they have been frequently found to be among the major spoilage organisms in
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MA storage when oxygen is excluded. The species associated with spoiled seafood is P. phosphoreum [37]. Sivertsvik et al. [38] have reviewed the relationship between this organism and MA storage.
28.4.8 The Genus Lactobacillus These organisms are Gram-positive nonsporeforming rods 2–9 mm long. All species of lactobacilli produce at least 1.0% lactic acid from glucose, are nutritionally fastidious, and are catalase negative. Members of the genus Lactobacillus do not predominate when seafood is stored under normal iced or refrigerated conditions. However, under conditions of MA storage, lactobacilli can dominate at the termination of storage. They are most readily enumerated from seafood products as dominant members of the prevailing flora with the use of Lactobacilli MRS Agar, which is designed to support luxuriant growth of all lactobacilli and is not a selective medium. Therefore, identification is based on the phenotypic properties of isolated colonies.
28.4.9
The Genus Vibrio
Members of the genus Vibrio are Gram-negative short asporogenous curved or straight rods, which are motile by means of polar flagella. They are all facultative anaerobes exhibiting fermentative metabolism in the absence of oxygen producing acid but no gas (H2 or CO2). They are cytochrome oxidase positive and are usually nonpigmented. All members of the genus are considered sensitive to the vibriostatic agent 2,4-diamino-6,7-diisopropyl pteridine (O/129) which is considered a diagnostic criterion for the genus. The molar G + C value for the genus ranges from 40% to 50%. Members of the genus are not considered spoilage organisms and are usually not found among the dominant flora of stale fish. The genus does have several species that are notable human pathogens such as V. cholerae, V. vulnificus, and V. parahaemolyticus that are associated with the consumption of raw seafood.
28.4.10 The Genus Aeromonas Members of the genus Aeromonas are straight rod shaped Gram-negative polarly flagellated cells. They are facultative anaerobes exhibiting fermentative metabolism in the absence of oxygen with the production of acid and gas (H2 + CO2). Isolates are proteolytic, produce extracellular DNase, are cytochrome oxidase positive, and insensitive to the vibriostatic agent O/129. The molar G + C content ranges from 57% to 63%. The genus contains several species pathogenic to fish such as A. hydrophila and A. salmonicida. Isolates of Aeromonas have on occasion been implicated in gastroenteritis.
28.5
The Microbiology of Modified Atmosphere Stored Seafood
MA storage of seafood results in a dramatic change in the bacterial flora that develop under refrigerated temperatures compared to the normal atmosphere of 79% nitrogen and 21% oxygen. An atmosphere of 20% CO2 and 80% air is commonly used, however studies involving the use
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of 50% CO2 and 50% nitrogen have indicated that the complete absence of oxygen in the storage atmosphere greatly influences the resulting dominant flora. Another factor that may influence the dominant flora at spoilage is fresh water farm raised fish versus ocean caught fish. The presence of 20% air is preferred to ensure that Clostridium botulinum does not develop. Mokhele et al. [39] found that with rock cod fillets stored in such a MA at 4°C for 21 days, the total bacterial population increased by only 2 log cycles, the fillets were not spoiled, and only Aeromonas-like (31%) and Lactobacillus (69%) isolates were recovered. In contrast, control fillets stored under conditions of a normal atmosphere at 4°C were spoiled after 7 days and had undergone an increase of 3–4 log cycles in total bacterial counts. Johnson and Ogrydziak [40] using an identical MA for the storage of rock cod at 4°C found that after 21 days only Lactobacillus (71%–87%) and tancolored Pseudomonas-like isolates were recovered. Their observations suggested that the tan isolates underwent mutation to enhanced tolerance to the MA. Some of the tan isolates produced H2S, strongly suggesting that they were S. putrefaciens. These authors also indicated that the tan isolates grew slowly under anaerobic conditions. S. putrefaciens does not have a fermentative metabolism but isolates are able to couple anaerobic growth to iron and manganese reduction, and are able to utilize an array of other electron acceptors anaerobically such as NO3−1, NO2−1, S2O3−2, S0, and fumarate; TMAO with most strains was able to utilize lactate, pyruvate, and some amino acids anaerobically [41]. Hovda et al. [36] found that when fresh water farm raised halibut were stored in a MA of 50%CO2:50%N2 that 16 days at 4°C were required for a 5 log increase in total counts to occur, whereas in a CO2:50%O2 MA no more than a 3 log increase occurred after 23 days, and that storage in air resulted in a 7 log increase in 16 days. 16S rDNA sequencing following denaturing gradient gel electrophoresis (16S rDNA-DGGE) indicated that in air Pseudomonas spp., P. putida, P. phosphoreum, and B. thermosphacta were dominant at the time of spoilage. With a MA of 50% CO2:50%N2 the predominant organisms at the time of spoilage were P. phosphoreum, and B. thermosphacta. S. putrefaciens was found only sporadically and in low numbers, while pseudomonads were not detected. In a MA of 50% CO2:50%O2 B. thermosphacta and Staphylococcus spp. dominated after 23 days. Rudi et al. [42] stored salmon and coalfish in a MA of 60% CO2:40%N2 for up to 18 days at 1°C and 5°C. The predominant organisms after 18 days on coalfish identified by 16S rDNA analysis were found to be Photobactrium spp., while those on salmon were found to be Carnobacterium spp. and Brochothrix spp. These authors made the interesting observation that Photobacterium spp. were underrepresented when identification was based on colony isolation compared to 16S rDNA sequencing following cloning which was presumably due to technical difficulties in culturing photobacterium. Their study also detected the presence of C. botulinum at the end of storage.
28.6
The Microbiology of Gamma-Irradiated Seafood
The organisms predominating at the time of spoilage on petrale sole subjected to gamma irradiation (4 kGy) and then stored in air at 0.5°C have been found to be members of the bacteria genera Moraxella and Acinetobacter (formerly Achromobacter) and the yeast Trichosporon [43]. When stored under conditions of vacuum packaging after irradiation (4 kGy) lactobacilli dominated [44]. These observations show that these organisms are more resistant to gamma irradiation than the other members of the bacterial flora such as the pseudomonads. Pseudomonads are seldom encountered with irradiation over 1 kGy.
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28.7 Determination of Varius Bacterial Counts from Seafood 28.7.1 Determination of Total Aerobic Plate Counts The conventional plate count method described in the following text for examining frozen and chilled foods conforms to the AOAC Official Methods of Analysis sec. 966.23 with procedural changes as indicated. The suitable counting colony range is 25–250. The frequently recommended diluent for dilution blanks consists of Butterfield’s phosphate-buffered dilution water (KH2PO4, 3.4%, pH 7.2 adjusted with NaOH) is not appropriate. The author has found that with seafoods, higher counts will result with dilution blanks prepared with 0.85% NaCl and 0.001 M MgCl2 in that organisms such as S. putrefaciens will readily undergo autolysis if placed into phosphate buffer [28]. Although standard methods recommend stomaching or blending 50 g of tissue with 450 mL of diluent, total aerobic counts from seafood tissue are as a rule sufficiently high so that 10 g of tissue can be added to 90 mL of diluent without compromising results. Dilutions should be prepared by transferring 10–90 mL of diluent and shaken 30 times, avoiding foaming. Reshake if dilutions stand for more than 3 min. One mL of each dilution is then transferred to duplicate petri dishes and 12–15 mL of Plate Count Agar (tryptone, 0.5%; yeast extract, 0.25%; glucose, 0.1%; agar, 1.5%; supplemented with 0.5% NaCl, pH 7.0) or Tryptic Soy Agar (tryptone, 1.7%; soytone, 0.3%; NaCl, 0.5%; K 2HPO4, 0.15%, agar, 1.5%, pH 7.3) at 45°C is added and the plates agitated on a flat surface by traversing a figure eight motion at least six times. The plates are then allowed to solidify before stacking. Alternatively, one can smear-plate 0.1 mL of dilutions onto prepoured agar plates, the surface of which have been allowed to dry for 1–2 days. Plates should be incubated at 20°C for 72 h. An incubation temperature of 20°C will yield approximately 10-fold higher counts than at 35°C [45] and will usually yield total counts that are within 70% of the true counts. However, if absolute maximum counts from stale fish are to be obtained, then it is necessary to incubate the plates at 3°C for 6 days, where with stale fish, counts have been found to be 30% higher than at 20°C [46]. This is due to the development of obligately psychrotrophic organisms (mostly vibrios) which do not grow above 15°C [46]. It is important to keep in mind that obligately psychrotrophic bacteria are extremely sensitive to elevated temperatures of 25°C–35°C [47]. Hence, for truly maximum counts one should use prepoured and solidified agar plates whose temperature is not allowed to rise above 10°C once in contact with the sample, and which are smear plated with 0.1 mL of decimal dilutions. Pipets, pipet tips, and dilution blanks must also be chilled. In addition, blending or stomaching must also be done under refrigerated conditions. Such low temperature plate counts are best achieved by performing all procedures in a refrigerated room or laboratory at about 2°C–4°C so that samples and supplies are never allowed to increase above this refrigerated temperature range [46].
28.7.1.1
Selective Enumeration of Members of the Genus Pseudomonas
The selective enumeration of essentially all pseudomonads from seafood tissue can be achieved with the use of Pseudomonas Isolation Agar. This medium contains: peptone, 20 g; MgCl2, 1.4 g; K 2SO4 1.5 g; Irgasan, 0.025 g; agar, 13.6 g; and deionized water, pH 7.0.
28.7.1.2
Enumeration of Fluorescent Pseudomonads
All culture media designed for the detection of fluorescent pseudomonads are high in magnesium and notably low in iron; the latter greatly suppressing fluorescence. The fluorescent pseudomonad
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agar medium of Sands and Rovira [48] is ideal for the selective isolation of only fluorescent pseudomonads. The basal medium consists of: proteose peptone no. 3, 20 g; Oxoid Ionagar no. 1, 12 g; glycerol, 8 mL; K 2SO4 1.5 g; MgSO4·7H2O, 1.5 g; distilled water, 940 mL, pH 7.2. Penicillin G (75,000 units, novobiocin, 45 mg; and cycloheximide, 75 mg are mixed together in 3 mL of 95% ethanol, and are then diluted with 50 mL of sterile distilled water, and added to the 940 mL of the melted basal medium at 45°C. The surface of the plates is allowed to dry overnight. Antibiotic activity will decrease significantly with prolonged storage of plates. Alternatively, the nonselective medium Pseudomonas Agar F can be used for distinguishing fluorescent Pseudomonas colonies from nonfluorescent colonies from seafood issue. However, total counts are frequently lower on this medium than on plate count or Tryptic Soy Agar. Pseudomonas Agar F contains: tryptone, 10 g; proteose peptone no. 3, 10 g; K 2HPO4, 1.5 g; MgSO4, Agar 15 g; and distilled water, 1000 mL, pH 7.0. Plates are viewed under a black UV lamp for detection of fluorescent colonies.
28.8 28.8.1
Useful Tests for Confirming the Identity of Seafood Spoilage Bacteria Genetic Transformation Assay for Confirming the Identity of Psychrobacter immobilis Isolates (Juni and Hyme [29])
1. The auxotrophic mutant A351-Hyx-7 (ATCC 43117) recipient culture requiring hypoxanthine is cultured onto a slant of brain heart infusion agar (BHI-A) or heart infusion agar (HI-A). Do not culture sequentially to prevent spontaneous reversion to prototrophy. 2. Colorless colonies of unknown organisms are picked to a slant of Nutrient Agar or Tryptic Soy Agar. 3. Several large loops of cell growth from an unknown slant are transferred to a vial of sterile lysing solution (0.05% sodium dodecyl sulfate in 0.15 M NaCl and 0.015 M trisodium citrate) and the cells are dispersed by vigorous agitation. The vials are then held at 65°C for 1 h to lyse the cells and achieve sterility. 4. Divide a BHI-A or an HI-A plate into four sections and aseptically transfer a full loop of crude DNA from each sample to a separate quadrant and smear a circular area of about 1 in. in diameter. Prepare a duplicate control plate for determining the sterility of the DNA samples. 5. To one set of BHI-A plates apply a loop of the recipient 135-Hyx-7 culture to each area smeared with DNA and leave the other plate as a sterility control. In addition, inoculate one quadrant of the BHI-A plate with just the recipient to detect spontaneous revertants. Incubate the plate overnight at 20°C. 6. Transfer a loop of cell growth from each area of the BHI-A plate that has grown up, to a quadrant of an M9A Agar plate by streaking the quadrant and incubate the plate at 20°C for 3 days and observe for the development of isolated colonies (genetic transformants) at the end of the streaks (see Figure 28.4). Discount solid areas of growth at the initial areas of the streaks, which are due to hypoxanthine carry over from the BHI-A plate. M9A Agar plates contain 0.8% vitamin free casein hydrolysate, 0.28% Na 2HPO4, 0.1% KH 2PO4, 0.5% NaCl, 0.045% MgSO4·7H 2O, 5.0 mL of 60% sodium lactate, 4.0 g of glucose sterilized separately, and 15 g of agar in a total volume of 1000 mL prepared with deionized water.
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Figure 28.4 Genetic transformation of P. immobilis. Growth of recipient A351-Hyx-7 genetically transformed by DNA from a fishery isolate of P. immobilis on an M9A plate lacking hypoxanthine. Quadrants without growth have been streaked with the recipient A351-Hyx culture without prior contact with exogenous DNA and serve as controls. Slight growth at the initiation of the control streaks can be observed because of nutrient carryover from the BHI plate.
28.8.1.1
Motility
The experience of the author has indicated that the most reliable assay for motility is to observe a young broth culture under the microscope. An additional advantage to microscopic observation is that with experience, the observer can distinguish motility of a peritrichously flagellated organism versus a polarly flagellated organism. Polarly flagellated organisms will frequently move at a much higher rate of speed than peritrichously flagellated organisms. In addition, polarly flagellated organisms tend to lift the flagellated end of the cell due to a rapid rotary swirling of the polar flagella. However, motility test agar may still be found useful by some and consists of tubes filled with 10 mL of: 1.0% peptone, 0.5% NaCl, and 3.5% agar. Inoculation is by stabbing. Motile cultures will exhibit turbid lateral growth, usually just below the surface. The flagella stain is notably time consuming and unreliable unless performed by an individual with extensive experience and technical expertise. However, it is still the only reliable method for determining the cellular location of flagella outside of electron microscopy. The flagella stain in the following text [49] has been found to be superior to several others and yields results amenable to photomicroscopy. 1. Cells from 18 h. Nutrient Agar slants are suspended in distilled water to yield a cloudy suspension. 2. A glass slide is scrupulously cleaned by scrubbing with a household diatomaceous earth cleaning powder and paper towel moistened with water to achieve a thick slurry. Blot the slide dry. Care should be taken not to handle the surface of the slide with bare fingers. 3. Several drops of the cell suspension are placed on the end of a cleaned glass slide and the excess fluid is allowed to run down and off the slide to facilitate orienting polar flagella in one direction. After air-drying, the slide is immersed in reagent I for 15 min. This step serves as both fixative and mordant. Reagent I consists of: deionized water, 100 mL; tannic acid, 5.0 g; FeSO4 (saturated solution), 1.5 mL; formaldehyde, 2.0 mL; and 1.0% NaOH, 1.5 mL. 4. Without rinsing the slide, it is then covered with reagent II until a brown color appears and is then washed with distilled water and air-dried. Reagent II contains: AgNO3, 5.0 g; distilled water, 100 mL; and NH4OH (several drops added to 10 mL of AgNO3 solution; when the precipitate clears add to 90 mL of remaining solution).
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28.8.1.2 The Oxidase Test The test for cytochrome oxidase is highly definitive for the distinction of various taxonomic groups of seafood spoilage bacteria. All members of the genera Pseudomonas, Vibrio, and Aeromonas in addition to P. immobilis are presently considered positive for cytochrome oxidase. Enteric organisms are all negative. The methods of Kovács [50] and Bovre and Henriksen [51] are both recommended to ensure against false negative reactions. The cytochrome oxidase test is performed as follows: moisten a sheet of filter paper with a 1% aqueous solution of tetramethyl-p-phenylenediamine [50], or dimethyl-p-phenylenediamine [51] and smear onto the surface bacterial cells from a slant or colony using a platinum loop (not Nichrome). A positive test is indicated by the development of a dark purple color within 20 s. The use of a metal loop with an iron content can result in weak false positive reactions derived from corroded iron debri shed from the loop.
28.8.2
Litmus Milk
Tubes of litmus milk inoculated with bacterial isolates from seafood afford the observer a variety of reactions for distinguishing cultures and facilitating identification. Reactions to be observed are: (1) peptonization or proteolysis resulting in the initially opaque tube becoming translucent, (2) an alkaline reaction derived from the deamination of amino acids and resulting in a blue coloration of the medium, (3) an acid reaction, derived from the utilization of lactose resulting in a pink coloration, (4) an acid clot resulting in precipitation of the casein, and (5) reduction of the litmus dye resulting in the tube turning white. Combinations of several reactions are also frequently encountered such as (a) reduction and proteolysis, and (b) proteolysis with an alkaline reaction. Litmus milk consists of 10% rehydrated dry skimmed milk and 0.5% litmus.
28.8.3
Proteolysis
There are several approaches for determining if cultures are proteolytic. Fish press juice offers a natural substrate for determining proteolysis of bacterial isolates. Fillets are placed into a press and the liquid expressed is collected. Alternatively, the fillets are first ground and then pressed. It is important to maintain the temperature of the fish juice as close to 0°C as possible to prevent thermal denaturation and precipitation of initially soluble proteins at or near room temperature. The juice can then be centrifuged at 4°C in a precooled rotor for 10 min at 10,000×g to sediment tissue particles. The juice is then sterilized by passage through a 0.25 m porosity filter membrane, maintaining the temperature as close to 0°C as possible. Sterile agar is then mixed with 9%–24% fish juice (final concentration 1.5%) and plates are poured. At room temperature, a significant amount of initially soluble proteins will precipitate out and impart a cloudy appearance to the agar. Inoculation of circular zones onto the agar surface and incubation at 20°C for 48 h will yield areas of growth surrounded by large zones of clearing by proteolytic organisms [52]. Alternatively, 15 mL of rehydrated skimmed milk (autoclaved separately) is added to 85 mL of sterile Nutrient Agar and plates are poured [52]. Gelatin deeps are used frequently for the detection of proteolysis. It should be kept in mind that gelatin is not as readily hydrolyzed by some proteases as is casein in skimmed milk so that an organism exhibiting definite proteolysis of casein may not yield a positive proteolysis result with gelatin. Nutrient gelatin deeps (10 mL) in tubes are prepared and contain peptone, 5%; beef extract, 3%; and gelatin, 12%; in distilled water, pH 7.0. Nutrient gelatin will liquefy at temperatures above 20°C. It is therefore important to incubate the tubes at 20°C and on removal from the incubator,
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it is necessary to place them in an ice bath to prevent temperature-induced liquefaction before they are read. Tubes of Nutrient gelatin are stab inoculated to the bottom of the tubes with an inoculating needle from a broth tube. For greatest sensitivity, the surface of a soft-agar-gelatin overlay plate can be inoculated [53]. This medium consists of bottom agar prepared with Nutrient broth to which is added MnSO4, 0.005%; NaCl, 0.8%; and agar, 1.5%, pH 7.0. Plates are poured and allowed to solidify. A soft-agar-gelatin overlay (3 mL) containing Nutrient broth, MnSO4, 0.005%; NaCl, 0.8%; and agar 0.8%, and 1.5% gelatin, pH 7.0 is poured onto the bottom agar. The surface is then carefully inoculated. After incubation, 5% acetic acid is applied to the plate to precipitate intact gelatin and clear zones surrounding areas of growth are noted. This procedure is ideally suited for direct enumeration of proteolytic organisms from seafood where the surface of a series of bottom agar plates is smear inoculated with 0.1 mL of decimal dilutions of the homogenized tissue. A soft-agar-overlay is then applied to each plate. If one desires to isolate the proteolytic organisms, a soft-agar-overlay prepared with skimmed milk can be used which does not require the application of acetic acid as with soft-agar-gelatin plates.
28.8.4 Detection of H2S Production Detection of H2S production by seafood isolates is most readily observed by stab inoculating tubes containing 10 mL of peptone-Fe-agar (peptone, 1.5%; proteose peptone, 0.5%; ferric ammonium citrate, 0.5%; sodium glycerophosphate, 0.1%; sodium thiosulfate, 0.008%; and agar, 1.5%, pH 6.7). Positive tubes will yield an intense black area of growth within 72 h along the stab resulting from FeS formation. It should be kept in mind that some isolates will be slow to form H2S, and may be weak H2S producers and that prolonged incubation, usually beyond 7 days, results in the fading of the intense black FeS due to slow oxidation by the penetration of atmospheric oxygen.
28.8.5
DNase Activity
The production of extracellular DNase activity by seafood isolates is most conveniently determined with the use of DNase test Agar w/methyl Green, which consists of tryptose, 2.0%; deoxyribonucleic acid, 0.2%; NaCl, 0.5%; methyl green, 0.005%; and agar, 1.5%, pH 7.3. Methyl green combines with high molecular weight DNA to impart a green coloration to the agar. When a DNase positive organism is streaked onto the surface, the extracellular DNase hydrolyzes the DNA immediately surrounding the area of growth yielding a colorless zone. Applying cells from an agar slant or broth tube to a circular area of about 0.5 cm allows a single plate to be used for five or six cultures. Detection of extracellular DNase is particularly useful in confirming the identity of S. putrefaciens isolates.
28.8.5.1
Molecular Techniques for Detection and Enumeration of Seafood Spoilage Bacteria
The minimum number of colony forming units (CFU) per gram of raw seafood tissue is about 1 × 104 unless the tissue is excised aseptically. On a more practical basis, commercially processed fresh fish fillets will usually have an initial CFU count of about 1 × 105 CFU per gram of tissue. Counts in the range of 107 to 108 per gram are usually associated with some degree of spoilage and poor quality. Universal primers have been successfully applied to the quantification of the total
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bacterial population on fish tissue [54–56]. The universal forward primer DG74 5′-AGG-AGGTGA-TCC-AAC-CGA-A-3′ and the universal reverse primer RW01: 5′-ACC-TGG-AGG-AAGAAG-GTG-GGG-AT-3′ primer [57] amplify a 370-bp sequence of the 16S rRNA gene derived from all bacteria. An extremely close linear relationship was found between the number of CFU per gram determined from plate counts and the total number of genomic targets determined by conventional and real-time polymerase chain reaction (PCR). In addition, this methodology has been extended to the use of the PCR for distinguishing the total number of dead and viable bacteria on fish tissue with the use of the selectively permeable DNA binding dye ethidium bromide monoazide [58]. Venkitanarayanan et al. [59] developed a pair of primers for the amplification of a 207-bp amplicon derived from the 23S rDNA sequence of meat spoilage bacteria. The assay was designed for detection of the following typical meat spoilage bacteria: P. fluorescence, P. putida, P. fragi, P. areofaciens, Acinetobacter calcoaceticus, Enterobacter liquefaciens, Flavobacerium breve, Moraxella osloensis, and Brochotrix teremosphacta. The assay should be equally applicable for the collective quantitative PCR enumeration of most of these spoilage organisms on seafood. However, these authors did not indicate the specificity of the assay. The sequence of the forward primer PF is 5′-AAG-CTT-GCT-GGA-GGT-ATC-AGA-AGT-GC and that of the reverse primer PR is CTCCGC-CCC-TCC-ATC-GCA-GT. Seafood isolates of S. putrefaciens can be confirmed as such by the PCR with the use of the primers SP-1: 5′-TTC-GTC-GAT-TAT-TTG-AAC-AGT AND SP-2r: 5′-TTC-TCC-AGC-AGATAA-TCG-TTC which amplify a 422-bp sequence of the Gyr B sequence [17]. The development of a primer pair specific for members of the genus Pseudomonas [60] has allowed the use of the PCR for the identification of Pseudomonas isolates. This PCR assay is ideally suited for the confirmation of presumptive isolates of Pseudomonas from seafood and has the potential to be used to quantify the total number of pseudomonads per gram of seafood tissue numerically. The assay is based on the presence of two Pseudomonas specific and conserved sequences, one at the middle of the 16S rDNA sequence and the other at the beginning of the 23S rDNA sequence. As a result, the amplified region includes the 3′-half of the 16S rDNA with the whole 16S–23S rDNA Internal Transcripted Spacer (ITS1) sequence in addition to the first 25 nucleotides of the 23S rDNA sequence from the 5′-end. The Pseudomonas specific primers generated amplicons of 1300-bp. The sequence of the forward primer fPs16S is 5′-ACT-GACACT-GAG-GTG-CGA-AAG-CG that of the reverse primer rPs23S is 5′-ACC-GTA-TGC-GCTTCT-TCA-CTT-GAC-C. All 33 Pseudomonas strains representing 14 species yielded amplicons while none of the 13 Gram-negative on Pseudomonas species with the exception of Azotobacter chroococcum. In addition, several of the Pseudomonas yielded two or three bands varying from 1100 to 1300-bp. The multiple bands are thought to reflect the number and variation in length of ITS1 sequences in a given species.
References 1. Castell, C. and Anderson, G. Bacteria associated with spoilage of cod fillets. J. Fish. Res. Board Can. 7, 370–377, 1948. 2. Georgala, D. The bacterial flora of the skin of North Sea Cod. J. Gen. Microbiol. 18, 84–91, 1958. 3. Tomiyama, T., da Sota, A., and Stern, J. A rapid vacuum distillation procedure for the determination of volatile acids and volatile bases in fish flesh. Food Technol. 10, 614–617, 1956. 4. Dyer, W. Report on trimethylamine in fish. JAOAC 42, 292–294, 1959.
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5. Dyer, W. and Mounsy, T. Amines in fish muscle. II. Development of trimethylamine and other amines. J. Fish. Res. Board Can. 6, 351–358, 1945. 6. Castell, C., Greenough, M., Rodgers, R., and MacFarlane, A. Grading of fish for quality. 1. Trimethylamine values of fillets cut from graded fish. J. Fish. Res. Board Can. 15, 701–716, 1958. 7. Hillig, F., Selton, L. Jr., Loughrey, J., and Bethia, S. Chemical indexes of decomposition in pollock and whiting. JAOAC 44, 499–507, 1961. 8. Proctor, B., Nickerson, J., Fazino, T., Ronsivalli, L., Smith, R., and Stern, Rapid determination of quality of whole eviscerated haddock. J. Food Technol. 13, 224–228, 1958. 9. Stanier, R., Palleroni, N., and Doudoroff, M. The aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43, 159–271, 1966. 10. Gennari, M. and Fragotto, F. A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water. J. Appl. Bacteriol. 72, 281–288, 1992. 11. Hugh, R. and Leifson, E. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66, 24–26, 1953. 12. Shewan, J., Hobbs, G., and Hodgkiss, W. A determinative scheme for the identification of certain genera of gram-negative bacteria, with special reference to the Pseudomonadaceae. J. Appl. Bacteriol. 23, 379–390, 1960. 13. Shewan, J., Hobbs, G., and Hodgkiss, W. The Pseudomonas and Achromobacter groups of bacteria in the spoilage of marine white fish. J. Appl. Bacteriol. 23, 463–468, 1960. 14. Castell, C., Greenough, M., and Dale, J. The action of Pseudomonas on fish muscle. 3. Identification of organisms producing fruity and oniony odours. J. Fish. Res. Board Can. 16, 13–19, 1959. 15. Castell, C., Greenough, M., and Jenkin, N. The action of Pseudomonas on fish muscle. 2. Musty and potato-like odours. J. Fish. Res. Board Can. 14, 775–782, 1957. 16. Ivanova, E., Kiprianova, E., Valery, M., Levanova, G., Garagulya, A., Gorshkova, N., Yumoto, N., and Yoshikawa, S. Characterization and identification of marine Alteromonas nigrifaciens strains and emendation of the description. Int. J. Syst. Bacteriol. 46, 223–228, 1996. 17. Venkateswaran, K., Moser, D., Dollhopf, M., Lies, D., Saffarini, D., MacGregor, B., Ringelberg, D., White, D., Nishijima, M., Sano, H., Burghardt, J., Stackebrandt, E., and Nealson, K. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. Int. J. Syst. Bacteriol. 49, 705–724, 1999. 18. Derby, H. and Hammer, B. Bacteriology of butter. IV. Bacteriological studies on surface taint butter. Iowa Agric. Exp. Stn. Res. Bull. 145, 387–416, 1931. 19. Long, H. and Hammer, B. Classification of organisms important in dairy products. III. Pseudmonas putrefaciens. Iowa Agric. Exp. Stn. Res. Bull. 285, 176–195, 1941. 20. Lee, J., Gibson, D., and Shewan, J. A numerical taxonomic study of Pseudomonas-like marine bacteria. J. Gen. Microbiol. 98, 439–451, 1977. 21. Baumann, L., Baumann, P., Mandel, M., and Allen, R.D. Taxonomy of aerobic marine eubacteria. J. Bacteriol. 110, 02–429, 1972. 22. MacDonell, M. and Colwell, R. Phylogeny of the Vibrionaceae and recommendation for two new genera, Listonella and Shewanella. Syst. Appl. Microbiol. 6, 171–182, 1985. 23. Levin, R.E. Correlation of DNA base composition and metabolism of Pseudomonas putrefaciens isolates from food, human clinical specimens, and other sources. Antonie Van Leeuwenhoek 38, 121–127, 1972. 24. Nozue, H., Hayashi, T., Hashimoto, Y., Ezaki, T., Hamasaki, K., Ohwada, K., and Terawaki, Y. Isolation and characterization of Shewanella alga from human clinical specimens and emendation of the description of S. alga Simidu et al., 1990, 335. Int. J. Syst. Bacteriol. 42, 628–634, 1992. 25. Vogel, B.F., Jørgensen, K., Christensen, H., Olsen, J.E., and Gram, L. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis. Appl. Environ. Microbiol. 63, 2189–2199, 1997.
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26. Sadovski, A. and Levin, R. Extracellular nuclease activity of fish spoilage bacteria, fish pathogens and related species. Appl. Microbiol. 17, 787–789, 1969. 27. Chai, T., Chen, C., Rosen, A., and Levin, R. Detection and incidence of specific spoilage bacteria on fish. II. Relative incidence of P. putrefaciens and fluorescent pseudomonads on haddock fillets. J. Appl. Microbiol. 16, 1738–1741, 1968. 28. Van Sickle, C. and Levin, R. Relative rates of autolysis of high and low GC isolates of Pseudomonas putrefaciens and other gram negative bacteria. Microbios Lett. 6, 85–94, 1978. 29. Juni, E. and Hyme, G. Transformation assay for identification of psychrotrophic achromobacters. Appl. Environ. Microbiol. 40, 1106–1114, 1980. 30. Chen, T. and Levin, R. Taxonomic significance of phenethyl alcohol production by Achromobacter isolates from fishery sources. Appl. Microbiol. 28, 681–687, 1974. 31. Juni, E. and Hyme, G. Psychrobacter immobilis gen. nov., sp. Non.: Genospecies composed of gramnegative, aerobic, oxidase-positive coccobacilli. Int. J. Syst. Bacteriol. 36, 388–391, 1986. 32. Rossau, R., Van Landschot, A., Gillis, M., and De Ley, J. Taxonomy of Moraxellaceae fam. Nov., a new bacterial family to accommodate the genera Moraxella, Acinetobacter, and Psychrobacter and related organisms. Int. J. Syst. Bacteriol. 41, 310–319, 1991. 33. González, C., Santos, A., García-López, M., and Otero, A. Psychrobacters and related bacteria in freshwater fish. J. Food Protect. 63, 315–321, 2000. 34. McMeekin, T. The adensonian taxonomy and the deoxyribonucleic acid base composition of some gram negative, yellow pigmented rods. J. Appl. Bacteriol. 35, 129–137, 1972. 35. Castell, C. and Mapplebeck, E. The importance of Flavobacterium in fish spoilage. J. Fish. Res. Board Can. 9, 148–156, 1952. 36. Hovda, M., Sivertsvik, M., Lunestad, B., Lorentzen, G., and Rosnes, J. Characterization of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-0DGGE. Food Microbiol. 24, 362–371, 2007. 37. Hovda, M., Lunestad, M., Sivertsvik, M., and Rosnes, J. Characterization of the bacterial flora of modified atmosphere packaged farmed atlantic cod (Gadus morhua) by PCr-DGGE of conserved 16S rrNA gene regions. Int. J. Food Microbiol. 117, 68–75, 2007. 38. Sivertsvik, M., Jeksrud, W., and Rosnes, T. A review of modified atmosphere packaging of fish and fishery products—Significance of microbial growth, activities and safety. Int. J. Food Sci. Technol. 37, 107–127, 2002. 39. Mokhele, K., Johnson, A., Barrette, E., and Ogrydziak, D. Microbiological analysis of rock cod (Sebasts spp.) stored under elevated carbon dioxide atmosphere. Appl. Environ. Microbiol. 45, 878– 883, 1983. 40. Johnson, A. and Ogrydziak, D. Genetic adaptation to elevated carbon dioxide atmospheres by Pseudomononas-like bacteria isolated from rock cod (Sebastes spp.). Appl. Environ. Microbiol. 48, 486– 490, 1984. 41. Nealson, K. and Saffarini, D. Iron and manganese in anaerobic respiration: Environmental significance, physiology, and regulation. Ann. Rev. Microbiol. 48, 311–343, 1994. 42. Rudi, K., Maudesten, T., Hannevik, S., and Nissen, H. Explorative multivariate analysis of 16S rRNA gene data from microbial communities in modified-atmosphere packed salmon and coalfish. Appl. Environ. Microbiol. 70, 5010–5018, 2004. 43. Pelroy, G., Seman, J. Jr., and Eklund, M. Changes in the microflora of irradiated petrale sole (Eopsetta jordani) fillets stored aerobically at 0.5 C. Appl. Microbiol. 15, 92–96, 1967. 44. Pelroy, G. and Eklund, M. Changes in the microflora of vacuum-packaged irradiated petrale sole (s) fillets stored at 0.5 C. Appl. Microbiol. 14, 921–927, 1966. 45. Silverrio, R. and Levin, R. Evaluation of methods for determining the bacterial population of fresh fillets. J. Milk Food Technol. 30, 242–246, 1967. 46. Makarios-Laham, I. and Levin, R. Isolation from haddock tissue of psychrophilic bacteria with maximum growth temperatures below 20°C. Appl. Environ. Microbiol. 48, 439–440, 1984.
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47. Haight, R. and Morita, R. Thermally induced leakage from Vibrio marinus, an obligately psychrophilic marine bacterium. J. Bacteriol. 92, 1388–1393, 1966. 48. Sands, D. and Rovira, A. Isolation of fluorescent pseudomonads with a selective medium. Appl. Microbiol. 20, 513–514, 1970. 49. Rosen, A. and Levin, R. Vibrios from fish pen slime which mimic Escherichia coli on Violet Red Bile Agar. Appl. Microbiol. 20, 107–112, 1970. 50. Kovács, N. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature (Lond.) 178, 703, 1956. 51. Bovre, K. and Hendriksen, S. Minimal standards for description of new taxa within the genera Moraxella and Acinetobacter: Proposal by the subcommittee on Moraxella and allied bacteria. Int. J. Syst. Bacteriol. 26, 92–96, 1976. 52. Kazanas, N. Proteolytic activity of microorganisms isolated from freshwater fish. Appl. Microbiol. 16, 128–132, 1968. 53. Levin, R. Detection and incidence of specific spoilage bacteria on fish. I. Methodology. Appl. Microbiol. 16, 1734–1737, 1968. 54. Lee, J. and Levin, R. Selection of universal primers for PCR quantification of total bacteria associated with fish fillets. Food Biotechnol. 20, 275–286, 2006. 55. Lee, J. and Levin, R. Direct application of the polymerase chain reaction for quantification of total bacteria on fish fillets. Food Biotechnol. 20, 287–298, 2006. 56. Lee, J. and Levin, R. Rapid quantification of total bacteria on cod fillets by using real-time PCR. J. Fisheries Sci. 1, 58–67, 2007. 57. Greisen, K., Loeffelholz, M., Purohit, A., and Leong, D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32, 335–351, 1994. 58. Lee, J. and Levin, R. Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction. J. Microbiol. Methods 67, 456–462, 2006. 59. Venkitanarayanan, K., Khan, M., Faustman, C., and Berry, B. Detection of meat spoilage bacteria by using the polymerase chain reaction. J. Food Protect. 59, 845–848, 1996. 60. Locatelli, L., Tarnawsi, S., Hamelin, J., Rossi, P., Aragno, M., and Fromin, N. Specific PCR amplification for the genus Pseudomonas targeting the 3′ half of 16S rDNA and the whole 16S–23S rDNAS spacer. Syst. Appl. Microbiol. 25, 220–227, 2002.
Chapter 29
Detection of Fish Spoilage George-John E. Nychas and E.H. Drosinos Contents 29.1 Introduction ..................................................................................................................537 29.2 Types of Spoilage...........................................................................................................538 29.2.1 Microbial Spoilage ...........................................................................................539 29.2.2 Ephemeral Spoilage Organisms........................................................................539 29.2.3 Physicochemical Spoilage—Fat Oxidation ..................................................... 544 29.2.4 Enzymatic Spoilage ......................................................................................... 544 29.3 Determination Methods ............................................................................................... 544 29.3.1 Sensory Analysis ..............................................................................................545 29.3.2 Total Volatile Bases (TVB) and Trimethylamine (TMA) Determination ........545 29.3.2.1 Solid-Phase Microextraction ............................................................545 29.3.2.2 HS/MS Analysis ............................................................................. 546 29.3.3 Detection of Fish Spoilage by Colorimetry—Texture Assessment ................... 546 29.3.4 Quantifying Biogenic Amines......................................................................... 547 29.3.5 Microbiological Analyses ................................................................................ 548 29.3.6 Rapid Methods for Detection and Quantification of Microorganisms ............ 549 29.3.6.1 Short-Wavelength Near-Infrared Spectroscopic Method (SW-NIR) ....................................................................................... 549 29.3.7 Predicting Fish Spoilage .................................................................................. 549 29.4 Conclusions ...................................................................................................................551 References ................................................................................................................................551
29.1
Introduction
Spoilage is a natural phenomenon that leads to the decomposition of a food substratum. In particular, the spoilage of fish can be considered as an ecological phenomenon that encompasses 537
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a series of changes in the available components (e.g., low molecular weight compounds) through (1) natural processes (e.g., chemical, enzymatic activity, or even autolytic process) and (2) microbial proliferation. A general feature of microbial spoilage is its relatively sudden onset, i.e., it does not appear to develop gradually, but more often as an unexpected and unpleasant revelation. This is a reflection of the exponential nature of microbial growth and its consequence that microbial metabolism can also proceed at an exponentially increasing rate. If a microbial product associated with spoilage, for example an off odor, has a certain detection threshold, the level will be well below this threshold for most of the product’s acceptable shelf life. Many efforts to delay this natural process encompass certain strategies that are determined by general factors of the society. Nowadays the mild preservation of foods and the effort to extend the food shelf life have changed the status of the food enterprises. Quality and food safety management systems are planned, established, and applied, operated and maintained to satisfy the consumers’ needs, expectations, and requirements. Monitoring and validation of the control measures applied are essential parts of the applied systems. Detection of spoilage and estimation of shelf life is of paramount importance during the operation of these systems [25]. It is important to determine the nature of the ephemeral spoilage organisms (ESO) growth and dominance in fish and fish products. Based on this information it is more convenient to establish indexes that reflect the microbiological quality of fish. In addition, microbiological quality will be interpreted in terms of spoilage [9,23,26,36,37,48]. Fish spoilage is a complex process in which physical, chemical, and microbiological mechanisms are implicated. Upon fish harvesting, the fish regulatory mechanisms, which prevent invasion of the tissues by bacteria, cease to function—bacteria or the enzymes invade the flesh of fish. This process produces toxic compounds in the fish and the fish becomes spoiled. The initial stages of fish spoilage is characterized by the loss of characteristic odor and taste are mainly due to autolytic degradation, while the final stages of fish quality deterioration is characterized by softening or toughening of flesh texture along with the production of unpleasant odors and flavors that are mainly due to microbial activity [4,17]. Another cause of fish spoilage is lipid oxidation and hydrolysis that leads to the development of rancidity, even with storage at subzero temperatures. This is due to the large amount of polyunsaturated fatty acid moieties found in fish lipids. In fact, this is a major cause of frozen fish spoilage [28]. During the spoilage, characteristic changes in sensory attributes relate to the appearance, aroma, taste, texture, and appearance will change [51]. Since fish is a very perishable commodity, it has drawn special attention. As most raw materials, fish consist of a large number of species of widely differing appearance and flavor so that customers are often unsure if particular species of products made from them are suitable and safe to eat. The public also becomes more demanding in respect of freshness, microbiological safety, free from pollutants, protection from damage, and convenience. Quality control and labeling of fish products depend on defining the appropriate criteria, which may be of different importance to the various parts of the supply chain in the fish sector. Freshness of fish is the key factor determining quality, but also seasonal variations, catching methods, handling, processing, and storage techniques influence quality [18,20].
29.2
Types of Spoilage
Spoilage of fish and seafood is caused by microbial, oxidative, and enzymatic activity which results in undesirable changes in odor, flavor, and texture [50]. pH of fish muscle is an important factor
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of spoilage. pH values higher than 7, observed when fish is exhausted in glycogen during netting operations, is favorable for microbial and enzymatic spoilage. Evisceration plays an important role in spoilage, e.g., small pelagics are not gutted, hence proteolytic enzymes along with microorganisms rapidly invade the fish’s tissues. Oily fish are susceptible to oxidative rancidity. Cold water species of fish, due to their microbiota psychrophilic, are predisposed to earlier spoilage under chill conditions. Type and rate of spoilage is multifactorial [45].
29.2.1 Microbial Spoilage Fish spoilage commences upon harvesting. At that time, fish come in contact with a totally different microbiota. Microbial activity is retarded, delaying but not inhibiting the spoilage of fish during storage conditions at the temperature of 0°C. Gram-negative, fermentative bacteria (such as Vibrionaceae) spoil unpreserved fish, whereas psychrotolerant Gram-negative bacteria (Pseudomonas spp. and Shewanella spp.) grow on chilled fish [40]. Fish, as a poikilothermic organism, possess a microflora influenced by the temperature of the water and by the microbiota dominating the bottom sediment of the catch area. Fish caught on a line may have lower bacterial counts than fish that are trawled by dragging a net along the bottom; the trawl net drags through the bottom sediment, which usually has high counts of microorganisms. Unlike other crustacean shellfish (i.e., lobster, crab, and crayfish) that are kept alive until preparation for consumption, shrimp die soon after harvesting. Decomposition of shrimp involves bacteria on the surface that originate from the marine environment or are introduced during handling and washing. Molluscan shellfish (i.e., oysters, clams, scallops, and mussels) are sessile and filter feeders and, as such, their microbiota depends greatly on the quality of water in which they reside, the quality of wash water, and other factors [48].
29.2.2
Ephemeral Spoilage Organisms
Studies have established that spoilage is caused only by an ephemeral fraction of the initial microbial association [48]. Th is concept has contributed significantly to our understanding of meat and seafood spoilage. The microbial associations developing on muscle tissues stored aerobically at cold temperatures are characterized by an oxidative metabolism. The Gram-negative bacteria that spoil fish are either aerobes or facultative anaerobes. Pseudomonas spp., Shewanella putrefaciens, and Photobacterium phosphoreum were found to be the dominant species on fish muscle stored at cold temperatures (Tables 29.1 and 29.2). Brochothrix thermosphacta also occur on chilled fish stored mainly under modified atmospheres in significant numbers that contribute to the microbial associations [9]. Lactic acid bacteria are considered to be important in spoilage when fish is stored under modified atmosphere packaging (MAP). Both lactic acid bacteria and B. thermosphacta are probably the most important causes of spoilage characterized by muscle souring. The other distinct type of muscle spoilage is characterized by putrefaction and is related to proteolytic activity and off-odor production by Gram-negative bacteria that dominate under aerobic conditions. Other common spoilage conditions and causative bacteria are listed in Table 29.3 [48,60]. In general, the metabolic activity of the ephemeral microbial association, which prevails in a muscle ecosystem leads to the manifestation of changes that are characterized as spoilage of meat. These undesirable spoilage changes are related to the type, composition, and population of the microbial association and the type and availability of substrates for energy production in
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Table 29.1
Ephemeral Spoilage Organisms (ESOs) in Seafood
Seafood Fresh and chilled, stored in air
Typical ESO S. putrefaciens-likea,b Pseudomonas spp.c
Fresh, chilled and stored in vacuum or modified atmosphere packaging
P. phosphoreumb Lactic acid bacteriac B. thermosphactac
Fresh and lightly preserved products stored at ambient temperature
Aeromonas spp. Vibrio spp. Enterobacteriaceae Enterococcus faecalis
Sous-vide cooked and chill stored
Gram-positive spore formers
Lightly preservedd and chill stored
Lactic acid bacteriae Enterobacteriaceaef P. phosphoreum Vibrio spp.
Semi-preserved, salt cured and chilled
Halobacterium spp., Halococcus spp., and osmotolerant molds and yeasts
Fermented and chilled
Molds and lactic acid bacteria
Sources: Based on Gram L. and Huss, H.H., The Microbiological Safety and Quality of Food, Lund, B.M. et al. (Eds.), Aspen Publishers, Gaithersburg, MD, 2000, 472–502; Gram, L. and Dalgaard, P., Curr. Opin. Biotechnol., 13, 262, 2002; Lambropoulou, K., The effects of varying extrinsic parameters and specific pretreatments in whole fish and prepared fish fillets, PhD Thesis, University of Lincolnshire & Humberside, Lincoln, U.K., 1999; Vogel, B.F. et al., Appl. Microbiol., 71, 6689, 2005; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140. a
b c d
e
Refer to S. putrefaciens, Shewanella baltica, and other closely related H2S-producing, Gram-negative bacteria. Typical of fishes from marine and temperate waters. Typical of fishes from freshwater and fishes from warmer waters. Include, for example, cold-smoked salmon, cooked and brined shrimps and brined roe products. Include, for example, Lactobacillus curvatus and Lactobacillus sakei.
fish (Table 29.4). Indeed the type and the extent of spoilage is governed by the availability of low-molecular-weight compounds (e.g., glucose, lactate) existing in fish; eventual muscle food changes and subsequent overt spoilage are due to catabolism of nitrogenous compounds as well as secondary metabolic reactions.
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Table 29.2 Main Low-Molecular-Weight Components of Beef and Fish Pre- and Post-Rigor Mortis (mg/100 g) Component
Pre
Post
Creatine phosphate
9.3a
0.2a
Creatine
ndb
nd
Betaine
nadc
100d
ATP
6.5a
0.2a
IMP
nd
nd
Glycogen
220
40
Glucose
220
40
Glucose-6-phosphate
21
32
Lactic acid
100
400
pH
7.3
6.5
Free amino acids
nad
250
TMAO
nd
350–1000
Carnosine and anserine
nd
100e
Sources: Huss, H.H., Quality and Quality Changes in Fresh Fish. FAO Fisheries Technical Paper, No. 348, FAO, Rome, Italy, 1995b; Jay, J.M., Modern Food Microbiology, Aspen Publishers, Gaithersburg, MD, 2000; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140. a b c d e
μmol/g. nd: not determined. nad: no available data. In some fish. Cod.
Bacteria are the major cause of spoilage of most seafood products. Their growth and metabolism results in the formation of amines, sulfides, alcohols, aldehydes, ketones, and organic acids with unpleasant and unacceptable off-flavors [49]. Especially ephemeral spoilage organisms (ESOs) give rise to the offensive off-flavors. Although they play only a minor part of the total microbiota, they are classified as active spoilers. These bacteria grow in fish juice producing spoilage off-odors described as fishy, rotten, or cabbage-like. The prominent characteristics of fish spoilage bacteria are an ability to reduce trimethylamine oxide (TMAO) and to produce hydrogen sulfide (H2S). However, ESO are not the same in every case and are dependent on
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Table 29.3 Production of Biogenic Amines by Muscle Microbial Biota in Muscle Foods and Broths Storage Condition
Biogenic Amine PUT
CAD
HI
Bacteria
T (°C)
Medium/Pack
Factors
1
vpa
1
Fish broth
H. alvei, S. liquefaciens, S. putrefaciens
1
vp
pH, lysine utilization
Proteus morganii, Kebsiella. Pneumoniae, H. alvei, Aeromonas hydrophila, Moraxella morganii, Photobacterim phosphoreum, S. putrefaciens
1.7
Fish in vp
Temperature, pH, histidine utilization
2.1
Fish in vp, fish broth
Hafnia alvei, Serratia liquefaciens, Shewanella putrefaciens
1
pH, ornithine (arginine) utilization
SPM
pH, SPD
SPD
pH, agmatine, arginine
TY
Lactobacillus sp., L. carnis, L. divergens, Enterococcus feacalis
Tryptamine
1
vp
20
airb
pH
pH
Sources: Bover-Cid, S. et al., Eur. Food Res. Technol., 216, 477, 2003; Chaouqy, N.E. et al., Sci. Aliment, 25(2), 129, 2005; Dainty, R.H. et al., J. Appl. Bacteriol., 63, 427, 1987; Emborg, J. et al., Int. J. Food Microbiol., 101, 263, 2005; Karpas, Z. et al., Anal. Chim. Acta, 463, 155, 2002; Lopez-Caballero, M.E. et al., Eur. Food Res. Technol., 215, 390, 2002; Luten, J.B. et al., Quality Assurance in the Fish Industry, Huss, H.H. et al. (Eds.), Elsevier, Amsterdam, the Netherlands, 1992, 427–439; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140; Veciana-Nogués, M.T. et al., J. Agric. Food Chem., 45, 2036, 1997. a b
Vacuum pack. Aerobic storage.
the climatic and storage conditions, the type of fish and even the place in which the fish was harvested [20,35,36]. The psychrotrophic nature and the ability of the S. putrefaciens to reduce TMAO to trimethylamine (TMA) explain its importance in spoilage of fi sh stored at low temperatures where the “fi shy” off-odor of spoiling fi sh is caused by the production of TMA. The bacterium also degrades sulfur-containing amino acids and produces volatile sulfides including H 2S [21]. H 2S-producing bacteria constitute only a minor fraction of the initial microbiota on newly caught fi sh, but Gram-negative, psychrotrophic species become dominant during iced storage, and H 2S-producing bacteria will typically grow to levels of 107 colony forming units (CFU)/g.
Detection of Fish Spoilage Table 29.4 Genera of Microorganisms Commonly Found on Seafood Microorganisms
Gram Reaction
Fish
Acinetobacter
−
X
Aeromonas
−
X
Alcaligenes
−
X
Alteromonas
−
X
Bacillus
+
X
Brochothrix
+
X
Chromobacterium
−
X
Corynebactenum
+
X
Bacteria
Cytophaga
X
Enterobacter
−
X
Enterococcus
+
X
Flavobacterium
−
X
Halobacterium
−
X
Lactobacillus
+
X
Microbacterium
+
X
Moraxella
−
X
Morganella
−
X
Photobacterium
−
X
Pseudomonas
−
XX
Shewanella
−
X
Staphylococcus
+
X
Streptococcus
+
X
Vibrio
−
X
Weissella
+
X
Yersinia
−
X
X = known to occur; XX = most frequently isolated.
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The appearance and physical properties of the fish body change due to action of the microorganisms. Besides odor and flavor the slime on skin and gills, initially watery and clear, becomes cloudy, clotted, and discolored. Skin loses its bright irridescent appearance, bloom and smooth feel becomes dull, bleached and rough to the touch [4,18].
29.2.3 Physicochemical Spoilage—Fat Oxidation Oxygen promotes several types of deteriorative reactions in foods including fat oxidation, browning reactions, and pigment oxidation. There is a wide surface area exposed to oxygen on fresh fish. Fish lipids are rich in the polyunsaturated fatty acids called omega-3 fatty acids. The lipids in oily fish are concentrated in a layer beneath the skin in contiguity with the dark muscle bands which contain powerful oxidizing enzymes such as cytochrome c. Oxygen reacts with this fat to produce rancidity, and undesirable strong odoriferous and flavorous compounds are produced. Fish spoil rapidly in air due to moisture loss or uptake, reaction with oxygen, and the growth of aerobic micro-organisms i.e., bacteria and moulds. Fat oxidation can be a serious problem for frozen fish stored for very long. This is one reason why fatty fish like bluefish do not remain in good condition during frozen storage unlike leaner fish like flounder [27].
29.2.4
Enzymatic Spoilage
Digestive enzymes may begin to digest the fish itself, causing belly burn or softening of the flesh around the gut. While the quantity of the enzymes rises, autolytic changes occur and flavor components make the flesh tasteless. This is especially likely if a fish is caught while feeding, since its digestive enzymes are already active. Other enzymes in fish muscle can also begin to affect the flavor and texture of the fillet. At warmer temperatures, the enzymes act more rapidly. But even at low temperatures, enzymatic processes may lead to a product showing a high degree of proteolysis. As a consequence of enzymatic decarboxylation of amino acids due to microbial enzymes and— to a lesser extent—to tissue activity, biogenic amines accumulate in fish, and other foods. Main biogenic amines usually found in fresh and processed meat products are putrescine (PUT), cadaverine (CAD), histamine (HI), and tyramine (TY) while natural polyamines (PAs) spermidine (SPD), and spermine (SPM) content slightly changes during storage or processing [24].
29.3 Determination Methods A well-established method for the evaluation of fish freshness is the sensory method while quality index method (QIM) is also used as a reference method [52]. QIM is based on well-defined characteristic changes of quality-related attributes (eyes, skin, gills, and smell) and corresponding score system of index points. The activity of micro-organisms is the main factor limiting the shelf life of fresh fish resulting in the degradation of the fish and the development of off-flavors. The estimation of the total viable counts and measurement of chemical indicators are used as acceptability indices in guidelines and specifications. Sensory and microbial analyses are lengthy procedures, so rapid chemical, biochemical, and physical methods are better solutions to measure the freshness of fish. These methods are based on e.g., nucleotide catabolism or production of amines, or physical properties e.g., electrical properties by handheld devices. However, none of these methods are widely used in fish industry [51]. In recent years, research has focused on developing new, rapid instrumental methods
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to detect the freshness of fish. Several new promising techniques have been developed and some of them have shown good correlation with traditional methods evaluating quality of freshness [22].
29.3.1
Sensory Analysis
Sensorial analysis employs several criteria, including the appearance of the skin, eyes, mucus and gills, the firmness of the flesh, and odor. These criteria are checked by trained specialists with evaluation equipment. They determine the samples using appropriate common, universal food sensory analysis. These are wholly dependent upon the human senses: sight, touch, odor, and flavor. Measure of a particular aspect of quality is usually compared to a standard, having gradations of quality (a wellknown scale). It is always possible to construct a scale showing change or incidence by the use of value words: slight, trace, medium, moderate, very highly. A further development of scales is to denote the different steps by numbered scores from 0 or 1 upwards. Human senses are better at recognizing complexities and are more discriminatory than instruments, but such determination of freshness is in part subjective, because of personal expressions. In addition, for transformed products, such as fillets, sensory analysis is less reliable because the number of assessment criteria diminishes [4,5].
29.3.2
Total Volatile Bases (TVB) and Trimethylamine (TMA) Determination
The procedure of this method is to extract nitrogen, the increase of which is one of the causes of fish spoilage, and then express the result as mg of nitrogen in 100 g of sample. During the extraction the sample must be homogenized first, then trichloroacetic acid (TCA) is added, and fi ltered. The extract is placed in a Conway plate and total volatile bases (TVB) are determined according to Conway’s microdiff usion method. The method is based on an osmotic mechanism and capillary electrophoresis. Volatile basic nitrogen (VBN) is collected with hydrochloric acid solution. Flow injection analysis (FIA), gas diff usion cell, and a laboratory-built photometer monitor TMA and total volatile bases (TVB) in fish sauce. Peak of TMA is influenced by sample matrix.
29.3.2.1
Solid-Phase Microextraction
Solid-phase microextraction (SPME) is a proven tool in volatile analysis and has previously been used for the analysis of flavor volatiles in seafood as well as in a variety of other foods. In general, SPME is both simple and cost-effective to use and can be used to analyze the levels of a wide range of volatile compounds. The method was developed for the analysis of salmon volatiles using SPME and gas chromatography (GC)–mass spectrometry (MS). The levels of several of the volatile compounds change during storage. There are several alcohols and aldehydes identified as potential markers for salmon freshness (e.g., cyclopentanol, hexanal). Some other volatiles (acetoin, ethyl benzene, propyl benzene, styrene, 3-methyl butanoic acid, and acetic acid) were identified as potential markers for salmon spoilage. The method can be used manually with any GC or GC/MS. GC can separate volatile and semivolatile compounds with great resolution, but it cannot identify them. MS can provide detailed structural information on most compounds and identify them, but it cannot readily separate them. This contributed to an idea of the combination of the two techniques. In both techniques, the sample is in the vapor phase, and both techniques deal with about the same amount of sample (usually less than 1 ng). The vapor phased sample is carried
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by the gas that is almost always a small molecule such as helium or hydrogen with a high diffusion coefficient. In MS method, organic molecules have much lower diffusion coefficients. GC effluent (the carrier gas with the organic analytes) is sprayed through a small nozzle because of the high diff usion coefficient; the helium is sprayed over a wide solid angle. And in MS spectrometry the heavier organic molecules are sprayed over a much narrower angle and tend to go straight across the vacuum region. Then the gas passes it to packed columns of the mass spectrometer. The higher-molecular-weight organic compounds are separated from the carrier gas, which is removed by the vacuum pump. The compounds of the sample are separated by the special separating material inside (e.g., separators made from glass drawing down a glass capillary). The particles escape from the column and are lodged in the spray orifice and stop (or at least severely reduce) the gas flow out of the GC column and into the mass spectrometer. All modern GC–MS data systems are capable of displaying the mass spectrum on the computer screen as a bar plot of normalized ion abundance versus mass-to-change (m/z) ratio (often called mass). Like the other parts of the GC–MS instrument, the data system must be calibrated [31]. FIA appears to be an adequate methodology for quality control in fish sauce production with the advantages of reduced sample volume, energy consumption, and cost. Unfortunately, there is a major incompatibility between the two techniques: The compound exiting the gas chromatograph is a trace component in the GC’s carrier gas at a pressure of about 760 torr, but the mass spectrometer operates at a vacuum of about 10−6 to l0−5 torr. This is a difference in the pressure of eight to nine orders of magnitude, a considerable problem.
29.3.2.2
HS/MS Analysis
Samples are equilibrated at room temperature by immersion in a water-bath for 30 min and homogenized for 1 min. The sample is washed and stored at −80°C before the analysis. To eliminate variations due to time spent in the automatic sample carousel, each vial is stored at 4°C and then equilibrated for 10 min at room temperature before analysis. To minimize the influence of the column of a gas chromatograph, the oven temperature is set at 220°C to permit simultaneous elution of compounds and direct observation of the abundance of ions.
29.3.3 Detection of Fish Spoilage by Colorimetry—Texture Assessment A colorimetry is a rapid method for evaluating the degree of bacterial degradation of finfish (such as codfish, catfish, and winter flounder). It can be carried out quickly by any lay person, with minimum training, and with portable supplies. It can be also carried out in any environment, e.g., on a fishing boat, without interference from the odor of the environment. What is very important—test results are objective and visually demonstrated by a color comparison which is clear to any lay person without the need for special training or evaluation equipment. A simplified colorimetric test process is based upon the discovery that triphenyl tetrazolium dye salts are colorless, ionized, water soluble, and capable of passing through the cell wall into a bacterial cell while undergoing a reduction reaction to form a nonionic, water-insoluble, and red-colored triphenyl tetrazolium formazan compound which is deposited within the bacterial cells. The intensity of the formed color is proportional to the concentration of the bacteria present, necessary to produce the reduction reaction, and therefore the visible color intensity provides a
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measure of the bacterial concentration which, in turn, provides an indication of the quality of the fish being tested. In the case of dark-flesh finfish, such as tuna fish, additional reactants such as an oxidizing or bleaching agent, preferably hydrogen peroxide and a defoamer are added to bleach the muscle pigments and lighten the color of the solution so that any color that appears during the assay is due to the reduction of the tetrazolium dye. The intensity of the color of the solution is compared by a visual colorimetric comparison with control color strips representative of known concentrations of formazan solution ranging from substantially colorless, for a product of excellent quality, light reddish color representing good quality, darker red color representing borderline quality, and intense red color representing unacceptable quality. These control colors are preliminarily determined by known traditional methods used to measure the bacterial content of finfish of excellent, good, borderline, and unacceptable freshness, such as total plate count determination, TMA determination, and sensory (odor) determination. Most of these traditional methods are not practical and need special, demanding environment. Ammonia is one of the compounds produced as fish or fish-products spoil. The study of the sensor response to standard ammonia gas provided a model of the response to total volatile base nitrogen (TVB-N). If amines and ammonia are produced during fish spoilage, the concentration TVB-N in the headspace is likely to be sufficiently high so that it can be detected by this proposed sensor (colorimetric sensor). In this method synthetic ammonia gas in nitrogen is used in the colorimetry to characterize the sensor. Further dilution of the ammonia gas with nitrogen is achieved through the use of mass flow controllers. The sensor is placed into a flow cell fitted with the optical scanner, which monitors in real time the sensor responses to changing ammonia concentration. A PC connected to the optical scanner log the data [53,54].
29.3.4
Quantifying Biogenic Amines
The determination of biogenic amines in fresh and processed food is gaining great interest not only for their potential risk for human health, but also because they could have a role as chemical indicators of unwanted microbial contamination and processing conditions. Biogenic amines are indicators of fish spoilage because their precursor amino acids are decarboxylated by bacterial enzymes (Table 29.3). We can monitor the presence of diacetyl by exposing an aromatic orthodiamine at acidic pH to an environment containing, or possibly containing, diacetyl and detecting any change in the absorption or reflection of electromagnetic radiation due to the ortho-diamine. The change may suitably, though not necessarily, be in the UV or visible region or both. At first a previous confirmation is necessary, and then an extraction with hydrochloric acid [5]. With high-performance liquid chromatography (HPLC), analytes are carried through a column filled with packing material (stationary phase) via a mobile phase (e.g., organic solvent). Individual analytes have varying affinities for the stationary phase and have characteristic retention times that dictate when each component will elute. Components coming off the column are visualized with a detector, e.g., fluorescence. This monitors eluents based on the ability of the sample compound to absorb light at a specific wavelength. Unfortunately, there are drawbacks of these methods that are related to precolumn, postcolumn, or on-column derivative process leading to an overall long analysis time, and low reproducibility owing to the stability of the derivatization products. Pre derivatization consists of a series of manual time consuming steps that may introduce imprecision but offers certain selectivity. Postcolumn derivatization has the advantage that it is in line, but it adds complexity to the instrumentation, and system must be set up in order to reduce the contributions to band widening. Changing of pH with a postcolumn system is simple, easy, and quick.
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The example of the chromatographic technique is ion-pair chromatographic method on a C18 reversed-phase column involving a postcolumn reaction with o-phthaldialdehyde (OPA) to form fluorescent derivatives with amines. For HI, European Community and Spanish regulations have fi xed a maximum average value of 100 mg/kg in a group of nine samples of fresh or canned fish and lower than twice this value for ripened fish products. The U.S. Food and Drug Administration have lowered the HI defect action level from 100 to 50 mg/kg.
29.3.5
Microbiological Analyses
Bacterial growth is a determinant factor in shell fish. The microbiota that is responsible for fish spoilage is formed by Gram-negative bacteria. Microbial spoilage is identified by the appearance of colonies. Certain spoilage metabolites can be used as quality indices. The shelf life of fish and shellfish is correlated to the level of specific spoilage bacteria and not to the count of total viable organisms, as it was thought before. Spoilage is organoleptically detectable when the number of sulfide producing bacteria exceeds 107 CFU/g of fish muscle (Table 29.5). The bacteriological production of hydrogen sulfide is a very common cause of spoilage in a variety of foods such as chilled fish. A number of attempts have therefore been made to detect the H2S-producing bacteria and various media have been developed. The example is a peptone iron agar (PIA) based on the detection of H2S-producing bacteria which appear as black/gray colonies due to the precipitation of sulfide complexes which are formed when H2S produced from thiosulfate reacts with metal ions such as Fe2+ or Pb2+. The number of black colonies on cysteine containing IA is, therefore, a good indication of the number of spoilage organisms in wet fish.
Table 29.5 Specific Spoilage Microbiota Dominating on Fresh Fish Stored at 0–4°C under Different Gas Atmospheres Gas Composition
Fish
Air
S. putrefaciens, Pseudomonas spp.
>50% CO2 with O2
B. thermosphacta, S. putrefaciens
50% CO2
P. phosphoreum, lactic acid bacteria
<50% CO2 with O2
P. phosphoreum, lactic acid bacteria,
100% CO2
B. thermosphacta lactic acid bacteria
Vacuum packaged
Pseudomonas spp.
Sources: Based on Gram, L. and Huss, H.H., Int. J. Food Microbiol., 33, 121, 1996; Huss, H.H., Assurance of Seafood Quality, FAO Fisheries Technical Paper, No. 334, United Nations Food and Agriculture Organization, Rome, Italy, 1995a; Nychas, G.-J.E. et al., Food Microbiology Fundamentals and Frontiers, Doyle, M.P. et al. (Eds.), ASM Press, Washington, D.C., 2007, 105–140.
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Rapid Methods for Detection and Quantification of Microorganisms
Detecting and enumerating sulfide-producing bacteria (SPB) is based on bacterial growth. SPB, such as S. putrefaciens, are present in seawater and on the surface of all living fish and shellfish, and are transferred to the flesh during catch and processing. Thus they are especially responsible for fish and fish products spoilage, even when the fish are stored on ice. A growth medium containing iron and sulfur is combined with the food sample forming an incubation mixture which is incubated for a period of time. SPB are determined in the sample if the fluorescence measurement initially increases and then decreases to form a fluorescence maximum (peak). The time to detect the fluorescence peak can be used with a correlation schedule to enumerate the SPB in the food sample. A visual test can also be used to identify color changes in the incubation mixture to provide a semiquantitative enumeration of SPB effective after incubation.
29.3.6.1 Short-Wavelength Near-Infrared Spectroscopic Method (SW-NIR) In this method, the key to determine fish spoilage and to quantify microbial loads is to use visible and short-wavelength near-infrared (SW-NIR: 600–1100 nm) spectroscopy. In the SW-NIR region, various fundamental molecular vibrations, including those from CH, O–H, N–H, C=O, and other functional groups can be detected [42]. If a fish sample is irradiated with NIR light, it absorbs the light with frequencies matching characteristic vibrations of particular functional groups, whereas the light with other frequencies will be transmitted or reflected. Therefore, the biochemical components of a food tissue determine the amount and frequency of absorbed light and the quantity of reflected or transmitted light can be used to infer the chemical composition of that food tissue [42].
29.3.7 Predicting Fish Spoilage The basic question to be addressed among scientists dealing with predicting modeling is “how wrong do they have to be not to be useful?” Mathematical models have been developed for the quantification of parameters, e.g., temperature, aw, pH, and carbon dioxide that affect growth and survival of spoilage bacteria such as B. thermosphacta, lactic acid bacteria, P. phosphoreum, pseudononads, S. putrefaciens, Listeria monocytogenes, and Salmonella in muscle foods [2,8,10,12,13,39,46,47,57,59]. However, the application of these models has not been focused on monitoring muscle food quality per se, but mainly as a management tool for shelf life and safety prediction and as a scientific tool to gain insight into muscle food spoilage. Different models (kinetic or stochastic) have been developed for various muscle products but still an accurate prediction of shelf life is not attainable [15,33,34,56,59,61,62]. There are limited, successfully validated models for predicting the growth of ESOs that have been included in application software and this has facilitated the prediction of food shelf life under constant and dynamic temperature storage conditions (Table 29.6). Measurement of enzyme synthesis and activity could be used to estimate shelf life, offering a completely different modeling approach [58]. The quality of the fish depends on how far the normal spoilage processes have progressed. These processes cannot be stopped, but the rate at which they occur can be controlled. The most
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Table 29.6
Available Shareware Software for Fish Spoilage
Seafood Spoilage and Safety Predictor (Shelf life of seafoods and growth of ESOs; Listeria monocytogenes in cold-smoked salmon) Dalgaard et al. [7]. Safety Monitoring and Assurance System (Koutsoumanis et al. [34,38]) (Greek predictive microbiology application software under development); software is based on kinetic data of spoilage bacteria derived from fish, meat and milk in situ. Gri-Gri*; shelf life modeling of fish stored under various storage conditions (*Greek for the fishermen setting sail in the evening) Software produced by K. Koutsoumanis (kkkoutsou@ auth.agr.gr) within G Nychas’s group (
[email protected]) of the Agricultural University of Athens, Greece. Other available software not specifically focused on fish GrowthPredictor (UK)—www.ifr.ac.uk/Safety/GrowthPredictor/(based on data previously used in the FoodMicromodel software; 18 models for growth of pathogenic bacteria; available free of charge). Sym’Previus—www.symprevius.net (French predictive microbiology application software under development). Pathogen Modelling Program (USA)—www.arserrc.gov/mfs/pathogen.htm (37 models of growth, survival and inactivation; frequently updated (version 7.0); Available free of charge during the last 15 years; ~5000 downloads per year. Sources: Based on Dalgaard, P. et al., Int. J. Food Microbiol., 73, 343, 2002; Koutsoumanis, K. et al., Int. J. Food Microbiol., 73, 375, 2002; Palleroni, N.J., The Prokaryotes, Balows, A. et al. (Eds.), Springer-Verlag, New York, 1992, 3071–3085. www.arserrc.gov/mfs/pathogen.htm; www.ifr. ac.uk/Safety/GrowthPredictor/.
important thing is to control the storage temperature—fresh, unfrozen fish should be kept as close as possible to 0oC, so the best way to do that is to pack fish in ice or ice water. The prediction can mean acidification or the addition of preservatives like sorbate and benzoate. Also drying or heavy dry-salting of fish eliminates bacterial growth. Recently preservation of fish shelf life has relied on the elimination or growth inhibition of spoilage organisms because the growth of bacteria is exponential. Respiratory Gram-negative bacteria are typically inhibited in fish products preserved by the addition of low levels of NaCl, a slight acidification and chill storage in vacuum packs. Under these conditions, the microflora typically becomes dominated by Lactobacillus spp. and Carnobacterium spp. with an association of Gram-negative fermentative bacteria such as P. phosphoreum and psychrotrophic Enterobacteriaceae. Growth of the ESOs can help in prediction from fish spoilage, but the technique that identifies the bacteria must be capable of detecting as little as 102 ESO/g of product. Shelf live can be predicted as the time ESO require to multiply from initial number to the minimal spoilage level (MLS). An example of the ESO’s technique is the CO2 packing of fresh fish. The respiratory spoilage bacteria (Shewanella and Pseudomonas) are inhibited then and the shelf life is markedly extended [14,18].
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Conclusions
Fresh fish is a product with a very short commercial life and high variability. After caching, unpreserved fish is spoiled by Gram-negative, fermentative bacteria (such as Vibrionaceae). Generally, the rates at which autolytic and microbial spoilage occur are dependent on the degree of microbial contamination and biota, storage temperature, and packaging. Fish spoils through the combined effects of chemical reactions, via the continuing activities of endogenous enzymes and lipid oxidation. The factors that influence the microbial contamination and growth include fish species and size, method of catch, on-board handling, fishing vessel sanitation, processing, and storage condition. The major cause of food spoilage is microbial growth and metabolism resulting in the formation of amines, sulfides, alcohols, aldehydes, ketones, and organic acids with unpleasant and unacceptable off-flavors [18]. To determine the level of fish spoilage, sensory and microbiological analyses are most widely used. Sensory analysis is appropriate for product development, but is costly and therefore not an attractive proposition for routine analyses. Microbiological methods give retrospective information which is satisfactory for product safety validation but unsuitable for product monitoring. Even when the ESOs are well established, there are instances where their enumeration is of limited value. Combining microbial ecology, molecular techniques, analytical chemistry, sensory analysis, and mathematical modeling allows us to characterize the ESOs and to develop methods to determine, predict, and extend the shelf life of products. Enumeration of ESO bacteria requires the development of rapid methods because 2–4 days are not reliable to determine the quality of a very perishable product such as fish. More rapid alternatives are being developed, e.g., epifluorescent microscopy, flow cytometry, and electrical impedance. Compared with microbiological methods, which are slow, chemical analyses may be significantly faster; however, for some compounds measurable concentrations are not present until close to spoilage. Smart packaging and use of field-validated indicators offer a new approach in detecting spoilage by the consumer [15,16]. The spoilage can be predicted in fish and fish products preserved by the most commonly used methods like low temperature storage, dehydration, canning, MAP, irradiation, the use of chemical and biological preservatives, and combinations of two or more of these methods. All these contribute to the improvement of quality and safety of seafood.
References 1. Bover-Cid, S., Hernandez-Jover, T., Miguelez-Arrizado, M.J., and Vidal-Carou, M.C. 2003. Contribution of contaminant enterobacteria and lactic acid bacteria to biogenic amine accumulation in spontaneous fermentation of pork sausages. European Food Research Technology 216: 477–482. 2. Bovill, R.A., Bew, J., and Baranyi, J. 2001. Measurements and predictions of growth for Listeria monocytogenes and Salmonella during fluctuating temperature II. Rapidly changing temperatures. International Journal of Food Microbiology 67: 131–137. 3. Chaouqy, N.E., Marakchi, E.I., and Zekhnini, A. 2005. Bacteria active in the spoilage of anchovy (Engraulis encrasicholus) stored in ice and at ambient temperature. Science Aliment 25(2): 129–146. 4. Connell, J.J. 1995. Control of Fish Quality, 4th edn. Fishing News Books, Oxford, U.K. 5. Dainty, R.H. 1996. Chemical biochemical detection of spoilage. International Journal of Food Microbiology 33: 19.
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6. Dainty, R.H., Edwards, R.A., Hibbard, C.M., and Ramantanis, C.V. 1987. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperature. Journal of Applied Bacteriology 63: 427–434. 7. Dalgaard, P., Buch, P., and Silberg, S. 2002. Seafood spoilage predictor – development and distribution of a product specific application software. International Journal of Food Microbiology 73: 343–349. 8. Devlieghere, F., Geeraerd, A.H., Versyck, K.J., Vandewaetere, B., Van Impe, J., and Debevere, J. 2001. Growth of Listeria monocytogenes in modified atmosphere packed cooked meat products: A predictive model. Food Microbiology 18: 53–66. 9. Drosinos, E.H. and Nychas, G.-J.E. 1996. Brochothrix thermosphacta, a dominant microorganism in Mediterranean fresh fish (Sparus aurata) stored under modified atmosphere. Italian Journal of Food Science 4: 323. 10. Duff y, L.L., Vanderlinde, P.B., and Grau, F.H. 1994. Growth of Listeria monocytogenes on vacuumpacked cooked meats: Effect of pH, aw, nitrite and ascorbate. International Journal of Food Microbiology 23: 377–390. 11. Emborg J., Laursen, B.G., and Dalgaard, P. 2005. Significant histamine formation in tuna (Thunnus alacares) at 2°C effect of vacuum – and modified atmosphere-packaging on psychrotolerant bacteria. International Journal of Food Microbiology 101: 263–279. 12. Farber, J.M., Cai, Y., and Ross, W.H. 1996. Predictive modeling of the growth of Listeria monocytogenes in CO2 environments. International Journal of Food Microbiology 32: 133–144. 13. Fernandez, P.S., George, S.M., Sills, C.S., and Peck, M.W. 1997. Predictive model of the effect of CO2, pH, temperature, and NaCl on the growth and survival of foodborne pathogenic bacteria. International Journal of Food Microbiology 37: 37–45. 14. Fraser, O. and Sumar, S. 1998. Compositional changes and spoilage in fish. Nutrition and Food Science 98: 275. 15. Giannakourou, M., Koutsoumanis, K., Nychas, G.-J.E., and Taoukis, P.S. 2001. Development and assessment of an intelligent Shelf life Desicion System (SLDS) for quality optimization of the food chill chain. Journal of Food Protection 64: 1051. 16. Giannakourou, M.C., Koutsoumanis, K., Nychas, G.-J.E., and Taoukis, P. 2005. Field evaluation of the application of time temperature integrators for monitoring fish quality in the chill chain. International Journal of Food Microbiology 102: 323. 17. Gill, T.A. 1992. Biochemical and chemical indices of seafood quality. In Quality Assurance in the Fish Industry. H.H. Huss, M. Jakobsen, and J. Liston (eds.), pp. 377–398, Elsevier, Amsterdam, London, New York, and Tokyo. 18. Gram, L. and Dalgaard, P. 2002. Fish spoilage bacteria – problems and solutions. Current Opinion in Biotechnology 13: 262. 19. Gram, L. and Huss, H.H. 1996. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33: 121–137. 20. Gram, L. and Huss, H.H. 2000. Fresh and processed fish and shellfish. In The Microbiological Safety and Quality of Food. B.M. Lund, T.C. Baird-Parker, and G.W. Gould (eds.), pp. 472–502, Aspen Publishers, Gaithersburg, MD. 21. Gram, L., Trolle, G., and Huss, H.H. 1987. Detection of specific spoilage bacteria from fish stored at low (0oC) and high (20oC) temperatures. International Journal of Food Microbiology 4: 65. 22. Haugen, J.E., Chanie, E., Westad, F., Jonsdottir, R., Bazzo, S., Labreche, S., Marcq, P., Lundby, F., and Olafsdóttir, G. 2006. Rapid control of smoked Atlantic salmon (Salmo salar) quality by electronic nose: Correlation with classical evaluation methods. Sensors and Actuators B 116: 72. 23. Herbert, R.A., Hendrie, M.S., Gibson, D.M., and Shewan, J.M. 1971. Bacteria active in the spoilage of certain sea foods. Journal of Applied Bacteriology 34: 41. 24. Hernández-Jover, T., Izquierdo-Pulido, M., Veciana-Nogués, M.T., Mariné-Font, A., and VidalCarou, M.C. 1997. Biogenic amine and polyamine contents in meat and meat products. Journal of Agricultural and Food Chemistry 45: 2780–2784.
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25. Hernández Palacios, M.R. 2001. Study of the quality management system and product traceability in a fish processing company. The United Nations University, Fisheries Training Programme, Iceland. 26. Hozbor, M.C., Saiz, A.I., Yeannes, M.I., and Fritz, R. 2006. Microbiological changes and its correlation with quality indices during aerobic iced storage of sea salmon (Pseudopercis semifasciata), LWT-Food Science and Technology 39 (2): 99–104. 27. Huss, H.H. 1995a. Assurance of Seafood Quality, FAO Fisheries Technical Paper, No. 334. United Nations Food and Agriculture Organization, Rome, Italy. 28. Huss, H.H. 1995b. Quality and Quality Changes in Fresh Fish. FAO Fisheries Technical Paper, No 348. FAO, Rome, Italy. 29. Huss, H.H., Dalgaard, P., and Gram, L. 1997. Microbiology of fish and fish products. In Seafood from Producer to Consumer, Integrated Approach to Quality, Development in Food Science, vol. 38. J.B. Luten, T. Borresen, and J. Oehlenschlager (eds.), pp. 413–430, Elsevier, New York. 30. Jay, J.M. 2000. Modern Food Microbiology, 6th edn. Aspen Publishers, Gaithersburg, MD. 31. Jónsdóttir, R., Ólafsdóttir, G., Chanie, E., and Haugen, J.-E. 2008. Volatile compounds suitable for rapid detection as quality indicators of cold smoked salmon (Salmo salar). Food Chemistry 109: 184–195. 32. Karpas, Z., Tilman, B., Gdalevsky, R., and Lorber, A. 2002. Determination of volatile biogenic amines in muscle food products by ion mobility spectrometry. Analytica Chimica Acta 463: 155–163. 33. Koutsoumanis, K.P. 2001. Predictive modeling of the shelf life of fish under non isothermal conditions. Applied and Environmental Microbiology 76: 1821–1825. 34. Koutsoumanis, K., Giannakourou, M., Taoukis, P.S., and Nychas, G.-J.E. 2002. Application of SLDs (Shelf life Decision system) to marine cultured fish quality. International Journal of Food Microbiology 73: 375. 35. Koutsoumanis, K., Lambropoulou, K., and Nychas, G.-J.E. 1999. Biogenic and sensory changes associated with the microbial flora of Mediterranean gilt-head seabream (Sparus aurata) stored aerobically at 0, 8, and 15°C. Journal of Food Protection 62: 392. 36. Koutsoumanis, K. and Nychas, G.-J.E. 1999. Chemical and sensory changes associated with microbial flora of Mediterranean boque (Boops boops) stored aerobically at 0, 3, 7 and 10°C. Applied and Environmental Microbiology 65: 698. 37. Koutsoumanis, K. and Nychas, G.-J.E. 2000. Application of a systematic experimental procedure in order to develop a microbial model for rapid fish shelf life predictions. International Journal of Food Microbiology 60: 171. 38. Koutsoumanis, K., Stamatiou, A., Skandamis, P., and Nychas, G.-J.E. 2006. Development of a microbial model for the combined effect of temperature and pH on spoilage of ground meat and validation of the model under dynamic temperature conditions. Applied and Environmental Microbiology 72: 124–134. 39. Koutsoumanis, K.P. and Taoukis, P. 2005. Meat safety, refrigerated storage and transport: Modeling and management. In Improving the Safety of Fresh Meat. J.N. Sofos (ed.), pp. 503–561, Woodhead/ Publishing, Ltd., CRC Press, Cambridge, U.K. 40. Koutsoumanis, K.P., Taoukis, P., Drosinos, E.H., and Nychas, G.-J.E. 2000. Applicability of an Arrhenius model for the combined effect of temperature and CO2 packaging on the spoilage microflora of fish. Applied Environmental Microbiology 66: 3528. 41. Lambropoulou, K. 1999. The Eff ects of Varying Extrinsic Parameters and Specific Pretreatments in Whole Fish and Prepared Fish Fillets. PhD Thesis. University of Lincolnshire & Humberside, Lincoln, U.K. 42. Lin, M., Mousavi, M., Al-Holy, M., Cavinato, A.G., and Rasco, B. 2006. Rapid near infrared spectroscopic method for the detection of spoilage in rainbow trout (Oncorhynchus mykiss) fillet. Journal of Food Science 71: S18. 43. Lopez-Caballero, M.E., Torres, M.D.A., Sanchez-Fernandez, J.A., and Moral, A. 2002. Photobacterium phosphoreum isolated as a luminescent colony from spoiled fish, cultured in model system under controlled atmospheres. European Food Research Technology 215: 390–395.
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44. Luten, J.B., Bouquet, W., Seuren, L.A.J., Burggraaf, M.M., Riekwel-Booy, G., Durand, P., Etienne, M. et al. 1992. Biogenic amines in fishery products: Standardization methods within EC. In Quality Assurance in the Fish Industry. H.H. Huss, M. Jakobsen, and J. Liston (eds.), pp. 427–439, Elsevier, Amsterdam, the Netherlands. 45. Luten, J.B., Oehlenschlager, J., and Olafsdóttir, G. 2003. Quality of fish from catch to consumer; labelling, monitoring and traceability. Wageningen Academic Publishers, Wageningen, the Netherlands. 46. McClure, P.J., Blackburn, C., Cole, M.B., Curtis, P.S., Jones, J.E., Legan, J.D., Ogden, I.D. et al. 1994. Modeling the growth, survival and death of microorganisms in foods: The UK food micromodel approach. International Journal of Food Microbiology 34: 265–275. 47. McMeekin, T.A., Olley, J.N., Ross, T., and Ratkowsky, D.R. 1993. Predictive Microbiology — Theory and Application. Research Studies Press Ltd., Taunton, U.K., p. 339. 48. Nychas, G.-J.E., Marshall, D., and Sofos, J. 2007. Meat poultry and seafood. In Food Microbiology Fundamentals and Frontiers, 3rd edn. Eds Doyle, M.P., Beuchat, L.R., and Montville, T.J. (eds.), pp. 105–140, ASM Press, Washington, D.C. 49. Olafsdóttir, G., Jonsdottir, R., Lauzon, H.L., Luten, J., and Kristbergsson, K. 2005. Characterization of volatile compounds in chilled cod (Cadus morhua) fillets by gas chromatography and detection of quality indicators by an electronic nose. Journal of Agricultural and Food Chemistry 53: 10140. 50. Olafsdóttir, G., Lauzon, H.L., Martinsdottir, E., and Kristbergsson, K. 2006. Influence of storage temperature on microbial spoilage characteristics of haddock fillets (Melanogrammus aeglefinus) evaluated by multivariate quality prediction. International Journal of Food Microbiology 111: 112. 51. Olafsdóttir, G., Martinsdottir, E., Oehlenschlager, J., Dalgaard, P., Jensen, B., Undeland, I., Mackie, I.M., Henehan, G., Nielsen, J., and Nielsen, H. 1997. Methods to evaluate fish freshness in research and industry. Trends in Food Science and Technology 8: 258. 52. Olafsdóttir, G., Nesvadba, P., Di Natale, C., Careche, M., Oehlenschläger, J., Tryggvadóttir S.V., Schubring R. et al., 2004. Multisensor for fish quality determination. Trends in Food Science and Technology 15: 86. 53. Pacquit, A., Frisby J., Diamond D., Lau K.T., Farrell A., Quilty B., and Diamond, D. 2007. Development of a smart packaging for the monitoring of fish spoilage. Food Chemistry 102: 466. 54. Pacquit, A., Lau, K.T., and Diamond, D. 2004. Development of a colorimetric sensor for monitoring of fish spoilage amines in packaging headspace. Sensors 1: 365. 55. Palleroni, N.J. 1992. Introduction to the family Pseudomonadaceae. In The Prokaryotes, 2nd edn. A. Balows, H.G. Truper, M. Dworkin, W. Harder, and K.-H. Schleifer (eds.), pp. 3071–3085, Springer-Verlag, New York. 56. Rasmussen, S.K.J., Ross, T., and McMeekin, T. 2002. A process risk model for the shelf life of Atlantic salmon fillets. International Journal of Food Microbiology 73: 47–60. 57. Ross, T. 1996. Indices for performance evaluation of predictive models in food microbiology. Journal of Applied Bacteriology 81: 501–508. 58. Sutherland, J. 2003. Modelling food spoilage. In Food Preservations Techniques. P. Zeuthen and L. Bogh-Sorensen (eds.), pp. 451–474, CRC Woodhead Publishing Limited, Cambridge, U.K. 59. Taoukis, P.S., Koutsoumanis, K., and Nychas, G.-J.E. 1999. Use of time-temperature intergrator and predictive modelling for shelf life control of chilled fish under dynamic storage conditions. International Journal of Food Microbiology 53: 21. 60. Tryfinopoulou, P., Tsakalidou E., and Nychas, G.-J.E. 2002. Characterization of Pseudomonas spp. associated with spoilage of gilt-head sea-bream fish stored under various storage conditions. Applied and Environmental Microbiology 68: 65. 61. Van Impe, J.F.M. and Bernaerts, K. 2000. Predictive Modelling in Foods — Conference Proceedings. KULeuven/BioTec, Belgium. 62. Vanderzant, C., Savell, J.W., Hanna, M.O., and Potluri, V. 1986. A comparison of growth of individual meat bacteria on the lean and fatty tissue of beef, pork and lamb. Journal of Food Science 51: 5–8, 11.
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63. Veciana-Nogués, M.T., Marine-Font, A., and Vidal-Carou, M.C. 1997. Biogenic amines as hygienic quality indicators of tuna. Relationships with microbial counts, ATP-related compounds, volatile amines, and organoleptic changes. Journal of Agricultural and Food Chemistry 45: 2036–2041. 64. Vogel, B.F., Venkateswaran, K., Satomi, M., and Gram, L. 2005. Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish marine fish. Applied and Environmental Microbiology 71: 6689.
Chapter 30
Detection of the Principal Foodborne Pathogens in Seafoods and SeafoodRelated Environments David Rodríguez-Lázaro and Marta Hernandez Contents 30.1 Introduction ..................................................................................................................557 30.2 Detection of the Principal Seafoodborne Pathogens ......................................................559 30.2.1 Detection of Pathogenic Vibrio Species in Seafoods and Seafood-Related Environments .................................................................................................. 560 30.2.1.1 V. parahaemolyticus ......................................................................... 560 30.2.1.2 V. vulnificus .....................................................................................565 30.2.1.3 V. cholerae ........................................................................................567 30.2.2 Detection of L. monocytogenes in Seafoods and Seafood-Related Environments .................................................................................................. 568 30.2.3 Detection of Salmonella spp. in Seafoods and Seafood-Related Environments ...................................................................................................570 References ................................................................................................................................572
30.1
Introduction
The importance of foodborne pathogens in public health is substantial. They cause more than 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths per year in the United States [88] 557
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with annual medical and productivity losses above 6,500 million dollars [23]. In England and Wales, the figures are similar, and they cause 1.3 million illnesses, 20,759 hospitalizations and 480 deaths each year [1]. The number of bacterial gastroenteritis associated to seafood products has been increased considerably during the last decades by the rapid globalization of the food market, the increase of personal and food transportation, and profound changes in the food consumption habits [66,88]. Among the bacterial pathogen that can produce gastroenteritis associated to seafood products, three can be considered as a primary threat: the enteropathogenic Vibrio, Listeria monocytogenes, and Salmonella spp. Three Vibrio species, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, are welldocumented human pathogens, specially associated to the consumption of raw or undercooked seafood products [67,87,103]. V. parahaemolyticus is an important seafoodborne pathogen worldwide [71]. It was first identified as a cause of foodborne illness in Japan in 1950 [35], and it has been reported to account for 20%–30% of foodborne illnesses in Japan [3] and a common cause of seafoodborne gastroenteritis in Asian countries [26,130]. In contrast, infections are occasional in Europe, and only sporadic outbreaks have been reported in Spain and France [113]. In the United States, V. parahaemolyticus is the leading cause of gastroenteritis associated with seafood consumption, and between 1973 and 1998 approximately 40 outbreaks were reported [24]. Consumption of raw or undercooked seafood, particularly shellfish, contaminated with V. parahaemolyticus may produce a self-limiting gastroenteritis involving symptoms such as vomiting, nausea, diarrhea with abdominal cramps, headache, and low-grade fever. V. parahaemolyticus is disseminated worldwide in estuarine, marine, and coastal water environments [65]. Some environmental factors such as the water temperature, salinity, zooplankton blooms, tidal flushing, and dissolved oxygen modulate its spatial and temporal distribution [95]. The increase of the prevalence of V. parahaemolyticus in raw shellfish is also correlated to the warm seawaters. The V. parahaemolyticus loads in oysters is usually lower than 103 cfu g−1 [70], but it can increase notably when the shellfish is cultivated in warmer seawater [28]. V. vulnificus produces one of the most severe foodborne infections, with a case-fatality rate greater than 50% [92]. It can cause fatal septicemia, wound infections, and gastroenteritis especially in immunocompromised individuals [11]. It was first isolated by the Center for Disease Control (CDC) in 1964 [112]. This organism is also disseminated worldwide in waters of different temperatures and salinities [131]. Environmental conditions such as water temperature and salinity modulate the variation in its prevalence [44]. Most of the outbreaks in United States have been reported during the summer generally associated to the consumption of raw seafoods [22,46,75,93]. V. cholerae is the causative agent of the cholera outbreaks and epidemics. There is a direct relationship between the consumption of raw, undercooked, contaminated, or recontaminated seafood and outbreaks produced by V. cholerae [30,34,67]. Foodstuff can be contaminated by this pathogen through contaminated irrigation water or human origin-fertilizer [30,91]. The O1 serogroup is the group predominantly isolated in cholera epidemics [34], and a new pathogenic serogroup, O139, has been also identified [4]. However, non-O1/O139 serogroups are sporadically involved in cholera-like diarrheal episodes, but infrequently in outbreaks [85,110]. Toxigenic V. cholerae O1 is rarely isolated and no isolations of serogroup O139 have been reported in western countries. In contrast, non-O1/O139 isolates are commonly found in estuarine water and shellfish [6]. Various O1 strains have become endemic in many regions in the world, including Australia and the U.S. Gulf Coast [21,123]. L. monocytogenes is an important foodborne pathogen, which usually (20%–50% of the cases) produces a fatal infection. It has been isolated from a wide range of sources, and seafood and seafood-related environments have been reported as important niches for this bacterium [105]. Cao et al. [17] reported the recurrent presence of this pathogen in shrimp samples and a frozen
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shrimp-processing line environment, without a positive correlation between its presence and the accompanying environmental microbiota. Farber [32] reported a low incidence of L. monocytogenes in imported seafood products between 1996 and 1998 (below 1%), and a complete absence in Canadian seafood products. Van Coillie et al. [118] studied the prevalence of L. monocytogenes in different ready-to-eat (RTE) seafood products on the Belgian market. The occurrence of L. monocytogenes was 23.9%, and the contamination levels were low in most cases (84% below 100 cfu g−1). The most prevalent serotype was 1/2a and serotypes 1/2b, 1/2c, and 4b were also present. In a longitudinal study in seafoods between 2001 and 2005 in France, Midelet-Bourdin et al. [90] observed similar findings (a prevalence of 28% with a low level of contamination). The presence of L. monocytogenes in tropical fish and shellfish in Mangalore, India was 17% and 12%, respectively [63]. Similar results were obtained by Nakamura et al. [96,97] in RTE seafood products commercially available or in a cold-smoked fish-processing plant in Osaka, Japan (13% and 7%, respectively). Its incidence was mainly in the summer and autumn, and it was only isolated in cold-smoked fish samples and in low numbers (below 100 cfu g−1). The serotype 1/2a was the most prevalent in both studies, and serotypes 1/2b, 3b, 4b, and 3a were also present. The consumption of seafoods and outbreaks of listeriosis is well documented [105]. For example, in a small human outbreak occurred in Ontario, Canada, the relationship between the presence of L. monocytogenes in seafood products (imitation crab meat) and the outbreak was clearly established [33]. Although all the foodstuffs obtained from the refrigerator of the two patients contained L. monocytogenes, three of them were heavily contaminated: imitation crab meat, olives, and salad. Molecular typing of the isolates by randomly amplified polymorphic DNA (RAPD) and pulsedfield gel electrophoresis (PFGE) typing demonstrated that the imitation crab meat and the clinical strains were indistinguishable. In addition, challenge studies performed with a pool of L. monocytogenes strains showed that imitation crab meat, but not olives, supported growth of this pathogen. Salmonella spp. is a major public health problem because of its large and varied animal reservoir, the existence of human and animal carrier states, and the lack of a concerted nationwide program to its control [42]. Furthermore, Salmonella is the main cause of documented foodborne human illnesses in most developed countries [18,117,124]. Of the outbreaks of foodborne illness recorded in the World Health Organization (WHO) report for 1993–1998, Salmonellae were most often reported as causative agent (54.6% of cases) [108]. Food items with a greater hazard include raw meat and some products intended to be eaten raw, raw or undercooked products, such as seafood and seafood products [31]. The presence of Salmonella spp. in tropical seafood products collected from different landing centers and open markets in Mangalore, India was studied by Kumar et al. [78]. The overall incidence of Salmonella spp. was 17%, suggesting that the contamination of seafoods with Salmonella may be occurring during postprocess handling and processing. A similar study was conducted in fish, shellfish, ice, and water obtained from the market and fish-landing center in Mangalore, India [109]. Twenty percent of the samples were positive using conventional methods, but the number of positives increased up to 52% when PCR was used, indicating the prevalence of Salmonella in seafood may be much more than that reported by conventional isolation techniques. The most prevalent serotype was Salmonella enterica serotype Weltevreden, and S. enterica serotype Worthington and S. enterica serotype Newport were also present.
30.2 Detection of the Principal Seafoodborne Pathogens As a consequence of the potential hazards described above, microbiological quality control programs are being increasingly applied throughout the seafood production chain in order to
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minimize the risk of infection for the consumer. Classical microbiological methods to detect the presence of those microorganisms involve enrichment and isolation of presumptive colonies of bacteria on solid media, and final confirmation by biochemical and/or serological identification. It is laborious and time consuming, and usually more than 3–5 days are needed for definitive results. Although remaining the approach of choice in routine analytical laboratories, the adoption of alternative techniques such as molecular-based methods in microbial diagnostics has become an alternative approach, as they possess inherent advantages such as shorter time to results, excellent detection limits, specificity, and potential for automation.
30.2.1
Detection of Pathogenic Vibrio Species in Seafoods and Seafood-Related Environments
30.2.1.1
V. parahaemolyticus
The most widely used methods for the detection of V. parahaemolyticus in foods are the International Organization for Standardization (ISO) standard 8914:1990 [51] and the most probable number (MPN) method described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) [72]. In the International Standard ISO 8914:1990, food samples are incubated at 35°C for 7–8 h in parallel in two enrichment broths (salt polymyxin B broth and alkaline saline peptone water or saline glucose culture medium with sodium dodecyl sulfate), and then streaked on two selective media (thiosulfate–citrate–bile salts–sucrose agar [TCBS] and triphenyltetrazolium chloride soya tryptone agar [TSAT]). After incubation for 18 h on TCBS or 20–24 h on TSAT, colonies being 2–3 mm, smooth, and green on TCBS or 2–3 mm, smooth, flat, and dark red on TSAT can be considered presumptive colonies of V. parahaemolyticus, and they must be confirmed by biochemical tests. Recently a new ISO standard (ISO/TS 21872-1:2007) has been published describing a horizontal method in food for detection of V. parahaemolyticus and V. cholerae [58]. In the FDA BAM method, after the MPN analysis, the tubes must be plated on TCBS selective medium and several presumptive isolates must be confirmed by biochemical testing. In both cases, these methods are cumbersome and laborious, and definitive results can be only obtained after more than 4–5 days. To overcome those disadvantages, different PCR methods have been developed for detection of V. parahaemolyticus in seafood products and seafoodrelated environments (Table 30.1). Some authors have reported PCR methods for the detection V. parahaemolyticus independently of the pathogenic capacity of the strains detected. For this purpose, different PCR targets and DNA protocols have used. Lee et al. [80] developed a PCR method based on a specific fragment, pR72H, cloned and sequencedv in that laboratory. To determine its selectivity, 124 V. parahaemolyticus and 50 non-V. parahaemolyticus isolates were assayed. The PCR assay was 100% selective. Finally, the applicability of the method was evaluated in oysters. Ten milliliters of oyster homogenate was inoculated with decreasing amounts of V. parahaemolyticus, and 1 mL of each homogenate was then mixed with 9 mL of tryptose soy broth (TSB) containing 2.5% NaCl and incubated at 35°C. After enrichment, the DNA was extracted following three different protocols (by heating; by addition of 10% Triton X-100 and heating; and by enzymatic digestion with lysozyme and proteinase followed by boiling). The limit of detection after 3 h enrichment, using enzymatic digestion and boiling was as few as 9.3 cfu g−1. Other gene marker used for V. parahaemolyticus-specific detection is the thermolabile hemolysin (tlh) gene. Wang and Levin [125] observed a linear relationship between the fluorescent intensity of the tlh PCR products in the agarose gel and the bacterial populations. Kaufman et al.
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Table 30.1 PCR-Based Method for the Detection of Pathogenic Vibrio Species in Seafood Products Organism
Method
Food Matrix
Reference
PCR
pR72H
Oyster
[80]
PCR
tdh
Oyster
[68]
PCR
gyrB
Shrimp
[120]
PCR
toxR
PCR
tdh
PCR
orf8
—
[94]
PCR
vmp
—
[83]
Multiplex PCR
tdh, trh
—
[114]
Multiplex PCR
tlh, th, trh
Oyster
[8]
tlh, tdh, trh
Seafoods
[84]
Real-time PCR
tdh
Oyster
[9]
Real-time PCR
tlh
Real-time PCR
tlh
Oyster
[69]
Real-time PCR
toxR
Clams
[115]
Real-time PCR
gyrB
Oyster
[15]
Multiplex real-time tlh, tdh, trh PCR
Mussels
[25]
Multiplex real-time tlh, orf8 PCR
Oyster
[104]
Multiplex real-time tlh, tdh, Trh, orf8 PCR
Oyster
[129]
Multiplex real-time tlh, tdh, trh PCR
Oyster
[98]
V. parahaemolyticus Multiplex PCR
V. vulnificus
Target Sequence
— Oyster
[73] [43]
—
[126]
PCR
vvhA
—
[45]
PCR
vvhA
—
[13]
PCR
gyrB
Oyster
[79]
Nested PCR
23 S rDNA
Fish
[1]
Multiplex PCR
vvhA
Oysters, shrimp
[128]
Multiplex PCR
vvhA
Oysters
[12]
Multiplex PCR
vvhA
Oyster
[82] (continued)
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Table 30.1 (continued) PCR-Based Method for the Detection of Pathogenic Vibrio Species in Seafood Products Organism
V. cholerae
Method
Target Sequence
Food Matrix
Reference
RT PCR
vvhA
Octopus
[81]
Real-time PCR
vvhA
Oyster
[16]
Real-time PCR
vvhA
Stools
[36]
Real-time PCR
vvhA
Oyster
[100]
Real-time PCR
vvhA
Clam
[126]
Real-time PCR
16 S rDNA
Real-time PCR
16 S rDNA
Oyster
[41]
PCR
ctxAB
Oyster, crab
[77]
PCR
ctxA
Oyster
[27]
PCR
Ctx
Oyster
[10]
—
[121]
[69] devised an alternative strategy for detection of V. parahaemolyticus in oyster. They used mantle fluids as food matrix instead of homogenized oyster tissues, since they observed that the levels of natural contamination of V. parahaemolyticus were similar in mantle fluids and oyster tissues. They developed a tlh-specific real-time PCR, which was 100% selective as determined using 37 V. parahaemolyticus, 27 other Vibrio, and 37 non-Vibrio isolates. A strong linear correlation between the PCR results and the concentration of cells inoculated into mantle fluids was observed, and the mantle fluid exhibited less PCR inhibition than the homogenized oyster tissue. Kim et al. [73] reported a PCR method based on the toxin transcriptional activator (toxR) gene. After testing 373 V. parahaemolyticus isolates and 290 isolates of other bacterial species, they concluded that the method was 100% selective. Similarly, Takahashi et al. [115] developed a toxR-based real-time PCR method. It was fully selective as tested 25 V. parahaemolyticus and 30 non-V. parahaemolyticus isolates. They also evaluated its applicability in shellfish. Twenty-five grams of short-neck clams was homogenized with phosphate-buffered saline (PBS), artificially contaminated with decreasing amounts of V. parahaemolyticus, and the DNA was extracted with the MagExtractor-Genome Kit (Toyobo). The real-time PCR detected as few as 100 cfu g−1. Venkateswaran et al. [120] reported a PCR method based on the B subunit of DNA gyrase (gyrB) gene. The selectivity of the method was evaluated using 117 strains of V. parahaemolyticus isolated from various environments, food, and clinical sources, and 150 isolates of other species. Twenty-five gram samples of shrimp were homogenized in 225 mL of alkaline peptone water (APW) and artificially contaminated with decreasing amounts of V. parahaemolyticus and V. alginolyticus, and incubated at 37°C. The homogenates were centrifuged and resuspended in 1 mL of sterile PBS. Ten microliters was used for PCR without extraction of DNA. The analytical sensitivity was as few as 1.5 V. parahaemolyticus cfu g−1 of homogenate. Similarly, Cai et al. [15] designed a gyrB-based real-time PCR. The selectivity was confirmed using 27 V. parahaemolyticus and 10 non-V. parahaemolyticus isolates. One gram oyster meat homogenate was artificially contaminated and 1 mL aliquot was used for the DNA extraction using the Wizard genomic DNA purification
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(Promega). The limit of detection of the method was 100 cfu mL−1 of oyster homogenates. When 300 seafood samples collected from local supermarkets in eastern China were tested, 32% of the samples were positive using the method. However, only 26% of the samples were positive using the conventional culture method. Interestingly, all culture-positive were also real-time PCR positive, indicating that the real-time PCR method was more sensitive that the conventional culture method. PCR methods have been also developed for the only specific detection of pathogenic strains of V. parahaemolyticus. Tada et al. [114] developed a PCR method based on the thermostable hemolysin (tdh) gene and tdh-related hemolysin (trh) gene. The selectivity was demonstrated using 263 V. parahaemolyticus and 133 isolates of other species. Karunasagar et al. [68] reported a PCR method for the detection of Kanagawa-positive strains in seafoods. The primers targeted the tdh gene. It was fully selective as tested in 4 Kanagawa-positive V. parahaemolyticus, 20 Kanagawa-negative V. parahaemolyticus, and 31 other Vibrio isolates. For the detection in seafoods, 50 g of samples was homogenized with 450 mL APW. One milliliter of homogenate was centrifuged at 100 × g, and the supernatant was again centrifuged, resupended, and lysed by heating. The analytical sensitivity was less than 10 cells of V. parahaemolyticus after 8 h enrichment. A real-time PCR method was also developed using the same molecular marker, tdh [9]. The sensitivity was demonstrated using 42 tdh+ V. parahaemolyticus isolates, 12 tdh− V. parahaemolyticus isolates, and 103 nontarget isolates. For detection of the pathogenic strains in oyster samples, a 50 mL aliquot of 1:1 oyster homogenate was added to 200 mL of APW and enriched overnight at 35°C. After the enrichment, 1 mL was boiled and 2.5 mL of the supernatant was used for PCR. The real-time PCR detected as few as 1 cfu per reaction. Finally, 131 natural oyster samples collected from Alabama, United States were analyzed by both conventional microbiological methods and real-time PCR. Forty-two percent of negative samples for the microbiological method were positive for the real-time PCR indicating a significantly higher detection rate (p < 0.05) and only a 20% of the samples positive for the microbiological method were negative for the real-time PCR method. Hara-Kudo et al. [43] optimized a PCR method using different DNA extraction procedures for the detection of the pathogenic V. parahaemolyticus in seafoods. The primers targeted the tdh gene, whose PCR selectivity had been tested previously [114]. Three different DNA extraction methods were evaluated: a silica membrane method using the NucleoSpin Tissue Kit (MachereyNagel), a glass fibber method using the High Pure PCR Template Precipitation Kit (Roche), or a magnetic separation method using the MagExtractor-Genome Kit (Toyobo). The use of the silica membrane and the glass fibber methods increased notably the analytical sensitivity. Taking in consideration the importance for public health of this pathogen, distinguishing between potentially pathogenic and nonpathogenic V. parahaemolyticus isolates is of critical importance. Bej et al. [8] reported a multiplex PCR method for the detection of total and hemolysinproducing V. parahaemolyticus in shellfish. The method targeted the tlh gene for the detection of all V. parahaemolyticus strains and the tdh and trh genes for the specific detection of the pathogenic strains. The selectivity of the method was evaluated using 111 V. parahaemolyticus isolates from different origins and 19 non-V. parahaemolyticus isolates. The tlh primers were 100% selective. Fifty-four percent of the V. parahaemolyticus isolates showed positive PCR amplification for the tdh primers and 39% showed amplification of the trh primers. Interestingly, three isolates showed no tdh- and trh-PCR amplification but were Kanagawa positive, and three other isolates were tdhPCR positive, but produced a negative Kanagawa reaction. Finally, 10 g of oyster homogenate was artificially contaminated with decreasing amounts of V. vulnificus strains with different tl/tdh/trh profiles, diluted in 350 mL of APW and incubated at 35°C for 6 h. DNA was extracted following
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a previously described method [38]. The limit of detection for all the three PCR primers was 100 cells for the tdh-primers, and 10 cells for tlh- and trh-primers. Using the same set of primers, Luan et al. [84] used a rapid MPN–PCR method for quantification of this pathogen in seafood samples purchased at local retail markets in Qingdao, China. Seventy-three percent of the samples were V. parahaemolyticus (tlh) positive with values higher than 719 MPN g−1, and 41.5% of samples were positive for tdh gene-possessing cells, indicating the presence of pathogenic strains. Nordstrom et al. [98] developed a multiplex real-time PCR method for detection of the total and pathogenic strains of this organism in oysters using the same targets: tlh, tdh, and trh genes, but this method included an internal amplification control (IAC). The IAC is a nontarget nucleic acid sequence present in every reaction, which is amplified simultaneously with the target sequence [106]. In PCR diagnostics, IACs are essential to identify false negative results [49] as in a reaction with an IAC, a control signal will always be produced when there is no target sequence present. The selectivity was evaluated using 117 V. parahaemolyticus isolates with different tlh/tdh/trh profiles and 36 isolates of other species of the genus Vibrio. A perfect correlation was shown between the results obtained for the V. parahaemolyticus isolates and the tlh/tdh/trh profiles, however 75% of the Vibrio hollisae strains gave a low positive signal for tdh. Twenty-seven natural oyster samples were collected at Alaska, and 1 g of homogenate was added to 10 mL of APW and incubated overnight at 35°C. After the enrichment, 1 mL aliquots were boiled and 2 mL of supernatant was used for PCR. Fortyfour percent, 44% and 52% of the oyster samples were positive for tlh, tdh, and trh, respectively. However, only 33%, 19%, and 26% were positive for tlh, tdh, and trh using conventional culture methods. Davis et al. [25] used a similar strategy to evaluate V. parahaemolyticus strains isolated from mussels and associated with a foodborne outbreak happening in 2002, in Florida, United States. The selectivity of the assay was confirmed using 20 V. parahaemolyticus isolates. The mussels were the only food sample with positive results. More than 21% of the mussels samples were positive for tlh indicating the presence of the V. parahaemolyticus in the samples, and almost 17% of the samples were positive for tdh, indicating the presence of pathogenic variants in those samples. The emergence of the O3:K6 serotype and its widespread distribution have fostered the development of detection methods to detect such pathogenic variants. Myers et al. [94] developed a PCR method for the specific detection of this serotype. The PCR target was the open reading frame 8 of phage f237 (orf8). They tested 37 V. parahaemolyticus O3:K6 serotype, 123 V. parahaemolyticus non-O3:K6 serotype, 114 isolates from other species, and they observed that the method was 100% selective. The method could detect down to 104 cells per 100 mL of water samples after the DNA purification using the FastDNA SPIN kit (Bio 101). Rizvi et al. [104] designed orf8 primers coupled with tlh primers for the simultaneous detection of total V. parahaemolyticus and pandemic O3:K6 serovar using a multiplex real-time PCR. The selectivity of the assay was evaluated using 37 V. parahaemolyticus O3:K6, 26 V. parahaemolyticus, 7 non-parahaemolyticus Vibrio, and 9 non-Vibrio isolates. All the V. parahaemolyticus and all the V. parahaemolyticus O3:K6 isolates were positive for the tlh- and orf8-PCRs, respectively, and none of the nontarget isolates was positive. One gram oyster tissue homogenates and Gulf water were artificially contaminated with V. parahaemolyticus O3:K6, and incubated at 37°C. After the enrichment, DNA extraction was performed using the Instagene matrix (Bio-Rad). The limit of detection of the real-time PCR method was 1 cfu of pandemic V. parahaemolyticus O3:K6 serovar per mL of Gulf water or 1 g of oyster tissue homogenate after 8 h enrichment. Ward and Bej [129] developed a multiplex real-time PCR assay for the simultaneous detection of V. parahaemolyticus using the tlh gene, pathogenic strains using the tdh and trh genes, and the pandemic O3:K6 serotype using the orf8. Detection of 1 cfu g−1 of oyster tissue homogenate was possible after overnight enrichment. Finally the method was applied to 33 natural samples from the Gulf of Mexico, Alabama (United States).
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Fifty-two percent of the samples were positive for tlh indicating the presence of V. parahaemolyticus in these samples, and 12% were positive for tdh indicating the samples contained pathogenic V. parahaemolyticus strains. Luan et al. [83] compared the performance of four PCR assays for the detection of V. parahaemolyticus. The PCR assays targeted the toxR [13], tlh, tdh, and trh [8], gyrB [120] and the V. parahaemolyticus metalloprotease (vpm) gene. Eighty-six V. parahaemolyticus and 16 non-V. parahaemolyticus isolates were tested with the four set of primers. All the four PCR assays were 100% selective. However the analytical sensitivity varied: the vpm-PCR assay detected as few as 4 pg of genomic V. parahaemolyticus DNA, whereas the toxR-PCR, tlh-PCR, and gyrB-PCR detected a minimum of 375, 100, and 800 pg, respectively.
30.2.1.2
V. vulnificus
The current guidelines recommended by the ISSC indicates that less than 30 cfu g−1 in postharvesttreated oysters is the threshold to consider a food item as safe for consumption [60]. The detection protocol approved by the FDA BAM method is based on the MPN enrichment series in APW coupled with isolation in selective medium and biochemical or molecular confirmation of V. vulnificus and on the direct isolation on minimally selective media followed by identification of V. vulnificus by colony blot DNA–DNA hybridization [72]. Recently the ISO/TS 21872-1:2007 standard has been published describing a horizontal method in food for detection of other potentially enteropathogenic Vibrio species than parahaemolyticus and V. cholerae [59], which is based in similar principles. In Table 30.2 are summarized the currently available selective media for V. vulnificus. As for V. parahaemolyticus, a battery of PCR-based methods have been devised to overcome the disadvantages of the microbiological culture methods (Table 30.1). Hill et al. [45] reported a PCR method based on the cytolysin gene (vvhA). The selectivity of the primers was evaluated by testing 5 V. vulnificus, 12 non-vulnificus Vibrio, and 10 non-Vibrio strains. The PCR method was fully selective. Using the vvhA gene as PCR target, Brauns et al. [13] confirmed the selectivity of the PCR assay testing one V. vulnificus, five non-vulnificus Vibrio, and nine non-Vibrio isolates. Campbell and Wright [16] developed a real-time PCR method based on the same gene. The selectivity of the assay was evaluated with 28 V. Vulnificus and 22 non-V. vulnificus isolates, showing to be 100%. Detection of V. vulnificus in pure cultures was possible down to 102 cfu mL−1. The applicability of this method for detection of V. vulnificus in oysters was evaluated using natural and artificially contaminated oysters. Thirty grams of oyster meat was 1:10 diluted in ASW and homogenized for 90 s. Ten milliliters of oyster homogenates was artificially contaminated with decreasing amounts of V. vulnificus. DNA was extracted using the QIAamp DNA minikit and concentrated with precipitation with ethanol. The results obtained by real-time PCR correlated well with plate counts based on colony blot hybridization enumeration. Similarly, another realtime PCR method using SYBR Green was developed targeting the vvhA gene [100]. The method was fully selective as 80 V. vulnificus isolates produced PCR signals and 47 isolates from other species did not produce any PCR amplification. One gram aliquots of oyster tissue homogenate were 10-fold serially diluted in sterile GWP-16 and artificially contaminated with V. vulnificus and incubated for 5 h at 37°C. After the enrichment, 5 mL-aliquots were used for DNA extraction using the Instagene matrix (Bio-Rad). The real-time PCR method detected as few as 1 cfu of V. vulnificus in 1 g of oyster homogenate. Using the same SYBR Green real-time PCR assay, Wang and Levin [126] optimized a DNA extraction protocol for clam samples. One gram homogenates were artificially contaminated with decreasing amounts of V. vulnificus. The aliquots were centrifuged at 1000×g for 5 min, and the supernatants were washed twice and lysed with TZ lysis.
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Table 30.2
Selective Culture Media for Isolation and Identification of V. vulnificus
Abbreviation
Incubation Temperature (°C)
Thiosulfate citrate bile salt agar
TCBS
37
V. vulnificus agar
Medium
Carbon Source
Colony Color
Reference
Sucrose
Green
[76]
VV
Salicin
Grey, dark center
[14]
SDS polymyxin sucrose agar
SPS
Sucrose
Blue with halo
[74]
Cellobiose polymyxin B colistin agar
COC
40
Cellobiose
Yellow
[86]
Modified cellobiose polymyxin B colistin agar
mCPC
40
Cellobiose
Yellow
[116]
V. vulnificus enumeration agar
VVE
37
Cellobiose, lactose, X-Gal
Blue green
[89]
Cellobiose colistin agar
CC
40
Cellobiose
Yellow
[48]
V. vulnificus medium
VVM
37
Cellobiose
Yellow
[19]
V vulnificus medium + colistin
VVMc
37
Cellobiose
Yellow
[20]
Source: Adapted from Harwood, V.J. et al., J. Microbiol. Methods, 59, 301, 2004.
The DNA was purified using Micropure EZ minicolumns. The real-time PCR detected as few as 100 cfu g−1 of clam tissue and 1 cfu g−1 after an enrichment step for 5 h at 37°C. Panicker and Bej [99] compared three previously reported sets of primers targeting the vvhA gene [16,36,100]. A TaqMan probe was developed for the first two sets of primers, and the probe previously described was used for the former [16]. The selectivity was evaluated using 81 V. vulnificus and 37 isolates from other species. The first two PCR systems were 100% selective, however the former was not fully selective as detected more than 32% of non-V. vulnificus isolates. Both PCR systems were used for detection of V. vulnificus in naturally and artificially contaminated oysters. For artificially contaminated oysters, 1 g aliquots homogenized samples were added to 50 mL of GWP-18 and the solution was artificially contaminated with decreasing amounts of V. vulnificus, and incubated at 37°C for 5 h. One milliliter aliquots were used for the DNA extraction using the Instagene matrix (Bio-Rad). The PCR methods detected as few as 1 cfu g−1. Other PCR targets have been used for the detection of V. vulnificus. Kumar et al. [79] developed a PCR method based on the gyrB gene. The PCR assay was 100% selective as tested with
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45 V. vulnificus and 49 other Vibrio isolates. The analytical sensitivity was evaluated using V. vulnificus pure cultures and artificially contaminated oyster meat. For artificially contaminated samples, 1 g of fresh homogenates was spiked with decreasing amounts of V. vulnificus, and lysed by heating. The PCR method detected as low as 3 V. vulnificus cfu mL−1 of pure cultures, and 300 cfu g−1 in artificially contaminated oyster homogenate without enrichment or 30 cfu g−1 after 18 h enrichment in APW. The method was also evaluated in 79 natural oyster samples collected from four different estuaries along the Mangalore coast, India. The homogenates were incubated for 0, 6, and 18 h. The best results were obtained after 18 h enrichment, where V. vulnificus was detected in 75% of natural oyster samples, while the conventional microbiological method (isolation on mCPC agar plates after 18 h enrichment) only detected V. vulnificus in 45.5% of samples. Vickery et al. [121] reported a real-time PCR method for the classification of V. vulnificus based on 16 S rRNA genotype (type A or B). A re-evaluation of the 67 U.S. isolates demonstrated that 45.5% of the isolates originally identified as 16 S rRNA type A were actually type AB, and 76% of clinical isolates tested were type B, 9% type A, and 15% type AB, and in contrast, 91% of nonclinical isolates were found to be of either type A or type AB, and only 9% type B. Other additional 18 strains were also examined, and all of the isolates were classified as type A, all the Biotype 3 strains isolated from an outbreak in Israel were type AB. Using a similar approach, Gordon et al. [41] distinguished V. vulnificus strains form environmental and clinical sources. In addition, no amplification was observed with any of the non-V. vulnificus isolates tested. Tissues from single oysters collected, in United States were 1:10 diluted in APW, artificially contaminated with V. vulnificus and incubated at 37°C for 4 and 24 h. After enrichment, the homogenates were 10-fold diluted. Two milliliters was boiled and 2 mL was used for PCR. The limits of detection were 103 and 102 cfu per reaction for type A and type B, respectively. Using this method, the authors described that the type A/B ratio of Florida clinical isolates was 19:17. The ratio in oysters harvested from restricted sites in Florida with poor water quality was 5:8, but it was 10:1 in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB.
30.2.1.3 V. cholerae The FDA BAM method for detection of V. cholerae in foods relies on the overnight enrichment in APW of 25 g of food samples at 42°C, the isolation on selective medium and final confirmation for biochemical and molecular tests [72]. Similarly the ISO Committee has developed a reference method for this pathogen, the ISO/TS 21872-1:2007 [58]. Another analytical approach is the screening of the samples for toxigenic V. cholerae with PCR assays targeting a portion of the ctx operon without or after enrichment (Table 30.1). Koch et al. [77] developed a PCR method, which targeted the cholera toxin operon, ctxAB. The selectivity was tested using 3 V. cholerae and 10 non-V. cholerae isolates, showing to be 100%. Analytical sensitivity was tested in artificially contaminated crab or oysters with V. cholerae before homogenization in APW. Ten percent APW homogenates were prepared and 1 mL aliquots were taken immediately and again after the 37°C incubation, boiled and 2–5 mL of supernatants was used for PCR. Crabmeat homogenates inoculated with as few as 4 × 104 V. cholerae cfu g−1 without further enrichment (equivalent to 10 cells in the reaction) and oysters homogenates artificially contaminated with as few as 10 V. cholerae cfu g−1 after 8 h enrichment produced positive amplification. DePaola and Hwang [27] evaluated the effects of dilutions, incubation times, and incubation temperatures on detection of V. cholerae by a ctxA-based PCR method. PCR detection of V. cholerae
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was significantly improved using oyster homogenates diluted 1:100 in APW and incubated at 42°C for 18–21 h. Blackstone et al. [10] developed a real-time PCR method for detection of toxigenic V. cholearae in seafood and seafood-related environments. The system targeted the cholera toxin (ctxA) gene, found in toxigenic V. cholerae strains. The real-time PCR assay was 100% selective as tested with 32 toxigenic V. cholerae and 59 non-V. cholerae isolates as well as DNA from different environments and eukaryotic organisms. The limit of detection of the method was less than 1 cfu per reaction in oyster. Finally, 6 shellfish and 10 related environmental samples collected in Mobile Bay, United States were evaluated. Twenty-five grams of oyster homogenate was added to 2475 mL of APW and incubated overnight at 42°C. A 1 mL aliquot of enrichment was boiled and 2–2.5 mL of the boiled aliquot was used for PCR. For environmental samples, 25 g of sediment and ballast water was added to 225 mL of APW and incubated overnight at 42°C. None of the seafood and environmental samples showed a positive signal for toxigenic V. cholerae.
30.2.2 Detection of L. monocytogenes in Seafoods and Seafood-Related Environments ISO has developed reference methods for detection and enumeration of L. monocytogenes: ISO 11290-1 and 11290-2, respectively [52,53,56,57]. In the ISO 11290-1, 25 g of food sample is homogenized in a primary enrichment medium (Half Fraser broth) and incubated at 30°C for 24 h. Subsequently, primary culture is plated on Agar Listeria according to Ottaviani and Agosti (ALOA) and in other selective medium (e.g., Oxford or PALCAM media) and incubated at 37°C for 24 h, and in parallel 0.1 mL primary enrichment aliquot is also transferred into a tube with 10 mL of the secondary enrichment medium, and incubated at 35°C or 37°C for 48 h. Afterwards, the secondary enrichment is also streaked on ALOA and other selective medium (e.g., Oxford or PALCAM media), and incubated at 37°C for 24 h. Finally, the typical L. monocytogenes colonies (green-blue colonies surrounded by an opaque halo in ALOA plates) are confirmed by biochemical tests. In the protocol for detection of L. monocytogenes recommended by the FDA [50], 25 g of seafoods is homogenized in 225 mL of buffered Listeria enrichment broth base containing sodium pyruvate without selective agents (BLEB), and incubated at 30°C for 4 h, and then the selective agents are added and incubated for 44 h more at 30°C. At 24 and 48 h, BLEB culture are plated onto one selective isolation medium such as Oxford agar, PALCAM agar, modified Oxford agar (MOX), and Lithium chloride–phenylethanol–moxalactam (LPM) agar fortified with esculin and Fe3+, and incubated at 35°C for 24–48 h for Oxford, PALCAM, or MOX plates or at 30°C for 24–48 h for fortified LPM plates. In addition primary cultures must be plated onto one L. monocytogenes–L. ivanovii differential selective agar (e.g., BCM, ALOA, RapidL’mono, or CHROMagar Listeria) after 48 h of enrichment (optionally at 24 h, too). Finally the typical L. monocytogenes colonies are confirmed by biochemical tests. In the ISO 11290-2, 10-fold dilutions of the seafood product homogenate are prepared and plated on ALOA, and incubated at 37°C for 24 h for the enumeration of L. monocytogenes. After the enrichment, the typical L. monocytogenes colonies are confirmed by biochemical tests. However, in the FDA protocol for enumeration of L. monocytogenes, only the positive food samples for presence of L. monocytogenes are tested by colony count on L. monocytogenes differential selective agar in conjunction with MPN enumeration using selective enrichment in BLEB with subsequent plating on ALOA or BCM differential selective agar.
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A study compared the reference ISO methods (ISO 11290-1 and 11290-2) with an in-house method in 543 seafood product samples collected from 21 different companies between 2001 and 2005 in France [90]. For the in-house method, 25 g of seafood product was homogenized with 225 mL of Listeria repair broth (LRB) [40,107], and left at room temperature up to 60 min. To enumerate L. monocytogenes, homogenates were spread over Listeria selective agar (LA) plates [64] and incubated at 37°C for 48 h. To detect L. monocytogenes, 0.90 mL of selective supplement LRB (Oxoid, U.K.) was added to the homogenate, and incubated at 30°C for 24 h, and subsequently streaked on ALOA and L. monocytogenes blood agar (LMBA) plates [64] and incubated 37°C for 48 h. For the second enrichment step, 0.1 mL of the 24-h culture was transferred to a tube with 10 mL of the Fraser broth, and the mixture was incubated at 37°C for 48 h. This second enrichment culture was streaked on ALOA and on LMBA plates and incubated at 37°C for 48 h. For each plate with suspect L. monocytogenes colonies, several colonies were spread on LA plates and incubated at 37°C for 48 h, and subsequently respread on Trypticase Soy Agar supplemented with yeast extract (TSAYE). Isolated colonies were taken into microcentrifuge tube containing 100 mL of sterile distilled water, and lysed by heating at 95°C for 25 min, then centrifuged and 3 mL of the supernatant was used for confirmation by PCR. Four sets of primers were used; one for the identification of Listeria spp. targeting the 16 S rRNA gene [47], and three specific for the identification of L. monocytogenes targeting the hly [7,101], and iap [47] genes. Twenty eight percent of the samples were positive by at least one of the methods and 16% were positive by both methods. The sensitivity of the methods was higher than 78%, being slightly higher than 79.5% in the case of the in-house method, and the efficiency of isolation was different depending on the nature of the seafood product. The international standard methods confirmed as positive more samples in smoked salmon and herb-flavored slices of smoked salmon, but the in-house method in carpaccio-like salmon, herb-flavored slices of raw salmon, and smoked trout. Agersborg et al. [2] were the first to develop a specific PCR method for the detection of L. monocytogenes in seafood products. They artificially contaminated 5 g of fish cakes, fish pudding, peeled frozen shrimps, salted herring, and marinated and sliced coalfish in oil with 500, 10, 5, and 1 L. monocytogenes cells. The seafood samples were homogenized in 20 mL of Tryptone Soy Broth or universal pre-enrichment broth (UPB) and incubated for 24 h. Afterwards, 0.5 mL aliquots were inoculated to 5 mL of UPB and incubated for other 24 h, and subsequently 1.5 mL aliquots were centrifuged for 10 min at 16,000×g, and submitted to bacterial DNA extraction. Three different protocols were used by the DNA isolation: the bacterial pellets were resupended (1) in 500 mL of double-distilled (dd-)water and treated by heating; (2) in 750 mL of dd-water and treated with lysozyme and proteinase K; (3) in 400 mL of dd-water and 400 mL of 2% Triton X-100 was added. In all the cases, the DNA solutions were centrifuged, and 10 mL of the supernatants was used by the PCR. The PCR systems targeted different regions of the hly [37,39] and iap genes [62]. Lysis by Triton X-100 was the most reliable DNA extraction procedure. After 48 h of incubation, samples inoculated with one to five L. monocytogenes cells were clearly positive for the three different set of primers. Isonhood et al. [61] developed an upstream processing method to facilitate the detection by PCR of L. monocytogenes in RTE (ready to eat) seafood salads. Eleven grams of the salads was diluted in 99 mL of sterile saline, and artificially contaminated with decreasing amounts of L. monocytogenes. After homogenizing, 80 mL of the filtrate was removed for a two-steps centrifugation, consisting of one centrifugation step (119×g for 15 min at 5°C) to remove large food particulates and a second centrifugation step (11,950×g for 10 min at 5°C) to concentrate the
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bacterial cells in the supernatant that was recovered after the first centrifugation. DNA extraction was done on the 1 g bacterial pellets using DNAzol (Invitrogen). The DNA was serially diluted and subjected to dilution series PCR amplification using a set of primers targeting the 16 S rDNA gene [111] and confirmed by chemiluminescent Southern blot hybridization. The mean recovery after the two-step method was 49.0%, and consistent PCR detection of L. monocytogenes was possible down to 103 cfu g−1. Destro et al. [29] combined RAPD and PFGE analysis to trace L. monocytogenes contamination in a shrimp-processing plant in Brazil, over a 5 month period (May to September 1993). Two random primers were used for the RAPD analysis, generating more than 10 different RAPD profiles, a lower number than reported previously. PFGE was performed using SmaI and ApaI restriction endonucleases, obtaining more than 12 restriction endonuclease digestion profiles (REDP), a number similar to previous studies. The combined profile generated when the two RAPD primers and the two PFGE enzymes were used, increased the discriminatory ability to detect differences among isolates of L. monocytogenes within serogroups. The combination of these two typing methods allowed tracking the origin of the isolates; i.e., natural isolates from inside the processing plant, and isolates introduced from outside the plant and restricted to the receiving area.
30.2.3
Detection of Salmonella spp. in Seafoods and Seafood-Related Environments
The International reference method for detection of Salmonella is the ISO 6579 [54,55]. In this standard, 25 g of food sample is homogenized with buffered peptone water (BPW), and incubated at 37°C for 18 h. Subsequently, a 0.1 mL pre-enrichment aliquot is transferred into 10 mL Rappaport-Vassiliadis (RV) medium with soya (RVS broth) and incubated for 24 h at 41.5°C and in parallel another 1 mL aliquot is transferred into 10 mL Muller–Kauffmann tetrathionate novobiocin (MKTTn) broth and incubated for 24 h are incubated at 37°C. After the 24 h-incubation, a loop of the RVS and MKTTn broths are streaked onto xylose lysine desoxycholate (XLD) agar and other selective medium, and incubate the plates at 37°C for 24 h. Afterwards, typical Salmonella colonies (pink colonies with or without black centers in XLD agar) are confirmed by biochemical (TSI agar test, urea agar test, l-lysine decarboxylation medium test, detection of b-galactosidase, Voges-Proskauer reaction, indole reaction), and serological tests. In the FDA protocol for detection of Salmonella [5] small differences can be noted. Twenty-five grams of food sample is homogenized in 225 mL sterile lactose broth. After 1 h at room temperature, 2.25 mL steamed Tergitol Anionic 7 or Triton X-100 are used, and the seafood homogenate is incubated for 24 h at 35°C. Subsequently, a 0.1 mL pre-enrichment aliquot is transferred into 10 mL RV medium and incubated for 24 h at 42°C and in parallel another 1 mL aliquot is transferred into 10 mL tetrathionate (TT) broth and incubated for 24 h at 35°C. Afterwards, the RV and TT enrichments are streaked on bismute sulfite (BS) agar, XLD agar, and Hektoen enteric (HE) agar, and the plates are incubated for 24 h at 35°C. Finally, typical Salmonella colonies (brown, grey, or black colonies; sometimes with a metallic sheen in BS agar, pink colonies with or without black centers in XLD agar; and blue-green to blue colonies with or without black centers in HE agar) are confirmed by biochemical or alternative tests. As for pathogenic Vibrio and L. monocytogenes rapid alternatives based on molecular methods have been also devised. The research group led by Bej at the University of Alabama developed a multiplex PCR method for the simultaneous detection of Escherichia coli, S. enterica serotype
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Typhimurium, V. vulnificus, V. cholerae, and V. parahaemolyticus [12]. The PCR primers targeted the E. coli uidA, S. typhimurium invA, V. vulnificus cth, V. cholerae ctx, and V. parahaemolyticus tl genes. The multiplex PCR was totally selective as each specific primer only detected the corresponding target. One gram of sterilized shellstocks from oysters obtained from local seafood restaurants was artificially contaminated with decreasing loads of these organisms. The sample was diluted in 30 mL of APW and incubated at 35°C for 6 h. After the enrichment, the oyster homogenates were centrifuged and the DNA was extracted using the Chelex 100 resin (Biorad). To achieve maximum sensitivity, a 5 mL aliquot of the initial multiplex PCR-amplified products was subjected to a reamplification by a second PCR. The minimum level of detection of each target in a single multiplex PCR was 100 cfu g−1. However, the detection limit was improved to 10 cells cfu g−1 using the second PCR round. The same research group improved the detection of S. enterica serotype Typhimurium, V. vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus using a multiplex PCR followed by DNA–DNA sandwich hybridization [82]. The target genes were the Salmonella hns and spvB, V. vulnificus vvh, V. cholerae ctx, and V. parahaemolyticus tlh genes. Oyster samples were processed according to standard methods and 1 g of oyster homogenates was diluted in 5 mL of APW and artificially contaminated with 10-fold dilutions of those four bacterial pathogens. The homogenates were enriched for 3 h at 37°C. The bacterial DNA extraction was performed as described above. The multiplex PCR allowed the detection of all four bacterial pathogens, and it was further confirmed by the nonradioactive and colorimetric CovaLinkk NH microtiter plate hybridization assay. The analytical sensitivity was down to 102 cells g−1 of oyster tissue homogenate. Vantarakis et al. [119] devised a multiplex PCR method for the simultaneous detection of Salmonella spp. and Shigella spp. in mussels. The multiplex PCR primers targeted specific nucleotide sequences of the Salmonella invA (215 bp) [122] and Shigella virA (275 bp) [102] genes. The PCR method was 100% selective as evaluated with six different Enterobacteriaceae genera. For the mussels analysis, 25 g of mussel meat was diluted in 90 mL of BPW. Decreasing amounts of Salmonella spp. and Shigella spp. were added to 1 mL of mussel homogenates and submitted to DNA extraction. Guanidine isothiocyanate was added to 1 mL homogenates and incubated at 65°C for 90 min, diluted and boiled for 5 min. The samples were cooled to room temperature, then sodium acetate was added to the samples, and centrifuged at 14,000×g for 10 min. The supernatants were transferred to new tubes and extracted twice with an equal volume of chloroform. Finally the DNA was precipitated with 95% ethanol and the DNA was resuspended in sterile distilled water. The PCR method detected less than 10 Salmonella cells mL−1 of homogenate. However the authors introduced a pre-enrichment step to increase the analytical sensitivity as well as to guarantee the only detection of viable cells. After a 22 h pre-enrichment in BPW, 10–100 cells of Salmonella spp. and Shigella per milliliter of homogenate were detected by the multiplex PCR. Wang and Yeh [127] developed a novel PCR method for the detection of Salmonella enteritidis, and evaluated its performance in different food samples, including seafoods. The PCR system targeted the Salmonella IE gene. All of the 24 Salmonella enteritidis strains generated positive PCR signals. Ninety-six non-enteritidis Salmonella and 40 non-Salmonella isolates including strains of the family Enterobacteriaceae such as E. coli, Shigella, and Citrobacter, did not produce any amplification signal, therefore, the PCR assay was 100% selective. The detection limit of the PCR assay was 102 cfu mL−1 of cell extracts prepared by heat lysis. For the analysis of seafood samples, the authors followed the FDA procedure, and 10 mL of the final enrichment was lysed by heating, and used for the PCR detection. None of the 15 samples were detected by either completed BAM method or by PCR.
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References 1. Adak, G.K., Long, S.M., and O’Brien, S.J. Trends in indigenous foodborne disease and deaths, England and Wales: 1992–2000, Gut, 51, 832–841, 2002. 2. Agersborg, A., Dahl, R., and Martinez, I. Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods, Int. J. Food Microbiol., 35, 275–280, 1997. 3. Alam, M.J. et al. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan, FEMS Microbiol. Lett., 208, 83–87, 2002. 4. Albert, M.J. Vibrio cholerae O139 Bengal, J. Clin. Microbiol., 32, 2345–2349, 1994. 5. Andrews, W.H. and Hammack, T. Chapter 5: Salmonella, in: Bacteriological Analytical Manual, 8th edn., Gaithersburg, MD: U.S. Food and Drug Administration, 2007. 6. Arias, C.R., Garay, E., and Aznar, R. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water, Appl. Environ. Microbiol., 61, 3476–3478, 1995. 7. Bansal, N.S. et al. Multiplex PCR assay for the routine detection of Listeria in food, Int. J. Food Microbiol., 33, 293–300, 1996. 8. Bej, A.K. et al. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh, J. Microbiol. Methods, 36, 215–225, 1999. 9. Blackstone, G.M. et al. Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR, J. Microbiol. Methods, 53, 149–155, 2003. 10. Blackstone, G.M. et al. Use of a real time PCR assay for detection of the ctxA gene of Vibrio cholerae in an environmental survey of Mobile Bay, J. Microbiol. Methods, 68, 254–259, 2007. 11. Blake, P.A. et al. Disease caused by a marine Vibrio. Clinical characteristics and epidemiology, N. Engl. J. Med. 300, 1–5, 1979. 12. Brasher, C.W. et al. Detection of microbial pathogens in shellfish with multiplex PCR, Cur. Microbiol., 37, 101–107, 1998. 13. Brauns, L.A., Hudson, M.C., and Oliver, J.D. Use of the polymerase chain reaction in detection of culturable and non-culturable Vibrio vulnificus cells, Appl. Environ. Microbiol., 57, 2651–2655, 1991. 14. Brayton, P.R. et al. New selective plating medium for isolation of Vibrio vulnificus biogroup 1, J. Clin. Microbiol., 17, 1039–1044, 1983. 15. Cai, T. et al. Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China, FEMS Immunol. Med. Microbiol., 46, 180–186, 2006. 16. Campbell, M.S. and Wright, A.C. Real-time PCR analysis of Vibrio vulnificus in oysters, Appl. Environ. Microbiol., 69, 7137–7144, 2003. 17. Cao, J. et al. Concentrations and tracking of Listeria monocytogenes strains in a seafood-processing environment using a most-probable-number enrichment procedure and randomly amplified polymorphic DNA analysis, J. Food Prot., 69, 489–494, 2006. 18. CAST. CAST Report: Foodborne Pathogens: Risks and Consequences. Task Force Report No. 122, Washington, DC: Council for Agricultural Science and Technology, 1994. 19. Cerda-Cuellar, M., Jofre, J., and Blanch, A.R. A selective medium and a specific probe for detection of Vibrio vulnificus, Appl. Environ. Microbiol., 66, 855–859, 2000. 20. Cerda-Cuellar, M. et al. Comparison of selective media for the detection of Vibrio vulnificus in environmental samples, J. Appl. Microbiol., 91, 322–327, 2001. 21. Colwell, R.R. et al. Occurrence of Vibrio cholerae O1 in Maryland and Louisiana estuaries, Appl. Environ. Microbiol., 41, 555–558, 1981. 22. Cook, D.W. et al. Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: A national survey from June 1998 to July 1999, J. Food Prot., 65, 79–87, 2002. 23. Crutchfield, S. and Roberts, T. Food safety efforts accelerate in 1990’s, USDA Economic Res. Service Food Rev., 23, 44–49, 2000. 24. Daniels, N.A. et al. Vibrio parahaemolyticus infections in the United States, 1973–1998, J. Infect. Dis., 181, 1661–1666, 2000.
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46. Hlady, W.G. and Klontz, K.C. The epidemiology of Vibrio infections in Florida, 1981–1993, J. Infect. Dis., 173, 1176–1183, 1996. 47. Herman, L.M.F., de Ridder, H.F.M., and Vlaemynck, G.M.M. A multiplex PCR method for the identification of Listeria spp. and Listeria monocytogenes in dairy samples, J. Food Prot., 58, 867–872, 1995. 48. Høi, L., Dalsgaard, I., and Dalsgaard, A. Improved isolation of Vibrio vulnificus from seawater and sediment with cellobiosecolistin agar, Appl. Environ. Microbiol., 64, 1721–2174, 1998. 49. Hoorfar, J. et al. Practical considerations in design of internal amplification controls for diagnostic PCR assays, J. Clin. Microbiol., 42, 1863–1868, 2004. 50. Hitchins, A.D. Detection and enumeration of Listeria monocytogenes in foods, in: Bacteriological Analytical Manual, 8th edn., Chapter 10: Gaithersburg, MD: U.S. Food and Drug Administration, 2003. 51. International Organization for Standardization (ISO). ISO 8914 General Guidance for the Detection of Vibrio parahaemolyticus, Geneva, Switzerland, 1990. 52. International Organization for Standardization (ISO). ISO 1190-1 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 1: Detection Method, Geneva, Switzerland, 1996. 53. International Organization for Standardization (ISO). ISO 1190-2 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 2: Enumeration Method, Geneva, Switzerland, 2000. 54. International Organization for Standardization (ISO). ISO 6579 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection of Salmonella spp., Geneva, Switzerland, 2002. 55. International Organization for Standardization (ISO). ISO 6579 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection of Salmonella spp., Technical Corrigendum 1, Geneva, Switzerland, 2004. 56. International Organization for Standardization (ISO). ISO 1190-1 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 1: Detection Method. AMENDMENT 1: Modification of the Isolation Media and the Haemolysis Test, and Inclusion of Precision Data, Geneva, Switzerland, 2004. 57. International Organization for Standardization (ISO). ISO 1190-2 Microbiology of Food and Animal Feeding Stuff s—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes. Part 2: Enumeration Method, AMENDMENT 1: Modification of the Enumeration Medium, Geneva, Switzerland, 2005. 58. International Organization for Standardization (ISO). ISO/TS 21872-1:2007 Microbiology of Food and Animal Feeding Stuff s. Horizontal Method for the Detection of Potentially Enteropathogenic Vibrio spp. Part 1: Detection of Vibrio parahaemolyticus and Vibrio cholerae, Geneva, Switzerland, 2007a. 59. International Organization for Standardization (ISO). ISO/TS 21872-2:2007 Microbiology of Food and Animal Feeding Stuff s. Horizontal Method for the Detection of Potentially Enteropathogenic Vibrio spp. Part 2: Detection of Species Other Than Vibrio parahaemolyticus and Vibrio cholerae, Geneva, Switzerland, 2007b. 60. Interstate Shellfish Sanitation Conference. (ISSC) Issue relating to a Vibrio vulnificus risk management plan for oysters. Proceedings of the Interstate Shellfish Sanitation Conference, Columbia, SC, 2003. 61. Isonhood, J., Drake, M.A., and Jaykus, L.A. Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads, Food Microbiol., 23, 584–590, 2006. 62. Jaton, K., Sahli, R., and Bille, J. Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples, J. Clin. Microbiol., 30, 1931–1936, 1992. 63. Jeyasekaran, G., Karunasagar, I., and Karunasagar, I. Incidence of Listeria spp. in tropical fish, Int. J. Food Microbiol., 31, 333–340, 1996.
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64. Johansson, T. Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environments, Int. J. Food Microbiol., 40, 77–85, 1998. 65. Joseph, S.W., Colwell, R.R., and Kaper, J.B. Vibrio parahaemolyticus and related halophilic Vibrios. Crit. Rev. Microbiol. 10, 77–124, 1982. 66. Käferstein, F.K., Motarjemi, Y., and Bettcher, D.W. Foodborne disease control: A transnational challenge, Emerg. Infect. Dis. 3, 503–510, 1997. 67. Kaper, J.B., Morris, J.G., and Levine. M.M. Cholera, Clin. Microbiol. Rev., 8, 48–86, 1995. 68. Karunasagar, I. et al. Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods, Int. J. Food Microbiol., 31, 317–323, 1996. 69. Kaufman, G.E. et al. Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix, J. Food Prot., 67, 2424–2429, 2004. 70. Kaysner, C.A. and DePaola, A. Outbreaks of Vibrio parahaemolyticus gastroenteritis from raw oyster consumption: Assessing the risk of consumption and genetic methods for detection of pathogenic strains, J. Shellfish Res., 19, 657, 2000. 71. Kaysner, C.A. and DePaola, A. Vibrio In: Compendium of Methods for the Microbiological Examination of Foods, 4th edn., Downes, F.P. and Ito, K., Eds., Washington, DC: American Public Health Association, 2001, pp. 405–420. 72. Kaysner, C.A. and DePaola, A. Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio spp., In Bacteriological Analytical Manual, 8th edn., Chapter 9: Revision A, Gaithersburg, MD: U.S. Food and Drug Administration, 2004. 73. Kim, Y.B. et al. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene, J. Clin. Microbiol., 37, 1173–1177, 1999. 74. Kitaura, T. et al. Halo production by sulfatase activity in V. vulnificus and V. cholerae O1 on a new selective sodium dodecyl sulfate containing agar medium: A screening marker in environmental surveillance, FEMS Microbiol. Lett., 17, 205–209, 1983. 75. Klontz, K.C. et al. Raw oyster-associated Vibrio infections: Linking epidemiologic data with laboratory testing of oysters obtained from a retail outlet, J. Food Prot. 56, 977–979, 1994. 76. Kobayashi, T. et al. A new selective isolation medium for vibrio group on a modified Nakanishi’s medium (TCBS agar medium), Jpn. J. Bacteriol., 18, 387–392, 1963. 77. Koch, W.H. et al. Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods, Appl. Environ. Microbiol., February, 556–560, 1993. 78. Kumar, H.S. et al. Detection of Salmonella spp. in tropical seafood by polymerase chain reaction, Int. J. Food Microbiol., 88, 91–95, 2003. 79. Kumar, H.S. et al. A gyrB-based PCR for the detection of Vibrio vulnificus and its application for direct detection of this pathogen in oyster enrichment broths, Int. J. Food Microbiol., 111, 216–220, 2006. 80. Lee, C.Y., Pan, S.F., and Chen, C.H. Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR, Appl. Environ. Microbiol., 61, 1311–1317, 1995. 81. Lee, J.Y., Eun, J.B., and Cho, S.N. Improving detection of Vibrio vulnificus in Octopus variabilis by PCR, J. Food Sci. 62, 179–182, 1997. 82. Lee, C.Y., Panicker, G., and Bej, A.K. Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization, J. Microbiol. Methods, 53, 199–209, 2003. 83. Luan, X. et al. Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction, Lett. Appl. Microbiol., 44, 242–247, 2007. 84. Luan, X. et al. Rapid quantitative detection of Vibrio parahaemolyticus in seafood by MPN-PCR, Curr. Microbiol., 57, 218–221, 2008. 85. Madden, J.M. et al. Virulence of three clinical isolates of Vibrio cholerae non O-1 serogroup in experimental enteric infections in rabbits, Infect. Immun., 33, 616–619, 1981.
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86. Massad, G. and Oliver, J.D. New selective and differential medium for Vibrio cholerae and Vibrio vulnificus, Appl. Environ. Microbiol., 53, 2262–2264, 1987. 87. McLaughlin, J.C. Vibrio, In Manual of Clinical Microbiology, 6th edn., Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., and Yolken, R.H., Eds., Washington, DC: ASM Press, 1995, pp. 465–474. 88. Mead, P.S., Slutsker, L., Griffin, P.M., and Tauxe, R.V. Food-related illness and death in the United States, Emerging Infect. Dis., 5, 607–625, 1999. 89. Miceli, G.A., Watkins, W.D., and Rippey, S.R. Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica), Appl. Environ. Microbiol., 59, 3519–3524, 1993. 90. Midelet-Bourdin, G., Leleu, G., and Malle, P. Evaluation of the International Reference Methods NF EN ISO 11290-1 and 11290-2 and an in-house method for the isolation of Listeria monocytogenes from retail seafood products in France, J. Food Prot., 70, 891–900, 2007. 91. Mintz, E.D., Popovic, T., and Blake, P.A. Transmission of Vibrio cholerae O1 in Vibrio cholerae and Cholera: Molecular to Global Perspectives, Wachsmuth, I.K., Blake, P.A., and Olsvik, O., Eds., Washington, DC: ASM press, 1994. 92. MMWR. Vibrio vulnificus infections associated with raw oyster consumption—Florida, 1981–1992, Morb. Mortal. Wkly. Rep., 42, 405–407, 1993. 93. Motes, M.L. et al. Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica), Appl. Environ. Microbiol., 64, 1459–1465, 1998. 94. Myers, M.L., Panicker, G., and Bej, A.K. PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico, Appl. Environ. Microbiol., 69, 2194–2200, 2003. 95. Nair, G.B. et al. Global dissemination of Vibrio parahaemolyticus serotype O3:K6 and its serovariants, Clin. Microbiol. Rev., 20, 39–48, 2007. 96. Nakamura, H. et al. Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan, Int. J. Food Microbiol., 94, 323–328, 2004. 97. Nakamura, H. et al. Molecular typing to trace Listeria monocytogenes isolated from cold-smoked fish to a contamination source in a processing plant, J. Food Prot., 69, 835–841, 2006. 98. Nordstrom, J.L. et al. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters, Appl. Environ. Microbiol., 73, 5840–5847, 2007. 99. Panicker, G. and Bej, A.K. Real-time PCR detection of Vibrio vulnificus in oysters: Comparison of oligonucleotide primers and probes targeting vvhA, Appl. Environ. Microbiol., 71, 5702–5709, 2005. 100. Panicker, G., Myers, M.L., and Bej, A.K. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR, Appl. Environ. Microbiol., 70, 498–507, 2004. 101. Paziak-Domanska, B. et al. Evaluation of the API test, phosphatidyl-inositol specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods, FEMS Microbiol. Lett., 171, 209–214, 1999. 102. Rahn, K.J., De Grandis, S.A., and Clarke, R.C. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella spp., Mol. Cell. Probes, 6, 271–279, 1992. 103. Rippey, S.R. Infectious diseases associated with molluscan shellfish consumption, Clin. Microbiol. Rev., 7, 419–425, 1994. 104. Rizvi, A.V. et al. Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes, FEMS Microbiol. Lett., 262, 185–192, 2006. 105. Rocourt, J., Jacquet, Ch., and Reilly, A. Epidemiology of human listeriosis and seafoods, Int. J. Food Microbiol., 62, 197–209, 2000. 106. Rodríguez-Lázaro, D. et al. Trends in analytical methodology in food safety and quality: Monitoring microorganisms and genetically modified organisms, Trends Food Sci Technol., 18, 306–319, 2007.
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107. Ryser, E.T. et al. Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media, Appl. Environ. Microbiol., 62, 1781– 1787, 1996. 108. Schmidt, K. and Tirado, C. WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe, 7th Report 1993–1998, Berlin, Germany: Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), 2001. 109. Shabarinath, S. et al. Detection and characterization of Salmonella associated with tropical seafood, Int. J. Food Microbiol., 114, 227–233, 2007. 110. Sharma, C. et al. Molecular analysis of non-O1 non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India, J. Clin. Microbiol., 36, 756–763, 1998. 111. Somer, L. and Kashi, Y. A PCR method based on 16 S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products, J. Food Prot., 66, 1658– 1665, 2003. 112. Strom, M.S. and Paranjpye, R.N., Epidemiology and pathogenesis of Vibrio vulnificus, Microbes Infect., 2, 177–188, 2000. 113. Su, Y.G. and Liu, C. Vibrio parahaemolyticus: A concern of seafood safety, Food Microbiol., 24, 549– 558, 2007. 114. Tada, J. et al. Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction, Mol. Cell. Probes, 6, 477–487, 1992. 115. Takahashi, H. et al. Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater, J. Food Prot., 68, 1083–1088, 2005. 116. Tamplin, M.L. et al. Enzyme immuno assay for identification of Vibrio vulnificus in seawater, sediment, and oysters, Appl. Environ. Microbiol., 57, 1235–1240, 1991. 117. Tirado, C., and Schmidt, K. WHO surveillance programme for control of foodborne infections and intoxications: Preliminary results and trends across greater Europe. World Health Organization, J. Infect., 43, 80–84, 2001. 118. Van Coillie, E. et al. Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market, J. Food Prot., 67, 2480–2487, 2004. 119. Vantarakis, A. et al. Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels, Lett. Appl. Microbiol., 31, 105–109, 2000. 120. Venkateswaran, K., Dohmoto, N., and Harayama, S. Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp, Appl. Environ. Microbiol., 64, 681–687, 1998. 121. Vickery, M.C.L. et al. A real-time PCR assay for the rapid determination of 16 S rRNA genotype in Vibrio vulnificus, J. Microbiol. Methods, 68, 376–384, 2007. 122. Villalobo, E. and Torres, A. PCR for detection of Shigella spp. in mayonnaise, Appl. Environ. Microbiol., 64, 1242–1245, 1998. 123. Wachsmuth, K. et al. Molecular Epidemiology of Cholera in Vibrio cholerae and Cholera: Molecular to Global Perspectives, Wachsmuth, I.K., Blake, P.A., and Olsvik, O., Eds., Washington, DC: ASM Press, 1994, pp. 357–370. 124. Wallace, D.J. et al. Incidence of foodborne illnesses reported by the foodborne diseases active surveillance network (FoodNet)-1997, J. Food Prot., 63, 807–809, 2000. 125. Wang, S. and Levin, R.E. Quantitative determination of Vibrio parahaemolyticus by polymerase chain reaction, Food Biotechnol., 18, 279–287, 2004. 126. Wang, S. and Levin, R.E. Rapid quantification of Vibrio vulnificus in clams (Protochaca staminea) using real-time PCR, Food Microbiol., 23, 757–761, 2006. 127. Wang, S.J. and Yeh, D.B. Designing of polymerase chain reaction primers for the detection of Salmonella enteritidis in foods and faecal samples, Lett. Appl. Microbiol., 34, 422–427, 2002.
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128. Wang, R.F., Cao, W.W., and Cerniglia, C.E. A universal protocol for PCR detection of 13 species of foodborne pathogens in foods, J. Appl. Microbiol., 83, 727–736, 1997. 129. Ward, L.N. and Bej, A.K. Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes, Appl. Environ. Microbiol., 72, 2031–2042, 2006. 130. Wong, H.C. et al. Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan, J. Food Prot., 63, 900–906, 2000. 131. Wright, A.C. et al., Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl. Environ. Microbiol., 62, 717–724, 1996.
Chapter 31
Parasites Juan Antonio Balbuena and Juan Antonio Raga Contents 31.1 31.2 31.3 31.4 31.5 31.6 31.7
Protozoa ........................................................................................................................581 Trematodes................................................................................................................... 582 Cestodes ....................................................................................................................... 587 Anisakid Nematodes .................................................................................................... 590 Other Nematodes ..........................................................................................................595 Acanthocephalans .........................................................................................................595 Seafood Safety .............................................................................................................. 596 31.7.1 Primary Production and Handling.................................................................. 596 31.7.2 Thermal Processing ......................................................................................... 596 31.7.3 Recommendations for Consumers and Restaurateurs.......................................597 31.7.4 Recommendations for Allergic and Immunosuppressed Persons ......................597 31.8 Further Developments ...................................................................................................598 Acknowledgments ....................................................................................................................599 References ................................................................................................................................599
The consumption of seafood has increased steadily over the last years. The yearly per capita consumption has augmented by 77% (from 9.14 kg in 1961 to 16.1 kg in 2003) [1]. As any other food, fish and shellfish are carriers of a wide range of parasites, but only a few have zoonotic significance. Nevertheless, their incidence on human health should not be neglected. For instance, of the ∼41 million people infected with foodborne trematodes, it is reckoned that ∼18 million cases correspond to fish trematodes [2,3]. Parasite infections in humans resulting from the consumption of fish and shellfish have been known for centuries. Indeed, the occurrence of fish helminths has been documented in ancient 579
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human remains in China [4] and Korea [5], and in pre-Columbian civilizations of both North [6] and South America [7,8]. Humans become infected mostly by eating raw, marinated, smoked, or undercooked seafood (fish, squid, oysters, shrimps, crabs, etc.) carrying larval stages of the parasites (Figure 31.1). These may or may not develop into adults in humans, but can lead to disorders whose severity varies depending of the species involved. Most seafoodborne parasites are metazoan helminths, particularly cestodes and, above all, trematodes. Infections with anisakid nematodes represent one of the most relevant emerging zoonoses worldwide. By contrast the significance of protozoan infections resulting from consumption of seafood is still poorly understood. Some helminth species can be detected visually by their size, color, and texture, which allow differentiation from fish tissues. Helminths located within the muscle or under the skin, can downgrade the product resulting in economic loss, but their detection is simpler, especially by candling of fish fillets. Other helminths, in contrast, occur in body cavities, viscera, or digestive tract. So detection by visual inspection under sanitary controls is more difficult and, thus, molecular or immunological assays are usually needed to reveal their occurrence. Diseases caused by parasites of fish and shellfish have been traditionally regarded as typical in communities with high-risk culinary traditions or in developing countries, where food hygiene and processing are limited. This is still so, but from the 1970s on, fish and shellfishborne zoonoses have increased progressively, in terms of both number of people and world regions affected. Demographic changes, market globalization, and improvement of transportation, which facilitate both food exports to almost every part of the world, and people movements to and from endemic areas, are factors accounting for this increment. The present chapter will review the main parasites with significance for human health of fish and shellfish from fresh, brackish, and marine waters, paying particular attention to the
Figure 31.1 raw fish.
Ceviche stand in Peru illustrates traditional consumption of dishes made with
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worldwide emerging anisakidoses. (Parasitic diseases mentioned in this chapter are named after the Standardized Nomenclature of Parasitic Diseases [9].)
31.1 Protozoa Information about protozoa of zoonotic relevance in fish and seafood is scanty. Most case reports concern species occurring in fecal water, which can contaminate all types of food. The pathogenic protozoa Cryptosporidium parvum and Giardia duodenalis (=G. intestinalis, G. lamblia) are well known for their capacity to produce waterborne disease outbreaks. Part of the life cycle of G. duodenalis occurs in the intestine of humans, as trophozoites and cysts. Although both forms are passed in stool (Figure 31.2), only the cysts can survive outside the host and are infectious to humans. Infections occur by ingestion of cysts contaminating drinking water, food, hands, or fomites (Figure 31.3). Fishborne transmission of G. duodenalis has been documented in the United States, via home-canned salmon, and in China, by means of koipla, a soup prepared with uncooked freshwater fish [10]. In addition, oocysts of Crystosporidium and cysts of Giardia spp. have been reported in different species of commercial marine bivalve mollusks [11–13]. Microsporidian spores have also been reported in fish and crustaceans. Moreover, it has been shown that spores of human-infectious microsporidians can accumulate in the Asian oyster, Crassostrea ariakensis [14,15]. Therefore, consumption of raw or undercooked bivalves can lead to protozoan infections in humans. Given that detection of G. duodenalis by stool analysis is difficult, enzyme-linked immunosorbent assay (ELISA) is the most usual approach for diagnosis in humans, whereas ELISA and immunofluorescent antibody analysis (IFA) are employed for detection in food [16]. The presence of Crystosporidium species in patients can be confirmed by both serological methods and stool analyses. The detection of oocysts in shellfish is relatively easy: the gills are removed, washed by vortexing and centrifugation, and oocysts present are examined and quantified by IFA. In addition, a polymerase chain reaction (PCR) protocol has been developed to genotype Crystosporidium oocysts in shellfish [17].
C
C
T
T 10 μm
Figure 31.2 G. duodenalis: trophozoites (T) and cysts (C) from human stool.
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3 a
b
c
1 2
Figure 31.3 Life cycle of G. duodenalis, a waterborne protozoan. (1) Cysts are shed in water along with feces. (2) Cysts contaminate water, food, hands, or fomites. (3) Humans become infected with cysts by consumption of contaminated water or food, or by contact with hands or fomites. (3a) Excystation occurs in the small intestine; two trophozoites emerge from each cyst. (3b) Trophozoites multiply asexually by binary fission. (3c) As they transit toward the colon, trophozoites form cysts, which are eventually passed in stool.
31.2 Trematodes Trematodes represent the most diverse group, in terms of number of species, of freshwater and seafood parasites infecting humans (Table 31.1). Most often, humans are accidental hosts of these trematodes, but in some species, such as Clonorchis sinensis or Paragonimus westermani, the parasites can attain sexual maturity and complete the life cycle in humans (Figure 31.4). The Paragonimidae represent a family of lung flukes, whose most pathogenic species belong to the genus Paragonimus and Pagumogonimus. Paragonimidosis is a severe lung disorder. The common symptoms are fever, cough, chest pain and, occasionally, subcutaneous nodules. Secondarily the disease can affect other organs, reaching the central nervous system and leading to meningitis. Exceptionally, paragonimidosis can be fatal. The most important species causing paragonimidosis are P. westermani and Paragonimus africanus, and Pagumogonimus skrjabini. China, Korea, Japan, and Thailand, in Asia, Cameroon and Liberia, in Africa and Venezuela, in South America, are countries where paragonimidosis is endemic [18]. Transmission to humans occurs by consumption of undercooked, marinated or raw crayfish or crab. It has been shown that metacercariae can survive outside crayfish, remaining viable for weeks on contaminated kitchen utensils [19]. Human infections can be diagnosed by chest x-ray, computer tomography, magnetic resonance imaging, sputum, and stool analyses (to reveal the occurrence of eggs), and by serological assays [18,20]. Apart from visual inspection of opened specimens, there are currently no other detection methods of metacercariae in crayfish. The species of intestinal flukes transmitted by fish belong to the families Heterophyidae and Echinostomidae. The heterophyids are fairly small and are not considered as highly pathogenic,
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Table 31.1 Main Fish and Shellfish Trematode Species Reported from Humans Species
Site
Intermediate and Paratenic Host
Other Definitive Hosts
Geographic Distribution
Acanthoparyphium tyosenense
Intestine
Estuarine bivalves and snails
Ducks
Korea
C. sinensis
Liver
Freshwater snails and fish
Carnivores, pigs, rats, buffaloes
Southeast and East Asia, Russia
Cryptocotyle lingua
Intestine
Marine fish
Piscivorous birds and mammals
Alaska, Greenland
Echinostoma hortense
Stomach, intestine
Freshwater snails and fish
Carnivores, rats, mice
Eastern Asia
Echinochasmus japonicus
Intestine
Freshwater snails and fish
Ducks, chickens
Eastern Asia
Echinochasmus perfoliatus
Intestine
Freshwater snails and fish
Carnivores, rats
Eastern Asia, Hungary, Italy, Rumania, Russia
Echinochasmus liliputanus
Intestine
Freshwater snails and fish
Carnivores
Middle East, China
Echinochasmus fujianensis
Intestine
Freshwater snails and fish
Carnivores, pigs, rats
China
Gymnophalloides seoi
Pancreas
Oysters
Wading birds
Korea
Haplorchis taichui
Intestine
Freshwater snails and fish
Carnivores, egret
Middle East, East and South Asia, North East Africa
Haplorchis pumilio
Intestinal
Freshwater snails and fish
Carnivores, pelicans
Thailand, Laos, China
Haplorchis yokogawai
Intestinal
Freshwater snails and fish
Carnivores, egret
Middle East and East and South Asia, Egypt
H. heterophyes
Intestine
Brackish water snails and fish
Carnivores, pelicans
Middle East and East Asia, North East Africa, Spain, Russia
Heterophyes nocens
Intestinal
Brackish water snails and fish
Cats
Eastern Asia (continued)
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Table 31.1 (continued) Humans Species
Main Fish and Shellfish Trematode Species Reported from
Site
Intermediate and Paratenic Host
Other Definitive Hosts
Geographic Distribution
Heterophyopsis continua
Intestine
Marine and brackish water fish
Cats, ducks, fish-eating birds
Eastern Asia
Metagonimus miyatai
Intestine
Freshwater snails and fish
Dogs, mice, rats, hamsters
Eastern Asia
Metagonimus takahashii
Intestine
Freshwater snails and fish
Dogs, mice
Eastern Asia
M. yokogawai
Intestine
Freshwater snails and fish
Carnivores, rats
Middle East, East and South Asia, Russia, Israel, Spain
Metorchis conjunctus
Bile ducts
Freshwater snails and fish
Carnivores
North America
Nanophyetus salmincola
Intestine
Freshwater and marine fish and snails
Carnivores
Northwest America, Eastern Siberia
Opisthorchis felineus
Liver
Freshwater snails and fish
Carnivore, pigs, rats, rabbits, martens, wolverines, seals
Eastern and South Europe, Russia, Caucasus
O. viverrini
Liver
Freshwater snails and fish
Carnivores, pigs, rats
South East Asia
P. skrjabini (=Paragonimus skrjabini)
Lungs
Freshwater snails and crabs
Carnivores
China, India
P. africanus
Lungs
Freshwater snails and crabs
Primates
Cameroon, Liberia
P. westermani
Lungs
Freshwater snails and crabs
Carnivores
East and South Asia
Paragonimus spp.
Lungs
Freshwater snails and crabs
Carnivores, pigs, rodents
South America
Pygidiopsis genata
Intestine
Brackish and freshwater fish
Domestic carnivores, piscivorous birds
Egypt
Parasites Table 31.1 (continued) Humans Species
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585
Main Fish and Shellfish Trematode Species Reported from Intermediate and Paratenic Host
Site
Other Definitive Hosts
Geographic Distribution
Pygidiopsis summa
Intestine
Brackish and freshwater fish
Domestic carnivores, piscivorous birds
Japan, Korea
Stellantchasmus falcatus
Intestine
Brackish water fish
Piscivorous birds
South East Asia, Hawaii
Stictodora fuscata
Intestine
Brackish water fish
Piscivorous birds
South East Asia
Stictodora lari
Intestine
Brackish water fish
Seagulls
Korea
Source: Based on Blair, D., in Marine Parasitology, Rhode, K., Ed., CSIRO Publishing, Collingwood, Victoria, Australia, 427, 2005; Chai, J.Y. et al., Int. J. Parasitol., 35, 1233, 2005.
6
5
1
2
4 3
Figure 31.4 The life cycle of C. sinensis illustrates the transmission of fishborne trematodes to humans. (1) Eggs are shed with feces in water. (2) Miracidia emerge from the eggs and swim freely to reach and penetrate into snails (first intermediate hosts). (3) Miracidia give rise to sporocysts and rediae, which multiply asexually in the snail. (4) Cercariae leave the snail and swim actively in search of the second intermediate host (fish and, more rarely, crustaceans). (5) Cercariae penetrate the host’s tissues and encyst as metacercariae. (6) Metacercariae develop into adults in the bile ducts of fish-eating birds or mammals (including humans).
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although certain species can produce important damage in the heart and central nervous system. Of the >30 heterophyid species that can infect humans, the most significant ones are Metagonimus yokogawai and Heterophyes heterophyes. The former is endemic in Korea and Japan, due to consumption of sweetfish, Plecoglossus altivelis. Human infections are also known in China, Taiwan, Siberia, and Europe. H. heterophyes is typical in Egypt, especially in the Nile Delta, where it is transmitted by consumption of salted or insufficiently baked grey mullets, Mugil cephalus. Human infections of this parasite have also been reported in Sudan and Saudi Arabia, and more rarely in Korea and Japan. The echinostomids usually occur in the digestive tract of their defi nitive hosts, birds and mammals, including humans, causing stomach, and duodenal ulcers. Echinostoma japonicus and Echinochasmus hortense are the most important of the numerous echinostomids reported in humans. Echinostoma japonicus is widely distributed in Korea and China, particularly in the Anhui, Fujian, Guangdong, Guangxi, and Jiangsu provinces. Echinochasmus hortense occurs mostly in Japan, Korea, and China, and it is transmitted by eating raw loach, Misgurnus anguillicaudatus, in Northeast China. Stool analyses often indicate mixed infections, making identification of the species involved more difficult [21]. Most liver flukes transmitted by fish and shellfish correspond to the family Opisthorchiidae. In addition, the pancreatic fluke Gymnophalloides seoi (family Gymnophallidae) is known to infect humans in Korea by consumption of oysters, Crassostrea gigas [22]. Opisthorchiosis can lead to severe inflammation of hepatic ducts and pancreatitis; chronic conditions are known in endemic regions. The most representative species are C. sinensis (Figure 31.5), especially abundant in China, with ∼12.5 million people infected [23], but also in Korea and Vietnam, Metorchis conjunctus, in North America (particularly among aboriginal people from Northern Canada and Greenland), Opisthorchis felineus, distributed from Southeastern Europe to Russia, and Opisthorchis viverrini, endemic in Thailand, Laos, Cambodia, and Vietnam. It has been pointed out that transmission of the latter species varies seasonally, increasing during the monsoon, because floods propitiate fecal contamination of water. A typical dish from northeastern Thailand and Laos, known as koi-pla, based on raw fish with garlic and vegetables, is the main source of infection of O. viverrini. Traditional eating habits, such as the morning congee with slices of raw freshwater fish in Southern China or slices of raw freshwater fish with red pepper sauce in Korea, are the main infection routes of C. sinensis. Detection of liver flukes in humans is based on stool analysis by cellophane tic smear or Kato-Katz techniques. ELISA assays are also used, and recently a PCR technique has been developed to detect O. viverrini in snails and fish [21]. Sometimes, mixed infections of liver flukes are reported. For instance, a 69-year-old man in Korea harbored 69,125 specimens of Gymnophalloides seoi, 328 of Heterphyes nocens, and 1 of Stictodora lari. The first mentioned species is the most pathogenic one, since it invades pancreatic ducts and leads to pancreatitis [24].
1 mm
Figure 31.5 C. sinensis, a liver fluke infecting fish-eating birds and mammals; whole mount of specimen extracted from the bile duct of a patient in Vietnam.
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Migrations have contributed a great deal to the extension of liver fluke infections. So, numerous reports of C. sinensis and Opisthorchis spp. in the United States and Canada can be linked to Asian immigrants. Tourism has also contributed to the expansion of infections. For instance, an outbreak of gastroenteritis produced by trematodes was reported in a group of American tourists returning from a trip to Kenya and Tanzania in 1983, and several similar cases have been reported from Canada [10].
31.3 Cestodes The main cestodes infecting humans transmitted by fish and seafood belong to the family Diphyllobotriidae, particularly to the genera Diphyllobothrium and Diplogonoporus (Table 31.2). Although only the life cycle of some species is known in detail, transmission occurs through aquatic food webs (Figure 31.6).
Table 31.2 Main Fish and Shellfish Tapeworm Species Reported from Humans Species
Site
Intermediate and Paratenic Hosts
Other Definitive Hosts
Geographic Distribution
Diphyllobothrium alascense
Intestine
Burbot, smelt
Dog
Alaska
Diphyllobothrium cameroni
Intestine
Marine fish
Seals
Pacific
Diphyllobothrium cordatum
Intestine
Marine fish
Dog, seals, walrus, sea lions
North Pacific, Arctic
Diphyllobothrium dalliae
Intestine
Freshwater fish
Dog, gulls
Alaska, Siberia
Diphyllobothrium dendriticum
Intestine
Freshwater fish
Fish-eating birds and mammals
Circumpolar, Switzerland
Diphyllobothrium elegans
Intestine
Marine fish
Seals, sea lions
North Sea, Greenland
Diphyllobothrium hians
Intestine
Marine fish
Seals
North Atlantic, Pacific Siberia
Diphyllobothrium klebanovski
Intestine
Salmonids
Unknown
Eastern Eurasia, Sea of Japan, Sea of Okhostsk
Diphyllobothrium lanceolatum
Intestine
Whitefishes
Dog, seals, porpoises
North Atlantic, North Pacific
Diphyllobothrium latum
Intestine
Burbot, pike, percids
Dog, bears
North and South America, Europe, Russia, Korea (continued)
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Table 31.2 (continued) Humans Species
Main Fish and Shellfish Tapeworm Species Reported from
Site
Intermediate and Paratenic Hosts
Other Definitive Hosts
Geographic Distribution
Diphyllobothrium nihonkaiense
Intestine
Pacific salmon
Unknown
Japan, Korea, Canada, France, Switzerland
Diphyllobothrium orcini
Intestine
Marine fish
Killer whale
Japan
D. pacificum
Intestine
Marine fish
Sea lions, fur seals
Alaska, Japan, South Eastern Pacific
Diphyllobothrium scoticum
Intestine
Marine fish
Sea lions, seals
South Atlantic Ocean
Diphyllobothrium stemmacephalum
Intestine
Marine fish
Toothed whales
North Atlantic, North Sea, Eastern Asia
Diphyllobothrium ursi
Intestine
Red salmon
Bears
North Eastern Pacific
Diphyllobothrium yonagoensis
Intestine
Salmon
Unknown
Japan, Eastern Siberia
Diplogonoporus balaenopterae (=D. grandis)
Intestine
Japanese anchovy
Baleen whales, sea lions, seals
Circumboreal, Antarctic, Spain
Source: Based on Blair, D., in Marine Parasitology, Rhode, K., Ed., CSIRO Publishing, Collingwood, Victoria, Australia, 427, 2005; Chai, J.Y. et al., Int. J. Parasitol., 35, 1233, 2005.
Diphyllobothriosis can be asymptomatic, but usual manifestations are abdominal pain, diarrhea, nausea, anorexia, and fatigue. Sometimes infections lead to pernicious anemia by depletion of vitamin B12. Diphyllobothrium latum and Diphyllobothrium pacificum (Figure 31.7) are significant representatives of this group. The former is typical in continental waters of the Holarctic region, and the latter occurs in marine waters along the Pacific coast of South America, where its abundance is influenced by El Niño event. Diphyllobothriosis occurs in communities where consumption of raw or little cooked fish is common. Dishes related to diphyllobothriosis include sushi and sashimi in Japan, gravlax in Scandinavia, strogonina in Eurasia, and ceviche, tiradito, and chinguirito in Peru, Ecuador, and Chile. Diphyllobothriosis is apparently declining worldwide, particularly in North America and Europe, as a result of effective public health policies. However, infections still persist in some endemic regions, such as the Russian Far East and Japan. In the last years, new cases have been reported in Chile, due to the introduction for angling of exotic freshwater fishes, such as rainbow trout, Oncorhynchus mykiss, and in Western Europe, owing to consumption of imported North Pacific salmon, Oncorhynchus keta [25,26].
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Figure 31.6 Life cycle of D. pacificum, a marine diphyllobothrid tapeworm infecting humans. (1) Eggs are shed in the feces of the definitive host (fur seals and sea lions). (2) Hatching occurs in water; emerging coracidia swim actively. (3) Coracidia are ingested by copepods (first intermediate hosts), where they develop into procercoids. (4) Copepods are preyed by fish (second intermediate hosts) and the procercoids become plerocercoids. (5) Second intermediate hosts can be preyed by larger fish (paratenic hosts). (6) Predation can occur several times, but plerocercoids undergo no further change. (7) Fish is eaten by the definitive hosts; plerocercoids develop then into adult worms. (8) Incidental infections occur by ingestion of plerocercoids in raw or undercooked fish.
2 cm
Figure 31.7
Proglottids of D. pacificum extracted from the intestine of a patient in Peru.
Detection in humans is based on standard stool analyses to reveal the occurrence of eggs or proglottids in feces. It is difficult to physically detect plerocercoids, in fish requiring meticulous analysis by specialized personnel. However, the presence of plerocercoids in fish elicits an immune response that can be detected by immunofluorescence techniques and ELISA [27].
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31.4 Anisakid Nematodes Anisakid nematodes are probably the most common parasites associated with seafood worldwide. Their larvae occur in fish and squid and their incidental ingestion by humans can cause anisakidosis and allergic reactions [28,29]. The most commonly reported anisakids causing disease in humans are Anisakis simplex and, to a lesser extent, Pseudoterranova decipiens [10,21,30], so that the more specific terms anisakiosis and pseudoterranovosis are used to designate infections with these species. Other larval anisakids, A. physeteris, Contracaecum osculatum, and Hysterothylacium aduncum (see Table 31.3), have been reported very rarely in humans [30]. Different genetic studies over the last 20 years have shown that A. simplex and P. decipiens in fact represent two respective complexes of sibling species, which exhibit some degree of geographic and/or definitive host differentiation [31,32]. The epidemiology and pathogenic manifestations in humans seem similar within each complex (although no formal study has been conducted to date in order to analyze potential interspecific differences) and, for convenience, in the present chapter the two species complexes will be referred to collectively as A. simplex and P. decipiens. A. simplex and P. decipiens utilize food webs for transmission to marine mammals (whales and seals, respectively), which act as definitive hosts. The life cycle of A. simplex has long been
Table 31.3 Main Fish and Shellfish Nematode Species Reported from Humans Species
Site
Intermediate and Paratenic Hosts
Other Definitive Hosts
Geographic Distribution
A. simplex
Stomach and intestine
Marine fish, squid
Cetaceans
Worldwide
Anisakis physeteris
Stomach and intestine
Marine fish, crustaceans, squid
Sperm whales
Worldwide
C. philippinensis
Intestine
Freshwater fish
Birds and monkeys experimentally
Southeast Asia
Gnathostoma spp.
Stomach and esophagus
Freshwater fish
Felids, pigs, weasels
Southeast and East Asia, India, Middle-East
Pseudoterranova decipiens
Stomach and intestine
Marine fish, crustaceans, squid
Seals, cetaceans
Cold waters worldwide
Contracaecum osculatum
Stomach and intestine
Marine fish, crustaceans
Seals, fisheating birds
Worldwide
Hysterothylacium aduncum
Stomach and intestine
Marine fish, crustaceans, squid
Marine fish
Worldwide
Source: Based on Ko, R.C., in Fish Diseases and Disorders. Vol. I. Protozoan and Metazoan Infections, Woo, P.T.K., Ed., CAB International, Oxon, 631, 1995; Nagasawa, K., in Marine Parasitology, Rhode, K., Ed., CSIRO Publishing, Collingwood, Victoria, Australia, 430, 2005.
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considered as pelagic, but recent studies have revealed differences between species within the complex, having pelagic, demersal, or benthic cycles [33]. In addition, the number and type of hosts used by each species varies depending on their availability and abundance in each geographic area [34]. However, a common trait is that the life cycle of A. simplex occurs offshore and, thus, A. simplex is virtually absent from estuarine and other brackish environments. Despite differences within the species complex, a generalized life cycle of A. simplex can be outlined (Figure 31.8). The life cycle of P. decipiens is similar to that of A. simplex, but the food web used is benthic or benthopelagic in order to target seals, instead of whales, as definitive hosts. The eggs sink to the bottom and the hatched larvae adhere to the substrate by their tails awaiting consumption by benthic crustaceans [35]. The bottom-up exploitation of marine food webs by anisakids (as illustrated in Figure 31.8) is very efficient to reach marine mammals because of their position as top predators. In particular, transmission is enhanced because the encapsulated third-stage larvae can use a wide range of hosts where they remain viable for long and, thus, accumulate over time in individual hosts [34,36]. So, in areas where marine mammals are abundant, anisakid larvae can be widespread, and common
8 7
1 6 2
4
3 5
Figure 31.8 Generalized life cycle of A. simplex. (1) Adult worms reside in the stomach of cetaceans (definitive hosts), where gravid females shed eggs that are passed in the host’s feces. (2) Free swimming larvae emerge from eggs. (3) Planktonic crustaceans (first intermediate hosts) ingest the larvae, which penetrate through the intestine into the crustacean hemocoel. (4) Third-stage larvae (L3s) occurring in planktonic crustaceans are infective and can already reach definitive hosts, such as baleen whales feeding on zooplankton. (5) Most often, infected crustaceans are preyed by fish, squid, or larger crustaceans, which in turn are consumed by larger predators. (6) Predation can occur several times and, at each instance, the ingested thirdstage larvae bore the intestinal wall to encapsulate in the visceral cavity of the new host. (7) When prey are eaten by cetaceans, the L3s molt three times to become fourth- and fifth-stage larvae and eventually adult worms. (8) Incidental infections occur by consumption of raw or undercooked seafood.
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10 mm
Figure 31.9 Third-stage larvae of A. simplex in the visceral cavity of blue whiting, Micromesistius poutassou. Note the characteristic larval print on the liver surface (arrow), which can prove the presence of the parasite even if larvae are inadvertently lost during examination.
among fish and squid of commercial size [33,37] (Figure 31.9), which accounts for the numerous incidental infections in humans. Incidental infections occur when the anisakid third-stage larvae are eaten with either raw or lightly cooked fish or squid. Given the physiological and anatomical similarity between humans and marine mammals, the larvae can survive and occasionally molt to fourth-stage larvae [38], but cannot mature and reach the adult stage. Some infections are asymptomatic; the larvae remain in the gastrointestinal tract without penetrating the tissues and can only be discovered when expelled by coughing, vomiting, or defecating. In other instances, the larvae release histolytic enzymes allowing them to penetrate into or through the stomach or intestinal mucosa (leading to gastric or intestinal anisakidosis, respectively) (Figure 31.10). More rarely, the larvae can invade other sites, such as the lung, liver, throat, and subcutaneous tissues [10,21,30]. The clinical manifestations of anisakidosis are varied and unspecific. The disease is characterized by a sudden onset of epigastric pain, sometimes accompanied by nausea and vomiting. Additional reported disorders include urticaria, pulmonary complications, allergic edema, hypersialorrhea, and polyarthritis. When the condition is chronic, histopathological examination reveals the larvae, with distinctive Y-shaped lateral chords, embedded in the gastrointestinal wall, accompanied by inflammatory infiltrate, forming eosinophilic abscesses or eosinophilic granulomas (Figure 31.11) [21,30]. The gastric condition can be confirmed by endoscopy, but clinical diagnosis of intestinal anisakidosis is extremely difficult because the symptoms can be easily attributed to common disorders, such as appendicitis, intestinal obstruction, or peritonitis [21]. Detection of anisakid larvae in fish is mostly made by visual inspection. Fillets and napes are usually inspected by candling on a light table and the larvae spotted are removed manually [35]. This procedure is less efficient with A. simplex (smaller and whitish larvae) than with P. decipiens [39]. Candling under ultraviolet (UV) light causes the larvae to fluoresce, but in A. simplex, it has been observed that fluorescent emission is only clearly seen in previously frozen fish [40]. In addition, worms embedded >0.5 mm deep in fish tissue are not visible [35]. Recently, PCR and ELISA methods have been developed to detect and quantify A. simplex larvae in seafood [41,42]. Some patients of anisakiosis show symptoms of urticaria and/or other allergic reactions, usually after manifestation of the digestive disorders, a condition known as gastroallergic anisakiosis. It has also become clear in recent years that the ingestion of A. simplex larvae can cause an immediate allergic response without showing further digestive symptoms [29]. The clinical symptoms range from urticaria, angioedema, and even arthralgia, to life-threatening anaphylactic shock [21,43]. Exposure by contact or inhalation of A. simplex allergens can also elicit allergic responses [29,43]. The strength of allergy to other anisakids is not yet known [21].
Parasites
◾ 593
Figure 31.10 A. simplex larva penetrating the small intestine wall, surrounded by a thick cuff of acute inflammatory cells (bar 1 mm). (From Takei, H. and Powell, S.Z., Ann. Diagn. Pathol., 11, 350, 2007. With permission.)
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LEC
DT
LEC
EG
M
Figure 31.11 Cross section through an A. simplex larva embedded in the small intestine wall. A thick cuff of acute inflammatory cells with numerous eosinophils surround the larva (bar 100 mm). M, muscle layer; LEC, epidermal chord; EG, excretory gland; DT, digestive tract. Note the characteristic Y-shaped LEC of A. simplex. (From Takei, H. and Powell, S.Z., Ann. Diagn. Pathol., 11, 350, 2007. With permission.)
The diagnosis of allergy to A. simplex is based on a compatible medical history (such as allergic symptoms after ingestion of fish) and immunological tests. Patients show positive skin tests and specific IgE against A. simplex, with a marked increase in total IgE and lack of reaction to proteins of fish [28,43]. However, specific IgE antibodies against A. simplex cross-react with those of other invertebrates, including dust mites, cockroaches, shrimps, and other helminths [43,44]. So a patient with, for instance, shrimp allergy might be misdiagnosed as allergic to A. simplex. An additional problem is that a considerable proportion of the population is sensitized to A. simplex, resulting from being exposed to the larvae without having developed allergy symptoms [45,46]. Therefore, the issue of distinguishing patients with clinical and subclinical sensitization has become highly relevant. In order to better understand the molecular bases of cross-reactivity and improve serological diagnosis of allergic anisakidosis, many studies have focused on molecular identification and characterization of A. simplex allergens [43–45,47]. To date, nine allergens of A. simplex have been molecularly characterized (www.allergen.org) [29,47], but the total number is probably much higher [48,49]. Somatic antigens (Ani s 2 and Ani s 3), obtained by homogenization of the whole larvae, seem to account for most cross-reactivity between A. simplex and other organisms [44]. So, whole-larva extracts used to detect specific IgE lack enough specificity for diagnosis of allergy to A. simplex. Excretory–secretory antigens, i.e., histolytic enzymes secreted by the parasite to penetrate the gastric mucosa, seem better suited for diagnosis [43]. In particular, Ani s 1 shows no or little crossreactivity with other allergens [43,50]. Ani s 7 is another major excretory–secretory antigen. Due to its sugar epitopes, it can cross-react with homologous glycoproteins of other organisms, but after o-deglycosylation, it is recognized by monoclonal antibody UA3 without false positive results [43]. Moreover, molecular characterization of A. simplex allergens and isolation of their encoding cDNAs is currently a very active field of research that is expected to facilitate diagnosis in the near future [29]. Anisakidosis is a health problem particularly important in countries and communities in which consumption of raw or undercooked fish is widespread. A. simplex accounts for the vast majority (∼97%) of human infections; P. decipiens represents only ∼2.7% of the total, whereas the remaining 0.3% corresponds to either unidentified anisakids or to other species [30].
Parasites
◾
595
Of the ∼20,000 human infections reported to date, ∼90% have been reported in Japan, where ∼2,000 new infections are diagnosed each year [21]. Anisakidosis in Europe is fairly common, with ∼600 cases reported mostly in France, Germany, the Netherlands, and Spain [39]. In Korea, 107 cases were reported between 1989 and 1992 [10], and ∼50 infections were known from the United States until the 1990s [51]. Other countries where anisakidosis has been reported sporadically include Canada, Chile, Egypt, Iceland, New Zealand, Oman, and Peru [21,52–54], and possibly China [23]. Evidence gathered over the last two decades shows that A. simplex is a frequent agent of foodrelated allergy. A serological survey performed in Japan on ∼2000 patients initially diagnosed with urticaria or food allergy revealed specific IgE against A. simplex in 29.8% of the subjects [55]. Another Japanese study has shown IgE responses to excretory–secretory antigens in 87.5% of patients with gastric anisakiosis, 75% of subjects initially diagnosed with fish-induced urticaria, 8.3% of individuals with idiopathic urticaria, and 10% of healthy controls [56]. Investigations carried out in Spain also point to a high prevalence of allergy to A. simplex. In a survey involving 868 subjects from three geographic areas, the prevalence among patients of urticaria or angioedema was 19.2%, and 13.1% of individuals without allergic symptoms were sensitized to the parasite [57]. Note that the above prevalence estimations might be inflated by cross-reactivity with other allergens and the large number of people manifesting subclinical sensitization [29]. However, a recent study using the more specific UA3-based ELISA assay revealed that the prevalence of sensitized individuals in Madrid was 12.4% [58], which is only slightly lower (15.7%) than that previously reported in the same city using a classical test [57]. Furthermore, current data from the Basque Country (Northern Spain) indicates that A. simplex accounts for 10% of anaphylaxis cases in adult patients, which is a figure similar to that of other food allergies combined [29]. Therefore, allergy to A. simplex should be a public-health concern in countries with high per capita consumption of fish. An interesting finding is that three HLA class II alleles are overrepresented in patients allergic to A. simplex [59]. Note also that sensitized subjects with repeated exposure to A. simplex will be at risk of developing acute anisakiosis with severe symptoms [46].
31.5 Other Nematodes Nematodes of freshwater fishes (Table 31.3), such as Capillaria philippinensis and Gnathostoma spp., can even be fatal for humans. C. philippinensis is typical in the Philippines, although it has extended to other countries, and it is currently considered an emerging zoonosis. Infections are common among farmers via ingestion of raw fish as lunch, during fishing activities in lagoons, lakes, and rivers. Another route of transmission is drinking basi (an alcoholic beverage) with raw food. Gnathostomiosis is mostly caused by four species: Gnathostoma hispidum, G. spinigerum, G. doloresi, and G. nipponicum. Thailand is one of the most affected countries, where G. spinigerum is widespread in the population of the country’s central regions by means of consumption of raw fish dishes, such as hu-sae, som-fak, and pla-som [60].
31.6 Acanthocephalans Human infections with acanthocephalans are extremely rare. However, several species use fish as paratenic hosts, thereby making incidental infections possible. Acanthocephalus rauschi and A. bufonis from the peritoneum of an Alaskan Eskimo and small intestine of an Indonesian man,
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respectively, represent two isolated case reports [61]. Other sporadic episodes concern members of the genera Bolbosoma and Corynosoma, which use cetaceans and seals as definitive hosts. They have been detected in an Eskimo from Alaska, and Japanese fishermen after consumption of sashimi [61,62].
31.7 Seafood Safety As any other food-related hazard, prophylaxis of parasitoses transmitted with seafood involves preventive steps at every level of the production–consumption chain [10].
31.7.1
Primary Production and Handling
The chances of human infections can be reduced by avoiding fishing and harvesting in particular areas, avoiding certain species or given sizes. In addition, control of feed in farmed species is highly relevant, since parasite infections are drastically reduced in species fed exclusively with artificial feed [39]. The occurrence of infective larvae in fish muscle is due to migration from the visceral cavity. Thus, evisceration on board reduces, but does not completely eliminate, the risk of human infections [37]. Note, however, that evisceration is not feasible in small species, such as sardines or anchovies. In addition, it has been suggested that viscera discarded over board can enhance transmission of parasites [33]. Trimming away the belly flaps of fish, candling and physically removing parasites detected visually can reduce the number of parasites. However, they do not completely eliminate the hazard, nor do they minimize it to an acceptable level [37,39,63]. Depuration is a standard safety procedure in which bivalve mollusks are placed in clean water under controlled conditions for varying periods of time in order to eliminate or minimize infective agents. The method is effective at reducing bacterial pathogens, but its efficacy with protozoa is currently unclear. Oocysts of Cryptosporidim sp. have been reported in bivalve samples with depuration times >72 h [11]. Industrial UV depuration protocols applied to spiked Pacific Oysters, Crassotrea gigas, resulted in a 13-fold increase in inactivation of C. parvum oocysts. However, low numbers of human-infectious oocysts still occurred in oysters after depuration [64]. In addition, it has been shown that depuration times of Cryptosporidim sp. oocysts and microsporidium spores in the Asian oyster are quite long (>33 days) [15]. Therefore, current depuration protocols are not enough to completely eliminate the risk of human protozoan infections.
31.7.2 Thermal Processing Thermal processing (either cooking or freezing) is the most effective way for eliminating the risk of parasitic disease from seafood. Other procedures (radiation, high pressure, acidification, salting, etc.) either are in experimental phase, affect the organoleptic properties of the product, or do not reduce risk to acceptable level [39,63]. Parasites are inactivated by heating until the inner part of the product reaches at least 63°C for 15 s or longer [10]. So, fully cooked, hot-smoked, and pasteurized seafood are safe from a parasitological point of view. When using conventional cooking, heating the food to 65°C for 10 min is recommended. Microwaving can be problematic, because microwave heating only acts on the outermost part of the product, so that heat is only transmitted further inside by conduction. Therefore, microwave cooking requires a higher safety temperature (74°C) [65].
Parasites
◾ 597
In the European Union, products to be consumed raw, cold-smoked, marinated, or salted must be frozen at a temperature of not more than −20°C, in all parts of the product for not less than 24 h [66]. This regulation is open to some misinterpretation, since the time needed to reach −20°C depends greatly on the type of freezer. For instance, a recent study [67] has shown that that it can take ∼10 h to attain −20°C inside fish placed in household freezers. Moreover, the same investigation showed that 50.5 h were required to kill all A. simplex larvae in fish kept at an internal temperature of −20°C [67]. This result suggests that the European regulation may not be stringent enough. The U.S. Food and Drug Administration recommendations [63], namely, freezing and storing at −20°C or below for 7 days or −35°C or below for 15 h, seem more in line with the above scientific evidence. The efficacy of proper freezing at preventing parasite infections in humans is unquestionable. Indeed, freezing is the only critical control point considered reliable in Hazard Analysis and Critical Control Points (HACCP) protocols for production of seafood [39]. In recent years, however, concern has been raised about the efficacy of thermal processing at preventing allergy to A. simplex. This issue is currently unsettled. Several studies suggest that allergic responses are only elicited by living larvae [39], at least in most patients [45]. However, other investigations have shown that A. simplex allergens are thermostable and, thus, cooking and freezing may not be protective enough [29,49]. The latter opinion is currently subscribed by the European Food Safety Authority [68].
31.7.3
Recommendations for Consumers and Restaurateurs
Information to consumers is of outmost importance to minimize the risks associated with parasites found in seafood. In order to avoid excessive public alarm, consumers should be aware that only a small portion of parasite species found in seafood are hazardous to public health. The following recommendations are largely based on information provided by the European Food Safety Authority [68], the Spanish Agency of Food Safety and Nutrition [39], and the U.S. Food and Drug Administration [51,63,65]. Fish and seafood should be purchased as fresh as possible. Medium and large fish should be bought eviscerated or be eviscerated immediately after purchase. The abdominal cavity should be washed and examined for parasites. Cook or freeze without delay. Raw, cold-smoked, marinated, or lightly cooked seafood should not be consumed, unless proof is given that the product has been kept frozen at −20°C or less, for at least 1 week. Seafood dishes should be properly cooked. Baking, boiling, and frying are safer than broiling and microwaving. In the latter case, setting the oven 14°C higher than the safety temperature (i.e., 74 °C + 14°C) is recommended. The use of cooking thermometers placed at the thickest part of the food is encouraged to guarantee that safety temperatures are attained. In addition, consumers and restaurateurs should always check that the dish is cooked throughout before serving or eating.
31.7.4
Recommendations for Allergic and Immunosuppressed Persons
In addition to the above, persons suffering from immunological deficiency or from allergy to A. simplex should consider to the following recommendations: Infections with waterborne protozoa can be fatal for immunosuppressed patients. Thus people with this ailment should by all means avoid consumption of raw or undercooked seafood [15].
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Evidence suggests that patients allergic to A. simplex react differently after ingestion of frozen fish [45,49]. So only a doctor can provide adequate dietary advice to each individual. However, most patients (between 80% and 90% [49]) show good tolerance to frozen fish. Fish eviscerated and blast-frozen on factory vessels are to be preferred, since the risk of larval emigration from the visceral cavity to the muscle is greatly reduced. Fish tails and marine farmed and freshwater fish are safer. Allergic patients should always avoid eating the hypaxial musculature of fish, as well as small whole fish [39].
31.8 Further Developments Seafood has played a major role in doubling animal protein consumption in developing countries over the last 30 years. In China, for instance, per capita fish consumption has quadrupled between 1970 and 2003 [1]. Although less dramatically, consumption has also increased in developed countries, driven by both globalization of culinary tastes and promotion of healthy food to prevent cardiovascular disease and obesity. Thus seafoodborne parasites are likely to remain a global issue in the years to come. Although some public health plans have proven successful to control and reduce some parasitic diseases (such as diphyllobothriosis in Scandinavia), worldwide the incidence of fishborne parasites is on the rise. One reason for this phenomenon is increased pressure on food sources to meet world population growth. Increasing poverty and malnutrition, coupled to limited public health services in many countries, propitiates the extension of seafoodborne parasitoses, particularly those caused by trematodes. In this context, steady growth of aquaculture as a source of inexpensive animal proteins (particularly in continental waters of Asia) will imply higher risk of seafoodborne zoonoses, if food habits and control policies remain unchanged [69]. Economy and trade globalization have fostered exports of seafood. The effects of food safety regulations imposed by developed countries to imports from developing countries, such as HACCP processes and technical barriers to trade, have already introduced high costs that tend to exclude small producers and processors from the export supply chain. Thus, future efforts should be directed at assisting and supporting fishermen, fish farmers, and processors in developing countries to adopt technology for efficient HACCP processes, and thereby realize benefit from global trade [70]. Low risk perception among consumers of developed countries of imported seafoodborne parasites favors uncommon human infections. For instance, case reports involving Diplogonoporus sp. and A. simplex in Zaragoza, inland in Spain, have been reported [71,72]. Many of these new human infections stem from cultural globalization by the adoption of cooking traditions based on dishes made with raw or little cooked seafood. The increment of human infections with H. heterophyes in North America [73] illustrates this aspect. Moreover, the increase of travel from and to endemic areas has facilitated the expansion of these parasitic zoonoses [21,74]. Therefore, the development of appropriate food safety programs poses new challenges to public health managers. In addition, improved diagnosis techniques and increased awareness among health personnel will result in detection of more cases that otherwise could go unnoticed. As in any other health hazard, better consumer information and education is of paramount importance for prevention. Providing the correct message and format to the different at-risk groups is a constant challenge to educational programs. However, providing alternative sustainable, affordable solutions to elicit behavioral change has also been a limitation of control programs [74].
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Acknowledgments The authors thank Drs. Jorge Manuel Cárdenas (Asociación Peruana de Helmintología e Invertebrados Afines), Angélica Terashima (Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia), Hidehiro Takei (Baylor College of Medicine, Houston), and Suzanne Z. Powell (The Methodist Hospital, Houston) for kindly contributing with photographs illustrating this review.
References 1. Food and Agricultural Organization of the United Nations. Food Balance Sheets. http://faostat. fao.org. 2. WHO. Control of Foodborne Trematode Infections. WHO Technical Report Series, 849. World Health Organization, Geneva, 1995. 3. Murrell, K.D. and Bernard F. Food-Borne Parasitic Zoonoses. Fish and Plant-Borne Parasites. Springer, New York, 2008. 4. Yang, W.Y. et al. Parasitologische Untersuchung einer alten Leiche aus der Chu-Dynastie der Streitenden Reiche aus dem Mazhuan-Grab Nr. 1, Kreis Jiangling, Provinz Hubei. Acta Acad. Med. Wuhan, 4, 23, 1984. 5. Seo, M. et al. Gymnophalloides seoi eggs from the stool of a 17th century female mummy found in Hadong, Republic of Korea. J. Parasitol., 94, 467, 2008. 6. Bathurst, R.R. Archaeological evidence of intestinal parasites from coastal shell middens. J. Archaeol. Sci., 32, 115, 2005. 7. Ferreira, L.F. et al. The finding of eggs of Diphyllobothrium in human coprolites (4,100–1,950 B.C.) from Northern Chile. Mem. Inst. Oswaldo Cruz, 79, 175, 1984. 8. Reinhard, K. and Urban, O. Diagnosing ancient Diphyllobothriasis from Chinchorro mummies. Mem. Inst. Oswaldo Cruz, 98, 191, 2003. 9. Kassai, T. Nomenclature for parasitic diseases: Cohabitation with inconsistency for how long and why? Vet. Parasitol., 138, 169, 2006. 10. Butt, A.A. et al. Infections related to the ingestion of seafood. Part II: Parasitic infections and food safety. Lancet Infect. Dis., 4, 294, 2004. 11. Freire-Santos, A.M. et al. Detection of Cryptosporidium oocysts in bivalbe molluscs destined for human consumption. J. Parasitol., 86: 853, 2000. 12. Gómez-Couso, H. et al. Contamination of bivalve molluscs by Crystosporidium oocysts: The need for new quality control standards. Int. J. Food Microbiol., 87, 97, 2003. 13. Gómez-Couso, H. et al. Detection of Crystosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR. Int. J. Food Microbiol., 91, 279, 2004. 14. Slifko, T.R. et al. Emerging parasite zoonoses associated with water and food. Int. J. Parasitol., 30, 1379, 2000. 15. Graczyk, T.K. et al. Recovery, bioaccumulation and inactivation of human waterborne pathogens by the Chesapeake Bay nonnative oyster, Crassotrea ariakensis. Appl. Environ. Microbiol., 72, 3390, 2006. 16. Sulaiman, I.M., and Cama, V. The biology of Giardia parasites, in Foodborne Parasites. Ortega, Y.R., Ed., Springer, New York, 2006, Chapter 2. 17. Xiao, L. and Cama, V. Cryptosporidium and cryptosporidiosis, in Foodborne Parasites. Ortega, Y.R., Ed., Springer, New York, 2006, Chapter 4. 18. Liu, Q. et al. Paragonimiasis: An important food-borne zoonosis in China. Trends Parasitol., 24, 318, 2008.
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19. Cross, J.H. Fish- and invertebrate-borne helminths, in Foodborne Disease Handbook, 2. Hui, Y.H., Sattar, S.A., Murrell, K.D., Nip, W.K., and Stanfield, P.D., Eds., Marcel Dekker Inc., New York, 2001, Chapter 12. 20. Adams, A.M. Foodborne trematodes, in Foodborne Parasites. Ortega, Y.R., Ed., Springer, New York, 2006, Chapter 7. 21. Chai J.Y. et al. Fish-borne parasitic zoonoses: Status and issues. Int. J. Parasitol., 35, 1233, 2005. 22. Chai J.Y. et al. Gymnophalloides seoi: A new human intestinal trematode. Trends Parasitol., 19, 109, 2003. 23. Zhou, P. et al. Food-borne parasitic zoonoses in China: Perspective for control. Trends Parasitol., 24, 190, 2008. 24. Chai J.Y. et al. Stictodora lari (Digenea: Heterophyidae): The discovery of the first human infections. J. Parasitol., 88, 627, 2002. 25. Yeraa, H. et al. Putative Diphyllobothrium nihonkaiense acquired from a Pacific salmon (Oncorhynchus keta) eaten in France; Genomic identification and case report. Parasitol. Int., 55, 45, 2006. 26. Wichta, B., de Marvalb, F., and Peduzzia, R. Diphyllobothrium nihonkaiense (Yamane et al., 1986) in Switzerland: First molecular evidence and case reports. Parasitol. Int., 56, 195, 2007. 27. Sharp, G.J.E., Pike, A.W., and Secombes, C.J. The immune response of wild rainbow trout, Salmo gairdneri Richardson, to naturally acquired plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825). J. Fish Biol., 35, 781, 1989. 28. Audicana, M.T. et al. Anisakis simplex: Dangerous—Dead and alive? Trends Parasitol., 18, 20, 2002. 29. Audicana, M.T. and Kennedy, M.W. Anisakis simplex: From obscure infectious worm to inducer of immune hypersensitivity. Clin. Microbiol. Rev., 21, 360, 2008. 30. Nagasawa, K. Anisakiasis, in Marine Parasitology. Rhode, K., Ed., CSIRO Publishing, Collingwood, Victoria, Australia, 2005, 430. 31. Mattiucci, S. et al. Genetic and ecological data on the Anisakis simplex complex with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae). J. Parasitol., 83, 401, 1997. 32. Zhu, X.Q. et al. SSCP-based identification of members within the Pseudoterranova decipiens complex (Nematoda:Ascaridoidea:Anisakidae) using genetic markers in the internal transcribed spacers of ribosomal DNA. Parasitology, 124, 615, 2002. 33. Abollo, E., Gestal, C., and Pascual, S. Anisakis in marine fish and cephalopods from Galician waters: An updated perspective. Parasitol. Res., 87, 492, 2001. 34. Klimpel S. et al. Life cycle of Anisakis simplex in the Norwegian Deep (northern North Sea). Parasitol. Res., 94, 1, 2004. 35. McClelland, G. The trouble with sealworms (Pseudoterranova decipiens species complex, Nematoda): A review. Parasitology, 124, S183, 2002. 36. Herreras, M.V. et al. Anisakid larvae in the musculature of the Argentinean Hake, Merluccius hubbsi. J. Food Prot., 63, 1141, 2000. 37. Adroher, F.J. et al. Larval anisakids (Nematoda:Ascaridoidea) in horse mackerel (Trachurus trachurus) from the fish market in Granada (Spain). Parasitol. Res., 82, 253, 1996. 38. Rosales, J. et al. Acute intestinal anisakiasis in Spain: A fourth-stage Anisakis simplex larva. Mem. Inst. Oswaldo Cruz, 94, 823, 1999. 39. Scientific Committee, Spanish Agency of Food Security and Nutrition. La alergia por Anisakis y medidas de prevención. Rev. Com. Cient. AESAN, 1, 19, 2005. http://www.informacionconsumidor. org/Documentacioacuten/tabid/57/Default.aspx?xspc = anisakis. 40. Tejada, M. et al. Scanning electron microscopy of Anisakis larvae following different treatments. J. Food Prot., 69, 1379, 2006. 41. Santos, A.T. et al. A method to detect the parasitic nematodes from the family Anisakidae in Sardina pilchardus, using specific primers of 18 S DNA gene. Eur. Food Res. Technol., 222, 71, 2006. 42. Arilla, M.C. et al. An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex. Parasitology, 135, 735, 2008. 43. Valls, A. et al. Anisakis allergy: An update. Rev. Fr. Allergol. Immunol. Clin., 45, 108, 2005.
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44. Guarneri, F., Guarneri, C., and Benvenga, S. Cross-reactivity of Anisakis simplex: Possible role of Ani s 2 and Ani s 3. Int. J. Dermatol., 46, 146, 2007. 45. Baeza, M.L. et al. Characterization of allergens secreted by Anisakis simplex parasite: Clinical relevance in comparison with somatic allergens. Clin. Exp. Allergy, 34, 296, 2004. 46. Toro, C. et al. Seropositivity to a major allergen of Anisakis simplex, Ani s 1 in dyspeptic patients with Helicobacter pylori infection: Histological and laboratory findings and clinical significance. Clin. Microbiol. Infect., 12, 453, 2006. 47. Rodríguez-Pérez, R. et al. Cloning and expression of Ani s 9, a new Anisakis simplex allergen. Mol. Biochem. Parasitol., 159, 92, 2008. 48. Arlian, L.G. et al. Characterization of allergens of Anisakis simplex. Allergy, 58, 1299, 2003. 49. Moneo, I. et al. Sensitization of the fish parasite Anisakis simplex: Clinical and laboratory aspects. Parasitol. Res., 101, 1051, 2007. 50. Ibarrola, I. et al. Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1. J. Investig. Allergol. Clin. Immunol., 18, 78, 2008. 51. Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. The Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. International Medical Publishing, McLean, VA, 2004, Washington, D.C., 1992, Chapter 25. http://www.cfsan.fda.gov/∼mow/ chap25.html. 52. Bhargava, D. et al. Anisakiasis of the tonsils. J. Laryngol. Otol., 110, 387, 1996. 53. Cabrera, R. and Trillo-Altamirano, M.P. Anisakidosis: ¿Una zoonosis parasitaria marina desconocida o emergente en el Perú? Rev. Gastroenterol. Perú, 24, 335, 2004. 54. Skírnisson, K. Hringormar berast í folk á Íslandi við neyslu á lítið elduðum fiski. Læknablaðið, 92, 21, 2006. 55. Kimura, S. et al. IgE response to Anisakis simplex compared to seafood. Allergy, 54, 1224, 1999. 56. Kasuya, S. and Koga, K. Significance of detection of specific IgE in Anisakis-related diseases. Arerugi, 41, 106, 1992 [in Japanese]. 57. Fernández de Corres, L. et al. Prevalencia de la sensibilización a Anisakis simplex en tres áreas españolas, en relación a las diferentes tasas de consumo de pescado. Relevancia de la alergia a Anisakis simplex. Alergol. Immunol. Clín., 16, 337, 2001. 58. Puente, P. et al. Anisakis simplex: The high prevalence in Madrid (Spain) and its relation with fish consumption. Exp. Parasitol., 118, 271, 2008. 59. Sánchez-Velasco, P. et al. Association of hypersensitivity to the nematode Anisakis simplex with HLA class II DRB1*1502-DQB1*0601 haplotype. Hum. Immunol., 61, 314, 2000. 60. Ko, R.C. Fish-borne parasitic zoonoses, in Fish Diseases and Disorders. Vol. I. Protozoan and Metazoan Infections. Woo, P.T.K., Ed., CAB International, Oxon, 1995, 631. 61. Schmidt, G.D. Acanthocephalan infection of man, with two new records. J. Parasitol., 57, 582, 1971. 62. Williams, H. and Jones, A. Parasitic Worms of Fish, Taylor & Francis, London, 1994. 63. Center for Food Safety and Applied Nutrition. Fish and Fisheries Products Hazards and Controls Guidance, 3rd edn., U.S. Food and Drug Administration, Washington, D.C., 2001, Chapter 5. http:// www.cfsan.fda.gov/∼comm/haccp4.html. 64. Sunnotel, O. et al. Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific Oysters (Crassostrea gigas). Appl. Environ. Microbiol., 73, 5083, 2007. 65. Center for Food Safety and Applied Nutrition. Food Code Report PB 2005-102200. U.S. Food and Drug Administration, College Park, MD, 2005, 75. http://www.cfsan.fda.gov/∼dms/fc05-toc.html. 66. European Parliament and Council of the European Unioñ. Regulation (EC) No. 853/2004 of 29 April 2004 laying down specific hygiene rules for on the hygiene of foodstuffs. Official Journal of the European Union, L 139/55, 30 April 2004, section VIII. http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri = OJ:L:2004:139:0055:0205:EN:PDF. 67. Adams, A.M. et al. Survival of Anisakis simplex in arrowtooth flounder (Atheresthes stomias) during frozen storage. J. Food Prot., 68, 1441, 2005.
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68. European Food Safety Authority. Opinion of the Scientific Committee on veterinary measures relating to public health—Allergic reactions to ingested Anisakis simplex antigens and evaluation of the possible risk to human health. April 27 1998. http://ec.europa.eu/food/fs/sc/scv/out05_en.html. 69. Thu, N.D. et al. Survey for zoonotic liver and intestinal trematode metacercariae in cultured and wild fish in An Giang Province, Vietnam. Kor. J. Parasitol., 45, 45, 2007. 70. Ahmed, M. Outlook for fish to 2020: A win-win-win for the oceans, fisheries and the poor? in Fish Aquaculture and Food Security, Sustaining Fish as a Food Supply, Record of a conference conducted by the ATSE Crawford Fund. Canberra, Australian Capital Territory, Australia, 11 August, 2004, 66. 71. Clavel A. et al. A live Anisakis physeteris larva found in the abdominal cavity of a woman in Zaragoza, Spain. Jpn. J. Parasitol., 42, 445, 1993. 72. Clavel A. et al. Diplogonoporiasis presumably introduced into Spain: First confirmed case of human infection acquired outside the Far East. Am. J. Trop. Med. Hyg., 57, 317, 1997. 73. Dixon B.R. and Flohr R.B. Fish- and shellfish-borne trematode infections in Canada. Southeast Asian J. Trop. Med. Publ. Health, 28, 58, 1997. 74. Macpherson, C.N.L. Human behaviour and the epidemiology of parasitic zoonoses. Int. J. Parasitol., 35, 1319, 2005.
Chapter 32
Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases Carlos Pereira Dopazo and Isabel Bandín Contents 32.1 32.2 32.3 32.4 32.5 32.6
Introduction: The Need for Diagnosis .......................................................................... 604 Diagnosis: Its Definition .............................................................................................. 604 Validation of Diagnostic Tests ...................................................................................... 605 Factors Affecting the Accuracy of Diagnosis: Sample Processing .................................. 606 Methods of Diagnosis for Aquatic Animal Diseases ..................................................... 608 Clinical, Histological, and Microscopical Techniques .................................................. 609 32.6.1 Gross Signs ...................................................................................................... 609 32.6.2 Histopathology ................................................................................................ 609 32.6.3 Immunohistochemistry ................................................................................... 609 32.6.4 Electron Microscopy ........................................................................................613 32.7 Isolation in Cell Culture................................................................................................613 32.7.1 Cell Lines and Cell Culture .............................................................................613 32.7.2 Selection of a Cell Line for Diagnosis...............................................................614 32.7.3 Viral Isolation in Cell Culture..........................................................................614 32.7.4 Performance of the Diagnostic Procedure ........................................................ 615 32.8 Serological and Immunological Techniques of Diagnosis ..............................................616 32.8.1 Scientific Basis..................................................................................................616 32.8.2 Advantages and Disadvantages of the Immunological Diagnostic Tools ..........616 32.8.3 Description of Immune Diagnostic Procedures ................................................619 603
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32.8.4 Antibody Detection Diagnosis .........................................................................621 32.9 Molecular Diagnosis..................................................................................................... 622 32.9.1 Scientific Basis: An Overview .......................................................................... 622 32.9.2 Performance of the Molecular Techniques of Diagnosis: Critical Steps and Critical Factors ................................................................................ 626 32.9.3 Molecular Methods of Diagnosis: Brief Description of Protocols .....................631 32.10 Nonlethal Methods of Diagnosis.................................................................................. 634 References ................................................................................................................................635
32.1 Introduction: The Need for Diagnosis Viral diseases cause important loses in fish and shellfish aquaculture. They are especially a worrying issue for fish farmers since they can cause either high mortality short after first symptoms are discovered in a stock, and quickly spread within the farm, or low but continuous deaths that end with high cumulative mortalities. In addition, the survivors of a viral disease will, in many cases, become asymptomatic carriers and spread the disease for a long time before they are detected. Therefore, although, at least to present knowledge, they do not represent a threat to human health, viral diseases in aquaculture can compromise the otherwise unstoppable worldwide development of this industry. On the other hand, those tools for controlling diseases caused by other agents are not available, poorly developed, or of low efficiency for viral disease. For instance, chemotherapy treatment, though available, is not affordable (for expensive) and its efficiency questionable. In addition, in spite of the important efforts in improving existing vaccines, and in the designing of new strategies for vaccination, this method for controlling viral diseases, though promising, is far from being effective. Therefore, control of fish and shellfish diseases caused by viral agents mostly relies on access to highly sensitive, rapid, and reliable diagnostic procedures. The importance of diagnostics is unquestionable. It is focused to two main roles in aquatic animal health: (1) to determine the cause of a disease previously detected in a culture facility or fish stock and (2) to be applied in specifically designed surveillance and monitoring programs. In the first case, diagnostic is demanded by the industry to identify the cause of mortality in the farm, and to provide the appropriate tools to reduce its effect on the production. The second role can be aimed to perform epidemiology studies to determine the origin of, and/or to eradicate, a certain infection, or to demonstrate freedom from a disease or infection in a certain population or geographic zone. In this former case, diagnostics is part of a strategy to reduce risk of spreading pathogens due to national and international trade of live fish, which is well described and widely employed by different National Administrations and International Organizations [1–5].
32.2
Diagnosis: Its Definition
Under a strict point of view, a diagnostic test is applied to determine the nature of a disease. Thus, the term diagnosis should only be employed if the test is applied to clinical diseased individuals, whereas when applied to asymptomatic fish those tests must be considered as for screening instead [6,7]. Considering that in most cases the same types of tests are applied for both purposes, such definition seems to be too strict. In fact, under a wider point of view, a diagnostic test might be
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defined as a method, procedure, or technique that is employed for the detection and the identification of a certain agent (a virus, for instance) in a given sample (e.g., a fish tissue) and/or to determine the health status of the corresponding fish. Definitions apart, as the final product of a diagnostic tool is the knowledge of the agent causing a disease in a fish or shellfish population, which will be the bases for important decisions regarding animal health control, that diagnostic tool must be reliable. This obvious remark (together with its applicability) is one of the most important aspects to be considered when selecting a diagnostic technique for any specific case. Therefore, parameters determining the performance of any diagnostic procedure must be defined and quantified.
32.3 Validation of Diagnostic Tests Any test of diagnosis should be validated before it is applied for a specific purpose, and under welldefined conditions. Several parameters can be used to quantify the accuracy and the reliability of a diagnostic test. For instance, sensitivity is one of the parameters most frequently employed. There are two ways of understanding this parameter [7]. Analytical sensitivity refers to the minimum amount of analyte that the test is capable to detect in a sample, and under the specific conditions assayed. It is equivalent to the detection limit (DL) mostly shown in reports on the design and the optimization of diagnostic methods. It provides information on the viral load threshold, independently from the level of infection of the animal. Diagnostic sensitivity or clinical sensitivity is defined as the percentage of diseased individuals that the test is capable to currently detect in a population. This kind of information is useful for field application and for surveillance programs statistics, and is therefore demanded by epidemiologists. However, its use has been scarce in the literature. As for sensitivity, there are two ways of defining specificity. For the pathologists and the designers of the analytical methods of diagnosis the term specificity deals with the premise that a diagnostic method must not yield false positive results, meaning that the test must detect the agent (i.e., the specific virus for which it has been designed) only if it is actually present in the animal [8]. This is known as analytical specificity, and is the reason why in most reports (if not all) on the design or the evaluation of diagnostic procedures negative controls are included to rule out false positives due to endogenous reactivity with other analytes or chemicals, and/or unexpected reactivity with other phylogenetically related or unrelated viruses. For an epidemiologist, however, specificity is defined as the probability to correctly detect healthy individuals in a population (diagnostic specificity). In this case, it deals with a second way of understanding specificity for a pathologist. Thus, if the intention is to detect diseased fish, the causative virus must be detected independently from the viral type. Therefore, the diagnostic method must be validated against the different types (serotypes or genotypes) known for that virus. Repeatability and reproducibility (R&R) are two parameters frequently misjudged. They are crucial to define the performance of a procedure since they are the only parameters quantifying its precision. Both deal with the probability to always obtain the same result (or the uncertainty of the obtained results). However, repeatability is defined as the precision determined under conditions where the same method and equipment are used by the same operator on a sample (equivalent to comparing results obtained from different replicas), reproducibility is the precision determined under conditions where the same method (with the same protocol and materials) but different equipment, in different days or laboratories are used by different operators. Although different statistical tests can be employed to quantify R&R [9], the most simple one is
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the use of the coefficient of variation between results when numeric values are yielded (e.g., viral load, or limit of detection (LD) ), or the percentage of identical results when presence/absence of the virus is determined. In spite of its importance, as we will further show, R&R values are rarely calculated and provided in reports on the evaluation of diagnostic tests, mainly due to the special effort that required repetitions represent for the laboratory. Other parameters frequently provided are dynamic range (range of viral concentration accurately detectable), analytical time required, cost, or applicability (which is a subjective, nonquantifiable parameter). On the other hand, other parameters (as predictive values or likelihood ratios), which are important for epidemiologist but rarely employed (or not at all) in the literature on the development of diagnostic procedures, will not be employed in the present chapter for the comparison of methods.
32.4 Factors Affecting the Accuracy of Diagnosis: Sample Processing The reliability of the result obtained from a diagnosis does not only depend upon the performance of the diagnostic test itself. There are previous steps that strongly influence the result: sampling procedure and type of sample, conditions of transportation and conservation of samples, and sample processing and concentration. These steps will be the subject of this chapter. In addition, after the application of the analytical test two final steps such as the confirmation and the interpretation of the results must be taken into consideration. Their description and influence on the result will be tackled for each specific diagnostic procedure. This chapter is on viral diagnosis, not on surveillance programs. Therefore, the description of the sampling methodology is not the scope of this chapter. The sampling procedure may influence the statistics of the health situation of a population under study, but not really, at least directly, the result of a test. For those really interested in sampling procedures, further reading is recommended [6,7,9,10]. We must remark that, no matter what is the kind of sample to be employed, it must be obtained from alive or moribund animals not from dead animals. On the other hand, if the viral load in a sample is critical for the efficacy of the diagnostic produce, symptomatic individuals must be chosen. For general purposes, the Diagnostic Manual of the Office International des Épizooties (OIE, World Organization for Animal Health; [7]) recommends the sampling of whole fish larvae, or head, kidney, spleen, and encephalon from fish or hemolymph and hepatopancreas from shellfish. However, at least theoretically, depending on its target organ, for each specific virus there should be an optimum tissue or organ to be sampled for diagnosis (i.e., the one with the higher viral load). Therefore, in the literature we can find the use of almost any kind of tissue with different levels of efficiency. The selection of a wrong organ or tissue to detect a specific virus in a specific fish or shellfish obviously influences the final efficiency of the diagnosis. For instance, using ovarian fluid to detect the viral hemorrhagic septicemia virus (VHSV, a virus which has not demonstrated vertical transmission) in an adult trout strongly reduces the chance (if any) to detect the virus, but seems to work properly for other viruses as infectious pancreatic necrosis virus (IPNV) [12] or infectious hematopoietic necrosis virus (IHNV; [11]). As another example, the use of certain organs may affect the performance of specific diagnostic tests. It is the case of using undiluted homogenates of liver or pyloric caeca to infect cell cultures—the toxicity of the homogenate might produce a false
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cytopathic effect (CPE) [12]—or the use of blood samples for viral detection by the polymerase chain reaction (PCR) or PCR-based methods (incorrect viral genome extraction may produce false negatives due to contamination with enzyme inhibitors). As published elsewhere, to avoid viral inactivation and drop viral load in the tissues, samples must be transported from their original location to the diagnostic laboratory in optimal conditions. The OIE [7] stipulates that they must be stored at 4°C for no longer than 24 h after sampling (though 48 h are also acceptable). In an interesting study, Hostnik et al. [13] demonstrated the effect of the temperature of conservation of fish tissues on the detection of IHNV in cell culture (CC) and by reverse transcription (RT)-PCR. They observed that at 4°C the virus could be detected at a maximum of 3d and 35d, respectively, whereas maximum periods were reduced to 1d and 8d, respectively, at room temperature (rT). Processing of samples is an important step with the objective of exposing the analyte to the detection system of a diagnostic test. In the case of solid samples, i.e., tissues and organs, processing begins with the homogenization of the tissue on a buffer specifically designed for cell culture. A variety of options are available but the most frequently used are Hanks’ balanced salt solution (HBSS) and Earles’ salt solution (ESS), which must be supplemented with antibiotics to eliminate microbial contamination (1000 mg/mL gentamicin, or 800 iu/mL penicillin plus 800 mg/mL streptomycin, and 400 iu/mL mycostatin or fungizone) [7]. For virus extraction, different methods of homogenization have been employed and published. The most frequently employed is the mortar and pestle [7,12,14–16]. Others have employed freezing and thawing [17] but its efficiency has proven not to be too high. In an old study, Agius et al. [14] demonstrated that the sonication of tissues or tissue homogenates favored the isolation of the virus in cell culture in comparison with the simple use of mortar, and more recently other authors reported similar results [12,16]. However, the application of sonication on large numbers of samples can be uncomfortable (or even harmful) for the operator, and additionally needs special equipment for protection. Other authors reported the use of trypsinization with high efficiencies of viral recovery, as demonstrated by isolation in cell culture, but the procedure can occasionally produce toxicity for the cell monolayer [12,18]. Finally, several devices have been designed with a performance as least as good as for regular homogenization with mortar [14,15], as Omnitron, Polytron, or Stomacher, which facilitate homogenization and reduce processing time for diagnosis. After homogenization, cell debris must be removed by centrifugation and the supernatant incubated for antibiotic treatment [7] before using the viral suspension for diagnosis. Other authors, however, have employed the filtration of supernatants (instead of antibiotic treatment), though it can sometimes retain part of the viruses and thus reduce the overall performance of the diagnosis [18]. In addition, for the application of molecular diagnostic tests, the homogenate pallets can also be employed. Other kind of samples that can be employed must be processed in different ways. For instance, to detect virus or viral components in blood, sera [19–22] or different cell fractions can be chosen [21,23–25]; mucus may be simply diluted for cell culture inoculation [24] as well as ovarian fluid [7] unless it includes cavity cells [11]. Finally, if a molecular technique of diagnosis is to be applied, nucleic acid extraction must be performed. Several methods have been published as proteinase K or pronase treatments, followed by phenol–chloroform extraction and ethanol precipitation [26–28]. Commercial methods that applied the old known system of lysis of tissues by guanidine-phenol and ethanol precipitation are available, such as RNAzol, Trizol, or similar products. In addition, other methods based on nucleic acid fi lter capture devices are available that considerably reduce the time required
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for the procedure. All these methods have been used and reported in the literature. However, their performance (i.e., reliability to recover viral nucleic acid) has never been compared. In an ongoing study (unpublished data), the authors have observed that some of these methods of nucleic acid extraction show really low R&R, being strongly influenced by the equipment or the operator. Therefore, to ensure the accuracy of the extraction and hence of the overall diagnosis, the diagnostic laboratory must perform previous validation of the method of extraction to be employed.
32.5 Methods of Diagnosis for Aquatic Animal Diseases Many analytical techniques can be applied for diagnosis in aquaculture. Some are based on the effect that the virus produces in the fish tissues or on cells; some are based on the detection of a viral component (protein or genome), and others depend on the host response to viral infection. In this chapter, we describe some of the techniques most employed in diagnosis of viral diseases of aquatic animals. They are described from a general point of view, but with references to their application to particular viruses. How to Select a Diagnosis Method?: The selection of a technique of diagnosis for a specific purpose should be based mainly on a deep knowledge (theoretical and practical) of the procedure and factors affecting its performance. In the literature, there is a large list of reports on optimized, modified, and even on the new methods of diagnosis. However, before introducing any in the routine of a diagnostic laboratory, previous evaluation and quantification of its reliability and accuracy should be carried out under different conditions, and on different fish or shellfish species and tissues. The quantitative knowledge of the performance of all methods available in a laboratory would allow us to choose the best method for any particular case based on objective criteria. Unfortunately, in most cases, such previous validation has not been performed, and only in few reports some data are provided. Frequently, the diagnostic techniques are grouped into “traditional” or “molecular,” the former includes histopathology (HP) and microscopy, CC isolation, and sero/immune techniques. Th is is perhaps because they have a longer history of application and therefore experts in diagnosis feel more confident on their performance. To a certain extent this is partially true, because although the so-named traditional methods are quite standardized and have been included for long time in the recommendations of international organizations such as the OIE [7], EU [3], FDA [29], or the Australian Administration [30], only few reports on their validation are available, or they are even absent in some cases. On the other hand, the molecular methods have a relatively shorter history (of around two decades), and thus they still need an important effort for standardization. However, in this case many reports do provide quantitative data that make comparisons with other techniques quite easy. Nevertheless, the knowledge of all the factors affecting their performance is still in an ongoing process, and this must be a serious consideration for any diagnostic laboratory. In this sense, as we will show in this chapter, these kind of techniques can be extremely sensitive, at least theoretically, but the risk of false positive and/or negative results from inexperienced hands is a real threat. In conclusion, in the absence of own objective criteria (based on experience), the best criteria is the use of the official recommendations of organisms as the OIE [7] in order to decide what technique to use, and how to apply it, for each specific case. Therefore, we will frequently reference to the OIE diagnostic manual, mainly for the traditional methods.
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Clinical, Histological, and Microscopical Techniques Gross Signs
The analysis of the clinical signs of the diseases (internal and external, including behavior of the affected individuals), and the consideration of the clinical history of the population, is the first step to be followed in diagnosis. Abundant information on clinical signs of each viral disease is available elsewhere [7,31–35], which may help the expert to decide what agents can be putatively affecting the population and thus representing an important support to decide what kind of analytical method must be selected. In general, this first step cannot be considered a diagnostic procedure by itself due to its low sensitivity (only disease situations are detected) and specificity: In most cases, different viruses can share similar symptoms. However, in some cases, the specificity of this method can be high because a specific virus can exclusively produce certain signs. This is the case of the white spots that appear in the body of the shrimp affected by the white spot syndrome virus (WSSV) [31,32]; the skin nodules that appear in the fish affected by the lymphocystis virus, or the spiral swimming of IPNV-infected salmonid fry [34,35].
32.6.2 Histopathology Viral replication in the host may provoke lesions in some tissues. There is a certain relationship between the viral group and the type of alteration of the affected tissues (Table 32.1). For some virus, the detection of the specific lesions may be determinant for their diagnosis [7]. However, in most cases its sensitivity does not reach acceptable values, and specificity might be excessively low [7,36–39]. Additionally, in some cases similar histopathological signs can be the consequence of noninfectious factors, thus yielding wrong diagnostic results. Therefore, the first condition for a laboratory to introduce HP-based tests in its routine diagnosis is to demonstrate sufficient skills and experience. The procedure for light microscope demonstration of tissue lesions is quite simple, and only requires two specific equipment: a dark field light microscope (available in any diagnostic laboratory) and a microtome (for those working with shrimp virus, this can even be avoided by employing squash mount preparations). The first step is the fixation of the tissues. Most frequently used fixative is 10% neutral buffered formalin, followed by 10% ethanol wash. For shrimp tissues, two fi xatives can be employed: Davidson’s AFA (alcohol, formalin, acetic) or nonacidic R-F (RNAfriendly) [7]. The fi xed fish tissues must be paraffin embedded and 5 mm sections hematoxylineosin stained.
32.6.3 Immunohistochemistry To confirm the histopathological analysis, the immunodetection of the agent can be applied on the tissues. For this purpose, different types of immune labeling are reported as fluorescein isothiocyanate (FITC) in an immunofluorescence antibody test (IFAT), or enzymatic labels as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The use of immunohistochemistry (IHC) procedures requires a previous reduction of the background due to endogenous activity [40]. Independently from the type of label employed, the most important factor is the use of specific antisera. Best results are obtained from the use of monoclonal antibodies (MAbs) [40–42]. However, in addition this kind of antibodies is not available for all laboratories (but we must remark that
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Table 32.1 Fish and Shellfish Viruses: Host Species, Gross Signs, and Diagnostic Tools Virus
Susceptible Species
Symptoms and Histopathology
Diagnostic Procedures
Channel catfish virus
Catfish (Ictalurus punctatus)
Renal tubules necrosis, exophthalmia, ascites accumulation, hemorrhages in muscle and fins.
CC, NT, IFAT, ELISA, PCR
Epizootic hematopoietic necrosis virus (EHNV)
Perch (Perca fluviatilis), rainbow trout (Oncorhynchus mykiss), sheatfish (Silurus glanis), catfish (Ictalurus melas)
Necrosis in liver, spleen, and kidney.
CC, IFAT, ELISA, EM, PCR
Infectious hematopoietic necrosis virus (IHNV)
Rainbow trout (O. mykiss), Atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus spp.)
Skin darkening, pale gills, edema, ascites and distended abdomen, exophthalmia, internal and external petechial hemorrhages, pseudolethargy. White color trailing fecal casts. Spinal deformities.
CC, NT, ELISA, NAH, IFAT (on imprints and on CC), RT-PCR
Infectious pancreatic necrosis virus (IPNV)
Susceptible species: Salmonid fish; host species: practically any.
Sudden and increasing mortality. Skin darkening, distended abdomen, black color trailing fecal casts, spiral swimming.
CC, NT, IFAT, ELISA, NAH, RT-PCR
Infectious salmon anemia virus (ISAV)
Atlantic salmon (S. salar)
Anemia, accumulation of ascites. Hepatic and kidney necrosis. Abnormally large and dark liver. Petechia in peritoneo.
CC, IHC, IFAT, RT-PCR
Lymphocystis disease virus (LDV)
Sea bass, grouper, sturgeon
Skin nodules. Cell hyperplasia.
EM, ISH, PCR
Oncorhynchus masou virus (OMV)
Pacific salmon and rainbow trout
Epithelioma around mouth and body surface. Skin ulcers. White spots in liver. Lethargy.
CC, NT, IFAT, ELISA.
Red sea bream iridovirus (RSIV)
Red sea bream (Pagrus major) and many other species of Perciformes and Pleuronectiformes
Lethargy, severe anemia, petechias in gills, spleen abnormally large.
CC, IFAT, PCR
Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases Table 32.1 (continued) Diagnostic Tools Virus
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Fish and Shellfish Viruses: Host Species, Gross Signs, and
Susceptible Species
Symptoms and Histopathology
Diagnostic Procedures
Salmonid alphavirus (SAV) (including sleeping disease (SD) and pancreas disease (PD) viruses)
Atlantic salmon, common trout, sea trout (Salmo trutta), rainbow trout
Lesions in pancreas and muscle. Anorexia, lethargy, reduced growth. Yellow-white trailing casts. Peripheral swimming.
HP, IFAT, PCR
Spring viremia of carp virus (SVCV)
Common carp (Cyprinus carpio carpio), koi carp (C. carpio koi), silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), grass carp (Ctenopharyngodon idella), goldfish (Carassius auratus), tench (Tinca tinca), Northern pike (Esox lucius), sheatfish
Degeneration of the gill lamellae, ascitic fluid containing blood, inflammation of the intestines. Hemorrhagic visceral organs. Petechia in swim bladder, muscle, and fat tissue.
CC, NT, IFAT, ELISA
Viral encephalopathy and retinopathy (VER) or nervous necrosis virus (NNV)
Sea bass (Lates calcarifer, and Dicentrarchus labrax), grouper (Epinephelus spp.), jack (Pseudocaranx dentex), parrotfish (Oplegnathus fasciatus), puffer (Takifugu rubripes), and flatfish (hallibut, Hippoglossus hippoglossus; Japanese flounder, turbot)
Retina and brain cells vacuolization. Neuronal necrosis. Abnormal swimming behavior (spiral whirling or upside down swimming).
CC, IFAT, RT-PCR
Viral hemorrhagic septicemia virus (VHSV)
Rainbow trout, pike, Japanese flounder (Paralychthys olivaceus), turbot (Scophthalmus maximus)
Skin darkening, exophthalmia, anemia (pale gills), skin, hemorrhages in fins and gills, distended abdomen, abnormal swimming. Lethargy. Rapid onset of mortality.
CC, IFAT, RT-PCR
(continued)
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Table 32.1 (continued) Diagnostic Tools Virus
Fish and Shellfish Viruses: Host Species, Gross Signs, and
Susceptible Species
Symptoms and Histopathology
Diagnostic Procedures
White sturgeon iridovirus (WSIV)
White sturgeon (Acipenses transmontanus)
Anorexia. Diffuse hyperplasia of skin. Abdominal hemorrhages.
CC, NT, IFAT
Infectious hypodermal and hematopoietic necrosis virus (IHHNV)
Shrimp (Penaeus stylirostris)
Irregular growth. Cuticular deformities. Anemia. Weakness, roll over movement. Motted appearance. Nuclear inclusion bodies, margination of chromatine.
Dot-blot, ISH
Taura syndrome virus (TSV)
White shrimp (P. vannaemei)
Lesions and necrosis in the epithelium of different body parts. Pale reddish coloration. Red tail. Soft shell.
HP, RT-PCR
White spot syndrome virus (WSSV)
Penaeid shrimp
White spots, anorexia, surface swimming. Hypertrophied nuclei.
HP, ISH, PCR
Yellow head disease virus (YHDV)
Penaeid shrimp
Yellowing of encephalothorax; clarified body. Systemic necrosis of ectodermal and mesodermal cells.
HP, RT-PCR
CC, Cell culture; ELISA, enzyme-linked immunosorbent assay; EM, electron microscope; HP, histopathology; IFAT, immunofluorescence antibody test; IHC, immunohistochemistry; ISH, in situ hybridization; NAH, nucleic acid hybridization; NT, neutralization; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR.
for certain viruses, MAbs can be purchased from some companies), and they can yield excessive specificity. Therefore, the use of polyclonal antibodies is not rejected (though in this case certain background can be produced). FITC, AP, and HRP protocols are quite similar, and can be applied on frozen [43] and dewaxed sections [44–46], imprints [40], squash tissues [7], or even directly on larvae [7,44]. The procedure begins in the treatment with a blocking agent, constituted by a solution of unspecific protein (normally skimmed milk), and followed by washes with buffer and incubation with the virus-specific antibody. New washes precede the treatment with the antispecific-labeled conjugate. Some authors have reported the use of biotinylated conjugates, employing in those cases, biotin-avidin AP [41], or streptavidin-FITC or HRP [46]. The final detection is performed by the
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addition of the corresponding substrate for AP [43,46] or HRP [40,41,45,47,48], or under UV light in a fluorescence microscope with the corresponding filters [41,43,46,48].
32.6.4 Electron Microscopy The application of this technology on fish tissues is not too frequent due to the need of special equipment (including ultramicrotome) and special skills for processing and interpretation of results. Few reports can be found that employ staining of ultra thin section for electron microscope (EM) diagnosis [49–52]. In most cases, the use of EM is focused to the preliminary identification of a virus isolated in cell culture.
32.7
Isolation in Cell Culture
32.7.1 Cell Lines and Cell Culture Since viruses are intracellular parasites, the unique procedure to detect them and simultaneously demonstrate that they are active and infective is to propagate them in an alive system. The old procedure (and still the only one for certain viruses) was the inoculation in experimental healthy animals (or avian eggs) to develop the disease. Later on, the use of primary cells from disaggregated tissues represented an important advantage in the study of viruses, and is still a useful tool in some cases. However, the revolution in virology (and fish virology) came from the production of CC of continuous line, or cell lines. The use of cell cultures simplifies the propagation of virus and hence their isolation from infected samples. The only requisite is that the cell line selected for the isolation of a specific virus must be susceptible to its replication, yielding alteration and/or cell lysis in the culture monolayers, ending with the development of a specific CPE easily detected under light microscopy. For fish viruses there is a large list of susceptible fish cell lines that have been described by many laboratories in the literature, and in most cases are available from the international culture type collection as the American Type Culture Collection (ATCC) or the European Collection of Cell Culture (ECACC). The cells can be bought in a ready-to-use monolayer, or in a frozen format (indications of the seller must be carefully followed to prepare the cell monolayers from the frozen vial). Working with CC is quite simple, and precise instructions can be found in specific manuals [53,54], but brief and useful indications can be found in the OIE diagnostic manual [7] and reported elsewhere. To culture cells, a laboratory needs (besides skills and experience) specific equipment and materials, as well as culture media and supplements. The equipment includes a sterile flow chamber and an inverted light microscope. Plastic flasks and plates specially treated to favor the adherence of cells are available from different companies with similar qualities; the best advice is to test different brands for different lines. There is a variety of culture media that can be chosen, all them sharing high concentration of basic nutrients. Most frequently employed media are the traditional Eagle’s minimum essential medium (EMEM) with Earle’s salt solution (ESS), and the Leibovitz L-15 medium. The OIE advises the use of amino acid and vitamin-enriched media, as the Stoker medium; however, other authors have reported the use of other media with good results. The media must be buffered with 0.16 M Tris or 0.02 M Hepes, and/or sodium bicarbonate (if closed flasks are employed). In all
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cases, the media must be additionally enriched with sera, normally fetal bovine sera (FBS), and supplemented with antibiotics to reduce the risk of bacterial (100 iu penicillin and 100 mg/mL streptomycin) and fungal (2 mg/mL fungizone or 50 iu/mL mycostatin) contamination.
32.7.2 Selection of a Cell Line for Diagnosis The most important issue in this method of diagnosis is the correct selection of the susceptible cell line or lines for the target. For each virus, the OIE recommends a short number of susceptible cells, but data from other authors might help us in the selection of the most appropriate one. For its recommendation, the OIE has selected the lines with demonstrated susceptibility to any strain or type of the specific virus. In this sense, in many cases, certain cells employed by some authors show low susceptibility for some viral types, which should be avoided to reduce the risk of false negatives. For instance, in spite of some authors reported the use of Chinook salmon embryo (CHSE-214) cells for the isolation of IHNV [55], others reported that those cells may fail in the isolation of some strains [56]. As another example, in a recent study, Ogut and Reno [57] reported that fathead minnow (FHM) and epithelioma papillosum cyprini (EPC) were suitable for the isolation of American types of IPNV; however, they have demonstrated failure to develop CPE with other strains. It is surprising the large variety of cell lines that the scientists use in the diagnosis of a specific virus in spite of the “officially” recommended one, or even in some cases using cells with demonstrated lower sensitivity than others. For example, although EPC, FHM, and CHSE-214 have been demonstrated to be of lower sensitivity to VHSV than the recommended BF-2 [7,58], some authors still report their use in diagnosis. In this sense, although they can be employed to propagate specific strains, their use is not advised in blind diagnosis, precisely to avoid false negatives due to excessive specificity. This does not mean that research on testing new and established cell lines for the isolation of virus under different condition is not advised. On the contrary, much effort must be focused on the validation of each available cell line for the detection of any viral type of each group. As a requisite, the introduction of a new cell line in the diagnostic routine of a virus must be preceded by its testing (preferable with all the corresponding types) to determine its optimal temperature and the range of permissiveness, characteristic of the CPE and time for its development at each temperature and for all strains, and range of viral titters yielded in each case. Unfortunately, in spite of the large number of reports based on this method of diagnosis, many fail to provide important data.
32.7.3
Viral Isolation in Cell Culture
For its isolation in a cell monolayer, the virus must be previously extracted, in a suspension, from the fish tissues by any of the procedures of homogenization described earlier. However, this is not actually a strict requisite, because some authors have reported the isolation of virus from monolayers cocultivated with fractioned, disaggregated, or trypsinized fish tissues [14,23,24,59,60] with different efficiencies. In addition, fluid samples as for crude virus (viral suspensions from infected monolayers) or sera can be directly inoculated. The cells must be inoculated before confluence. For this purpose, the culture medium must be removed, and the viral suspension incubated on the monolayer, at the corresponding optimum temperature, for an adsorption period of around 1 h. The remaining inoculum must be removed and the monolayer covered with the same culture medium but supplemented with
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lower percentages of FBS (normally 2%) to reduce advance of the cell growth (favoring the replication of virus in low loads or with slow replication). Afterward, the infected monolayers must be incubated at the selected optimum temperature, and daily visualized for the detection of characteristic CPE. Although there are some CPEs quite specific for a virus, in most cases different viruses can share the cytopathic alteration. Due to that specificity failure, this diagnostic procedure must be considered exclusively for viral detection and should be followed by a method of identification. This is also the general rule in the official diagnostic procedure in most of the cases. Thus, the OIE stipulates, for each virus, an initial protocol for viral isolation followed by a group of recommended techniques of identification (Table 32.1). A variety of techniques can be employed which will be further described. In some cases, the identification procedure can be directly applied onto the infected cells, even before a clear CPE is visualized. Those are the IHC-type techniques, as immunoperoxidase (IP) or immunofluorescence [43,44,61–63], and in situ hybridization (ISH) [59,61]. Frequently, the identification is performed on the isolated virus, using crude, concentrated, or, less frequently, purified virus. The most simple, though not conclusive, identification method is the visualization of the size and the morphology of the isolated and concentrated virus under electron microscopy [44,51,63]. However, the most frequently employed are viral neutralization [59,61,63–65] or the enzyme-linked immunosorbent assay (ELISA) in liquid or solid (immunodot-blot) phase [42,44,62,63,66–70], using specific polyclonal or monoclonal antisera. Molecular techniques such as nucleic acid hybridization (NAH) and, in the last decade, the PCR and PCR-based procedures have been introduced as a complement of the CC isolation for identification of the isolate. [27,71–74].
32.7.4
Performance of the Diagnostic Procedure
In fish virus diagnosis, the isolation in CC is still considered, after many decades, a method of reference, not indeed due to its sensitivity/specificity but because it is the only technique that simultaneously detects the virus and confirms its infectivity. In this sense, although it is the gold standard for official organizations as the OIE [7], the EU [3,75], and the American Fisheries Society [29], and is theoretically considered to have a limit of detection (LD) of one viral particle, much is still to be known on its real sensitivity (in quantitative terms), specificity, and R&R. Regarding the first one, sensitivity of this procedure is really a “still to know” parameter. In fact, there are few reports with a real quantification of the sensitivity in term of LD, and in most cases, the supported data actually apply to the identification method: CC plus ELISA (101 or 103.5 TCID50/mL; [69,76]), CC plus electropherotyping (105 TCID50/mL; [77]), CC plus ISH (0.5–1 × 103 TCID50/ mL; [17,61]) or CC plus PCR (100 TCID50/mL; [78]). Several authors have reported the improvement of the sensitivity of viral isolation by the use of certain substances as polyethylene glycol (PEG; [79]) or certain patented proteins [80]. However, their use is not extended and therefore more data are needed before being introduced into a routine diagnosis. In viral isolation, all those parameters are in fact closely related. In a Delphi panel study, Bruneau et al. [81] interviewed a set of experts in diagnosis from reference laboratories on the sensitivity and specificity (in probabilistic terms) of the method for diagnosis of IPNV and IHNV, and the result was really worrying. Thus, not only the authors concluded that the sensitivity of the method is far from perfect but also most remarkable is the list of factors that the experts believe that can strongly influence sensitivity. Among them not only are included, as
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expected, the sampling procedure and sample processing, or the level of infection of the sample, but other factors that will also affect R&R, as the cell line and cell line age, materials, the interpretation of the results, and the staff involved in the procedure. Similar inconveniences of the method have been reported by other authors [8,82,83]. Additional factors that may strongly affect the specificity of the technique have been published as the development of defective interference particles, presence of neutralizing factors in tissue homogenates, or tissue toxicity that can yield false negative or positive results [18,84,85].
32.8 Serological and Immunological Techniques of Diagnosis 32.8.1 Scientific Basis In the present section, we will describe the methods of diagnosis that are based on a specific antigen–antibody reaction, allowing the detection (and identification) of any of both: the antibody, by means of the sero-diagnostic techniques, or the antigen, by the immune-diagnostic techniques. Nevertheless, the procedures described here are similar independently from their application to sera- and immune-diagnosis. The techniques included in this chapter are the neutralization test (NT), the IFAT, and the immunoenzymatic assays (IEA) (including the immunodot (ID), AP and IP, and the ELISA). Although all the methods share similar scientific basis (all of them depend upon a specific reaction between the virus and its specific antibody) they show a big difference in the way used to detect such specific reaction. In this sense, in NT the antibody binds to the cell-attachment specific epitopes of the virus, therefore blocking its capacity to infect a susceptible cell (i.e., being neutralized) (Figure 32.1). Th is is an important difference with the remaining techniques which, to detect the specific reaction, use a label linked to the specific antibody (direct methods) or to an anti-antibody known as conjugate (indirect procedures) (Figure 32.2). This difference is one of the causes for the different levels of sensitivity and specificity between both types of techniques, because the number of putative binding sites for fish antibodies is broader for the label-linked immune techniques in comparison with the few neutralizing epitopes generally present in a virus [86,87]. The second difference relies on the substrate employed to perform the diagnostic procedure. Whereas IFAT, IP, and AP-IEA are applied onto infected cells or tissues, ID uses nitrocellulose membranes, and ELISA microwell plastic plates. Therefore, although similar, the procedures of these label-linked-based techniques and their applicability obviously differ. In Section 32.8.2, all these techniques are approached as a unique group to analyze their advantages and disadvantages, and their application in sera- and immune-diagnosis. Section 32.8.3 focuses on the description of each method.
32.8.2
Advantages and Disadvantages of the Immunological Diagnostic Tools
The detection of a specific antigen is the objective of the immunological diagnostic tools, which use, for such purpose, a homologous antibody. The better specificity of those antibodies implies the highest specificity of the reaction, and thus the best reliability of the diagnosis. Therefore, the first critical factor in these methods is the type of antisera and the procedure to obtain it.
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Virus + homologous Ab
Susceptible cell
Susceptible cell
Neutralized virus
Cell lysis
Figure 32.1 Viral neutralization. After mixing the virus with a homologous antiserum, the viral receptors are blocked by the antibodies, preventing those from attaching to the cell receptors. Therefore, the cell monolayer will not show the cytopathic effect developed in the absence of the specific antibodies.
There are two types of antisera. Polyclonal antisera are produced by means of inoculation of the virus in an animal. In fish virology, this is normally a rabbit (young New Zealand type). In using this system to obtain the antisera, the most important concern is the use of purified virus to reduce the production of nonspecific antibodies against any kind of contaminants that can be present in crude viral supernatants (i.e., cellular antigens and proteins from bovine sera). In addition, for the highest production and the quality of the polyclonal antisera, the inoculation schedule must be carefully chosen for each viral type. The description of those procedures is not the scope of this chapter. Therefore, the readers must refer to the extensive literature available. A polyclonal antiserum is actually constituted by a pool of antibodies specific for all antigenic epitopes present in the viral particles, including the neutralizing antibodies. In addition, even if the sera have been obtained from purified virus, before use they must preferably be absorbed on cell monolayers to remove unspecific antibodies [88]. On the other hand, MAbs (obtained by means of the hybridoma technology) are epitope specific. Therefore, many scientists opine that they are superior to polyclonal antisera for many applications [89] since they improve the sensitivity and the specificity of the immune-assays. However, this assumption must be carefully considered for each technique and case. In fact, some authors have reported lower sensitivity of some MAbs, or even cross-reactivity with close-related virus [90,91]. In addition, the high specificity of MAbs can actually represent a handicap because certain strains of a viral type might be miss-detected [82]. This is the case reported by Ariel and
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Detection Direct
Detection
Indirect
Detection
Sandwich
Specific antibody
Figure 32.2
Anti-antibody
Primary antibody
Label
Scientific bases of the immunolabel diagnostic techniques.
Olesen [92], from the Community Reference Laboratory for Fish Diseases, who tested a set of commercial kits (based on ELISA or IFAT using MAbs) for the detection of IHNV, IPNV, Spring viremia of carp virus (SVCV), and VHSV, in comparison with their own reference methods. They observed that the only reliable one was the kit for IHNV detection, the remaining being nonspecific or too specific for some strains. However, Dixon and Longshaw [93], who tested two commercial kits for the detection of a series of fish rhabdovirus: SVCV, grass carp rhabdovirus (GCRV), pike fry rhabdovirus (PFRV), and tenca rhabdovirus, observed that, whereas the IFAT-based kit (employing a monoclonal antibody) specifically detected SVCV, the ELISA kit, that used a rabbit polyclonal antiserum, did not discriminate among the four viruses. Many authors consider that the immunological techniques of diagnosis exhibit a level of sensitivity at least as high as the isolation in cell culture. This is especially true for those techniques that are strictly employed to identify a previously isolated virus (e.g., the neutralization test). However, in some cases these techniques can be applied on the infected cell, and detect the virus before CPE is visualized [89,90,94,95], which itself represents a clear advantage. In terms of LD, in general those techniques have shown LD values between 103 and 105 TCID50/mL for IPNV and VHSV, or 104 –105 for IHNV [66,87,88,95–101]. For other viruses the values do not differ: 103.5 TCID50/mL for epizootic hematopoietic necrosis virus (EHNV) [69] or 103 –104 TCID50 for betanodavirus [102], although some authors have reported really low DLs of 102 TCID50/mL for yellow head disease virus (YHDV) by ID [103] or ever 10–50 TCID50/mL for VHSV by ELISA [83]. Nevertheless, for Davis et al. [66], those values are understandable from the physical limitations of binding assays, and thus, those reported DLs of 102/mL should be treated with caution.
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32.8.3
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Description of Immune Diagnostic Procedures
Although all these procedures are well documented in the OIE manual [7], in this section we present a summary from scientific reports employing the techniques. Neutralization: This technique is widely employed in fish virology and thus there are many reports describing the procedure for both, identification and typing of viruses [104,105], with minor differences. Briefly, 0.1 mL of the isolated virus, diluted to around 103 –104 TCID50/mL must be mixed with the serial dilutions of the specific neutralizing antisera (normally polyclonal antisera are employed) and incubated for 1 h at rT to 37°C. The dilutions are then incubated in replicate monolayers and daily visualized for the detection of CPE. Positive control reactions (reference virus assayed with the same antisera) are necessary to confirm the neutralizing activity of the sera on the isolated virus and to calculate the neutralization ratios [104]. Negative controls (with heterologous virus) are employed to ensure specificity. The technique can also be employed with a unique precalculated antiserum dilution, yielding only a qualitative result (positive or negative identification). Immunodot: This method can be applied for identification and typing of viral isolates, and has been employed for both fish [101,104,106,107] and crustacean [103,108] viruses. Although time consuming, due to the high number of steps required, this procedure is quite simple, and no special apparatus is required. Most frequently, the “indirect” version is employed, using an enzyme-labeled anti-immunoglobulin (conjugate) specific for the virus-specific antibodies. Some authors, however, have employed the “direct” version, which reduces one-step but requires the virus-specific antibodies to be previously linked to the labeling enzyme [107]. In addition, for best performance virus-specific MAbs should be used. However, polyclonal rabbit antisera obtained from purified virus and adsorbed onto cell monolayers have also been employed with good results [104]. For the procedure, activated nitrocellulose membranes are loaded with the virus (either by pipetting or by using a vacuum filtration multiwell device). After drying, the membrane is immersed for 1 h at rT to 25°C in a blocking solution containing nonspecific protein in buffer. This solution may be 2%–5% skim milk or 1%–1.5% bovine seroalbumin (BSA) in PBS or Tris buffer. After 3–5 washes with 0.05% Tween 20 in the same buffer, the membrane is immersed for 1 h (rT to 25°C) in the virus-specific antisera, and then washed again. The conjugate (normally with HRP) is added and incubated as earlier (this step is avoided if the “direct” version is chosen), the membrane washed again and developed by the corresponding chromogenic substrate (for HRP-conjugate: 4-Cl-a-nafol plus H2O2, or carbazol plus H2O2; for substrate preparation uses the referenced reports). The results are visualized as defined dark spots in the membrane. The method can be quantitative if reference concentrations are assayed and special software and hardware are available. Immunohistochemistry of infected cells: Labeling of the viral antigen directly on the infected cells opens the opportunity of detecting and identifying the virus in situ. The procedure is quite simple on infected monolayers [45–46,109–111], but has also been applied on infected lymphoid primary cells [112], imprints [40], and frozen [43] and deparaffinized tissue sections [45,105,108]. The initial step for tissue sections is the deparaffinization and rehydration. Infected cell cultures must be fi xed using acetone (−20°C) for 10 min or Canay’s solution (acetic acid:methanol; 1:3). After washing, the elimination of endogenous enzyme activity is recommended mainly if peroxidase conjugate will be further employed (treat 30 min with blocking solution: 0.3% H2O2 in methanol). Rinse with buffer (phosphate buffer saline, PBS, or Tris buffer saline, TBS, can be employed) and immerse the cells or tissue in serum blocking solution (10% normal goat serum, 5% skim powdered milk, or 1.5 BSA) for 20 min to 1 h, at rT to 37°C. After washing with 0.05%
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Tween 20 in PBS or TBS, the primary antibody is added (most frequently, the “indirect” version is employed, though the “direct” version, using HRP-labeled specific antibody can be chosen; [111]). For best performance, mouse MAbs must be employed, though polyclonal rabbit antisera can also be used. The only requisite is the right selection of the corresponding secondary antibody (antimouse or antirabbit IgG conjugates, respectively). After the incubation of the primary specific antibody for 1 h at rT to 37°C, and three to five washes, the secondary antibody (conjugate) must be added and incubated for 1 h. The cells are washed again and the chromogenic/ substrate supplemented. Different systems have been reported. Perhaps the more convenient way is using the commercial systems available, as Vector Red (Vector) or True Blue (Kirkegaard and Perry laboratories, KPL), which use AP, HRP, or biotinylated conjugates followed by the specific chromogen/substrate provided by he manufacturer. However, the traditional procedure (using AP or HRP conjugate and the corresponding substrate) can also be employed. Immunofluorescense antibody test: This is a technique well standardized [7] and widely employed for most laboratories. It can be applied on infected CC [47,71,103,114,115], purified cells [24], imprints, [110,116], or fi xed tissues [19,113], and allow the detection of the virus in the infected cells time before any CPE is visualized [94], and with a sensitivity at least as high as that of the isolation in CC [115]. In addition, it has also been designed and employed for viral quantification by the fluorescent foci counting method [117]. The procedure begins with the fi xation of cell monolayers or tissues by cold acetone and rinsing with PBS or TBS buffer. Although some authors skip the blocking step, it is recommended. Therefore, the cells are immersed in a solution of unspecific protein (normally 5% skim milk) for 1 h at rT. After several rinses, the primary antibody (preferably a MAb, though polyclonal Ab can also be employed) is added for 1 h at rT to 37°C. After rinsing, the secondary Ab (FITC-conjugate anti-Ig) is added. The cells are then rinsed, mounted with glycerol, and visualized in a UV light microscope for brilliant green light foci specifically localized in the infected cells on a black or dark background. Enzyme-linked immunosorbent assay: This is a well-recognized, standardized, and in many cases a validated method of diagnosis. It is well described for most viruses in the OIE manual [7], and has been widely described for fish and shellfish viruses in the literature [67,69,99–102,118–126]. Therefore, we recommend the reader to consider the following description of the procedure as a summary, not as a ready-to-follow protocol. Most frequently, the procedure employed is the antigen capture or sandwich ELISA (swELISA), which provides higher sensitivities and specificities than the indirect ELISA (iELISA). Moreover, frequent is the employment of MAbs as primary antisera, as it reinforces specificities. Microwell ELISA plates are employed. In the swELISA, the wells are coated with antivirusspecific antiserum (normally polyclonal, though MAbs have also been applied by some authors) for 2 h at 37°C, or overnight (o/n) at 4°C. Then, the wells are rinsed with any of the following ELISA buffer: TNE (0.05 M Tris, 0.15 NaCl, 0.001 M EDTA), 0.1 M carbonate–bicarbonate buffer (pH 9.6), or most frequently PBS (supplemented with 0.05 Tween 20). Afterward, and before applying the viral suspension (crude virus or fish tissue extracts), the wells are blocked with ELISA buffer supplemented with unspecific protein (1.2% BSA or 3%–5% skim milk) for 1–2 h at rT to 37°C. After washing, the viral sample is added and incubated as earlier. In the iELISA version, the wells are coated with the viral sample, diluted in buffer with BSA or skim milk, by incubation o/n at 4°C. Then, the wells are rinsed and blocked as mentioned earlier. In both versions, the protocol continues with the rinsing of the wells and incubation with the specific antivirus antiserum, for 1–2 h at rT to 37°C. The wells are rinsed again and incubated with the conjugate (1–2 h/rT to 37°C), rinsed again and covered with the corresponding substrate. Two types of conjugate can be employed, which use different chromogenic substrates: AP, which uses pNPP (p-nitrophenyl
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phosphate) as chromogen substrate, or peroxidase, which can be complemented with different chromogens (orthophenylene diamine; tetramethylenediamine; 5-aminosalicylic acid or ABTS [2,2′-azino-di-3-ethylbenzthiazoline-6-sulphonic acid]) supplemented with H2O2 as the substrate for the enzyme. After incubation for 15 min to 1 h at rT, the reaction is stopped and optical density (OD) measured in a ELISA plaque reader at 450 nm (for AP) or 498 nm (for peroxidase).
32.8.4 Antibody Detection Diagnosis In general, the capacity of antigen detection by the immune-techniques is over the viral loads characteristic of asymptomatic carrier fish [66,95]. The immediate solution relies on the previous amplification in cell culture. However, another approach is the application of sero-diagnostic techniques to detect specific antibodies in the fish sera. Crustaceans do not produce humoral response to infection, thus making these methods useless. In fish, infection with most viruses yield a humoral immune response that, in spite of the lower complexity than in higher vertebrates, can be easily detected by means of the same techniques employed for immune diagnosis [95,97], such as plaque NT (PNT) [22,37,86], IFAT, and ELISA [36,127–131]. Nevertheless the level of antibodies in the fish depends on many factors, including the general status of the fish, stress situation, and time post-infection, which can really compromise the relative sensitivity of these methods of diagnosis [88]. On the other hand, although recognizing that these methods can be helpful in certain occasions, the OIE does not accept the use of direct diagnostic methods arguing that the serological methodology is insufficiently developed or validated to ensure the detection of specific antibodies in fish sera. In the following text, the most frequently used methods for antibody detection will be described. Since IFAT for antibody detection has been poorly reported, only PNT and ELISA will be approached here. Plaque neutralization test: This procedure for antibody detection has been described for IHNV and VHSV [86,132,133]. As an initial step, the reference virus (against which specific antibodies are to be diagnosed in fish sera) must be freshly titrated by the plaque assay method. In addition, the heat inactivated fish sera must be mixed with fish complement for 30 min at 15°C–18°C. Then, the reference virus is added to a final concentration of 2 to 8 × 103 pfu/mL and incubated for 30 min (to o/n). After quantifying the nonneutralized virus by the plaque assay counting method, the 50% PNT is calculated as the reciprocal value of the highest serum dilution yielding 50% reduction of the average pfu with respect to the control titration. ELISA for fish antibody detection: Two versions of the procedure have been employed [86,128,130,131,134,135]: iELISA and antigen-capture ELISA or sandwich ELISA (swELISA). In both cases, immunosorbent 96 wells plastic plates are employed. In the swELISA, the plate is initially coated with antivirus immunoglobulin (normally from rabbit) diluted in carbonate [86] or borate [132] buffer, incubated for 1–2 h, and washed with 0.05% Tween 20 in ELISA buffer (EBT) then, the procedure follows same steps as in the iELISA. Different ELISA buffer have been reported, but the most frequent is PBS. For iELISA, the procedure starts with the coating of the well’s bottom with the referencespecific virus, normally o/n at 4°C or at rT. Exclusively for iELISA, some authors have developed recombinant viral coat protein that can be used for this first step [134,135]. After washing three times with EBT, the remaining binding sites are blocked with unspecific protein for 1–2 h between rT and 37°C. Different blocking solutions have been reported with similar performance: 1% (w/v) gelatine, 1.0%–1.5% BSA, or 2%–5% skim milk. After washes, the test fish serum (previously treated 30 min at 45°C) is added (diluted in ELISA buffer) to each well and incubated rT to 37°C
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1–2 h (some authors have reported shorter incubation). The wells are washed and covered with anti fish Ig (preferable monoclonal, although polyclonal antiserum has also been employed with good results). After washing, the conjugate must be added and incubated as before. For instance, if the former anti fish Ig has been obtained in mouse, a rabbit-anti-mouse-enzyme labeled conjugate must be employed. Two types of enzyme label have been employed: HRP and AP. The wells are then washed and covered with the corresponding chromogen/substrate (e.g., orthophenylenediamine and H2O2 x for HRP conjugate, or pNPP for AP-conjugate). After incubation at rT to 37°C (incubation time varies from 15 min to 1 h, depending on the chromogen), the reaction must be stopped (e.g., with 1 M H2SO4 for HRP, or 3 M NaOH for AP) and absorbance readings measured as OD at 405–492 nm in an ELISA reader.
32.9
Molecular Diagnosis
The techniques based on the detection of a specific sequence of the viral genome or mRNA (those known as molecular techniques of diagnosis) were first applied in aquaculture in the eighteens of the twentieth century. They were initially thought to substitute the traditional and sero-/ immune-diagnostic tests since they were expected to yield better performance. Nevertheless, nowadays, more than two decades later, they are far from being the reference methods in fish and shellfish disease diagnosis. They are still theoretically of higher sensitivity and specificity, but other parameters such as R&R must be improved throughout the standardization of the procedures. In this chapter, we approach to the scientific basis of this kind of techniques, their advantages and disadvantages, the parameters defining its performance, and the critical steps affecting it. Finally, a description of the different procedures is also included.
32.9.1 Scientific Basis: An Overview In broad terms, molecular diagnostic techniques can be classified into two groups: those strictly based on the detection of a specific sequence, and those that additionally include the amplification of the target and/or signal. In the first group are included the NAH and NAH-derived procedures (e.g., ISH) and the hybridization based arrays (DNA chips). The second group is constituted by the PCR and PCR-based procedures, the nucleic acid sequence based amplification (NASBA), and the loop-mediated isothermal amplification (LAMP). There are other molecular techniques that will not be included in this chapter since they are more devoted to viral typing than to diagnosis: electropherotyping, T1 ribonuclease fingerprinting, ribonuclease protection assay (RPA), restriction fragment length polymorphism (RFLP), or genome sequencing are among them [136]. Nucleic acid hybridization (NAH): The NAH-derived techniques are based on two singlestranded nucleic acid molecules hybridize just if they exhibit a minimum sequence homology. In its use for diagnosis, the procedure includes a specific probe, which will hybridize with a homologous viral sequence if present in a sample. The probe is constituted by a nucleotide genome sequence complementary to a target genome or mRNA, and is linked to a reporter molecule (biotine, fluorophor, or isotope). The probe can be obtained by cloning procedures or by PCR. The sample under examination must be processed to extract the viral genome and/or mRNA, which will be subjected to hybridization with a specific probe. At the end of the process, the presence of the reporter (demonstrated from a colorimetric reaction, fluorescent emission, or radioactivity
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detection) allows simultaneous detection and identification. The procedure can be performed on a nylon membrane (dot-blot hybridization; DB) or on infected cells or tissues (ISH). The ISH is a useful technique to detect viruses in tissue imprints and squashes, and is therefore extensively applied to those viruses that cannot be grown and isolated in cell culture, as the crustacean viruses [7,137–141]. It has also been applied to fish viruses as aquatic birnaviruses [142,143], infectious salmon anemia virus (ISAV; [144]), iridovirus [145], or herpesvirus [146], in fish tissues, and also directly on infected monolayers. Regarding its application on cell culture, although the procedure is time consuming in comparison with other techniques [145], it has been demonstrated to yield less background than immunohistochemistry [142], and to let the detection of the virus in infected monolayers at shorter times p.i. than with immunofluorescence [143]. A more frequent procedure among the hybridization-derived techniques is the dot blot. In this case, the technique can be applied just for the identification of previously isolated virus [27,55,71,72,147,148] or to detect viral genomes directly from infected fish tissues [27,149–152]. In any of both cases, the extracted target nucleic acid is denatured and blotted onto a positively charged nylon membrane, which is immersed in the following solutions. Finally, the visualization of a dot is interpreted as a positive detection and identification. PCR and PCR-based diagnostic techniques: PCR technology was developed in 1983 by the Cetus Corporation [153] and soon introduced in diagnostic laboratories. The procedure is based on the amplification of a sequence (of the viral genome or mRNA) in between a selected pair of primes which hybridize in specific positions to yield a fragment of known size. The complete process is constituted by a series of cycles consisting of a sequence of three steps corresponding to denaturing, re-annealing, and DNA polymerization. In each cycle, two size-classes of amplified products are produced: the specific fragment of the expected size, and a larger intermediate (Figure 32.3). Whereas the number of intermediate molecules increases arithmetically (Ic + 1 = Ic + 1, where I is the number of intermediate molecules and c the cycle number), the number of doublestranded molecules of the specific fragment increases in an exponential mode (Fc + 1 = 2Fc + Ic, where F is the number of specific fragments amplified). In this way, as shown in Table 32.2, amplification from a unique target molecule would yield around 1010 specific fragments (amplicons), all the process in about 1–3 h. Such a quantity of molecules is easily visualized, by means of a simple intercalating dye, in agarose gels. This is, together with the confirmation of the size of the fragment (by comparison with molecular size standards) the simplest way to develop the result, and for some authors it is enough for diagnosis in most cases [154]. However, to avoid the failure of the specificity due to false positives, an additional confirmatory final step is advisable. Several approaches have been reported: Nested PCR (Nt-PCR) is the most frequently employed since it simultaneously increases sensitivity [18,21,23,62,149,155–160]. It consists in a second round of PCR applied to the amplicon yielded in the first PCR, but using a second set of primers hybridizing in the internal positions of the first specific fragment. The detection of the second amplicon in a gel is considered as confirmatory of the diagnosis, which comes from a secondary amplification from a nonspecific primary amplification is of extremely low probability. A second approach also commonly employed is based on the use of specific probes. They are usually employed for confirmation of the specificity of the band in agarose gel by Southern blot (SB; blotting of the band in a nylon membrane, and application of NAH with a labeled probe; [21,23,26,158,161–164]), but have also been employed to avoid gel electrophoresis by the detection of the PCR product by dot blot hybridization [149], or even by using a miniarray system with colorimetric detection of the amplicon [165]. Others have reported the use of ELISA detection of DIG-labeled amplicon (labeled during PCR amplification; [166]).
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Figure 32.3 Specific sequence amplification by polymerase chain reaction. In the first cycle, extension from the primers produce two intermediate (I) chains of longer length than the specific fragment. In the following cycles, more intermediate chains are produced from the target (T) DNA in an arithmetical kinetics. From the second cycle, the specific fragments (F) will be produced in a geometrical-type kinetics. *: number of expected molecules of each type (T, I, and F) after each cycle.
Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases Table 32.2 Number of Fragments Amplified by PCR after n Cycles from a Unique Target DNA
◾
625
Another alternative is the sequencing of the amplified fragment, which additionally provides phylogenetic information [120,136,139,167]. Finally, some authors have reported the use of RFLPs, for confirmatory purpose of PCR results [138,154]. However, the procedure is time consuming and cumbersome. Number of If the selected target is RNA, the procedure requires an initial Amplified reserve transcription step to produce the cDNA to be subjected to Cycle Fragments amplification by PCR. This can be performed in a separate reac1 0 tion (2-step RT-PCR) or in a single tube (1-step RT-PCR). The performance of the diagnostic will not be necessarily affected by the 2 1 procedure chosen. In addition, each version has its own advantages 3 4 and thus, it is a matter of personal choice (based on experience). For instance, the 1-step RT-PCR has the advantage of reducing the 4 11 risk of contamination due to manipulation. On the other hand, 5 26 the 2-step version allows the use of the same RT product for PCR application for different virus. 6 57 Real-time PCR (Rt-PCR) and quantitative PCR (Qt-PCR): 7 120 Although this should be included in the former section since it is also a PCR-based method of diagnosis, its peculiarities 8 247 makes it to deserve a specific section. Although of quite recent 9 502 development, this technology is being increasingly employed in 10 1013 fish and shellfish diagnosis. It is based on the labeling of the amplicon with a fluorescent reporter. The detection of the fluo4 15 1.7 × 10 rescent signal (and its intensity) therefore provides information 20 3.8 × 105 (in real time) on the level of production of specific fragments. In addition, the level of monitored signals depends on the original 25 1.2 × 107 quantity of the target DNA, which allows to use the method 30 3.9 × 108 not only for diagnosis, but also for quantitative purposes. The disadvantage of this method of diagnosis is the need of special 35 1.2 × 1010 equipment (not affordable for any laboratory) and skills. How40 3.9 × 1011 ever, it is being increasingly employed in shellfish [20,168–170] and fish virus diagnosis including RNA viruses such as IHNV and VHSV [171–173], ISAV [143], salmonid alphavirus (SAV) [174–176], betanodavirus [163,177,178], or DNA viruses such as herpesvirus [179], and iridovirus [180,181]. Two types of procedures, depending on the method of labeling, are available. The simplest one is SYBR-Green Rt-PCR, which uses a dye (SYBR-Green) that unspecifically binds dsDNA-molecule. Therefore, the amplification must be complemented by determining the melting temperature of the amplicon to confirm if it coincides with that of the expected fragment. Th is procedure is easy to introduce for PCR-expert laboratories, and has produced good results in fish virus diagnosis [143,182–184]. Another method uses internal probes labeled with the fluorofor, and which is only activated throughout the hybridization of the probe to the specific amplified fragment. In this case, two alternatives exist, depending on the kind of probe employed: Molecular Beacon or TaqMan probe, though the latter one has been most frequently applied for fish and shellfish diagnosis [169,171,172,175,176,181,183,184]. The advantage of using probes is that it eliminates the need of any additional test for confirmation of the detection, thus reducing manipulation and time.
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Nucleic acid sequence based amplification (NASBA): Th is technology was first described by Compton in the early 1990s [185]. It is also based on amplification, which ensures low DLs as for PCR, but has the advantage that the procedure is performed at constant temperature and thus the process for diagnosis is shorter because it does not waste time in raising/lowering reaction temperatures. However, it has not been frequently employed in fish viral diagnosis [186,187]. As for PCR, NASBA uses a pair of primers though one of them (secondary primers) is complemented with a T7 promoter. The amplification is based on the coordinate activity of three enzymes: reserve transcriptase, RNase H, and T7 RNA polymerase. The first step is the synthesis of a cDNA chain, and throughout the formation of the intermediate hybrid DNA– RNA, the original target RNA chain is eliminated by means of the RNase H activity. The enzyme responsible of the cycling amplification is the T7 RNA-polymerase, which synthesizes a single-strand RNA (using the secondary primer) of opposite polarity to that of the original target. The process is performed in the presence of a molecular beacon as specific internal probe. Similarly to its use for PCR, the probe is linked to two molecules, a reporter and a quencher. In its hybridized form, the probe shows a stem-loop structure and thus the fluorescence of the reporter is quenched due to this close proximity. The open form allows the emission of the fluorescence, which allow the detection of the signal and thus of the production of the specific RNA chain. Loop-mediated isothermal amplification (LAMP): Recently a very novel technique has been developed and described [188], which allows amplification of a target DNA from a few copies to 109 in just 1 h. It is also based on amplification but has three main advantages: (1) it is performed under isothermal condition (at 60°C–65°C, depending on the selected primers), and therefore special reaction equipments are not needed, (2) it uses a set of four specific primers that recognize a total of six sites on the target DNA, which increases specificity, and (3) do not need secondary test or special equipment to interpret the result, because it just depends on the appearance of a white precipitate corresponding to magnesium pyrophosphate [189]. It is based on a sequence of synthesis and the displacement of strands (http://loopamp.eiken.co.jp/e/lamp/principle.html) generating different classes of stem-looped DNA molecules that can be separated in multiple bands of different sizes in agarose gel electrophoresis. The main requirement is the right selection of the set of four primers using specific software (http//primerexaplorer.jp/lamp3o.ol): A forward inner primer (fip), with sequences of the sense and antisense strand; a forward outer primer (fop); a backward inner primer (bip), with sequences of the sense and antisense strands; an outer backward primer (bop) (Figure 32.4). The process is based on the principle of autocycling strand displacement DNA synthesis, and is performed by a special DNA-polymerase, the Bst polymerase, which exhibits a strong strand displacement activity [189]. In its short history, this technology has been developed for diagnosis of a few fish [180,190–193] and shellfish [194,197] viruses, of both DNA [180,190,194] and RNA [191,193–195] genomes.
32.9.2 Performance of the Molecular Techniques of Diagnosis: Critical Steps and Critical Factors The molecular methods of diagnosis share theoretical high sensitivities and specificities. However, they are considered to yield different results among laboratories [196], which seem to indicate low reproducibility. For all these techniques, a number of critical steps and factors can compromise one or several performance parameters.
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Recognition sequences 5΄
f3
f2
3΄
f1
b1c
f1c
b1
3΄
b2
5΄
b3
Specific primers Primers fip 5΄
Primers fop 5΄
f1c
f3
f2
3΄
Primers bip 5΄
Primers bop 5΄
b2
b3
b1c
3΄
3΄
Figure 32.4 Specific primers and recognition sites in the isothermal loop-mediated amplification (LAMP) diagnostic technique.
Extraction of viral nucleic acid: Independently from the method selected, all molecular diagnostic techniques (except perhaps ISH) have in common the first step. In this sense the viral genome or mRNA must be extracted from the infected cell or fresh tissues. Even for those techniques with a theoretical DL of one viral genome, if only one molecule is actually present in the sample, the method of extraction should have an absolute efficiency to facilitate its detection. There are different types of methods. The traditional ones were based on lysis by guanidine thiocyanate [197] or by proteinase K treatment [27,198] followed by phenol–chloroform extraction and ethanol precipitation. These procedures are constituted by many steps and are time consuming; therefore, nowadays they have been substituted by commercial kits of different types [69,82]. The most economical are those based on the adaptation of the traditional methods, such as the RNAzol and TriReagen LS from SIGMA, or the Trizol and Trizol LS Reagent from Invitrogen. A second group is based on lysis by guanidimicin thiocyanante and precipitation by Cs-triofluoroacetate-LiCl solutions. The third type is constituted by those methods that use lysis with guanidium thiocyanate followed by ethanol precipitation and resin-capture by silico membranes. In a long study performed in the author’s laboratory (unpublished data) to validate the methods of extraction, they have studied the recovery, the purity, and the R&R of a selection of methods and kits under different conditions to analyze the effect of (1) the storage conditions of the kit components, (2) the experience of the technician, and (3) the time of the day (at the beginning and at the end of the day), and the day of performance. From the results the authors can only conclude that there is not a unique method to advice. The traditional-based methods give the best recoveries in terms of quantity of nucleic acid, as spectrophotometrically measured. However in some cases, as when applied by inexperienced technicians, the purity of the extracted nucleic acid is quite different, and the failure of techniques as PCR is probable. On the other hand, the resin-based methods exhibit, probably due to their significant reduction of steps, higher purities and better R&R, although the recoveries are significantly lower. Critical steps and factors in the diagnostic procedures: Once the optimum extraction method has been selected, the detection method must be also subjected to optimization. Regarding hybridization, the first parameter to optimize is the probe. In this sense, oligonucleotide probes are usually employed [55,199] because they are easy to obtain and usually provide good sensitivities.
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However, other authors prefer the use of longer probes, to ensure specificity though reducing sensitivity [27,72]. A critical step in HAN is the hybridization solution; therefore, special care must be evinced in testing the different reported solutions to introduce in routine diagnosis. Finally, the selection of the probe label is also important. The radioactive probes were originally used as they provided higher sensitivities than biotinylated ones [27]. However, the new chemiluminescence labeling provide high sensitivities using x-ray plaques, or even higher with special reader devices. The number of critical steps is even higher in PCR-based techniques. The choice of primers is the first concern. The selection of primers should not be performed on a unique reference sequence, but on multi-alignments to search for conserved regions. In addition, the selected sets should be tested against a broad variety of strains from different origins and hosts, and against not related viruses. Both approaches will ensure the specificity of the primers by reducing the risk of false positives and false negatives. In the case of Rt-PCR, the selection of the set of primers can even be complicated if an internal probe must be used. Special software must be employed that does not allow the use of multi-alignments. Therefore, different sets of primers and probes should be tested. Nevertheless, to ensure detection of any strain of a virus multiplex method might be required. There are several commercial kits available for PCR and RT-PCR, and it is not possible to select one by its theoretical characteristics because most of them have been demonstrated to perform appropriately. The best advice is the laboratory to test them, and select the one showing best performance. Once a kit has been validated, it should not be substituted for a different one before its efficiency is also confirmed. To quantify the performance of the procedure [82,154,200] and to facilitate quantitative epidemiology [200], the RT and PCR conditions, including the temperature and the time of RT and extension steps, the concentration of target nucleic acid, primers and Mg2+, the number of cycles, and the length and the temperature of the steps in each cycle should be tested for each kit and virus. Another important step is the development and the confirmation of the result. Although for some scientists visualization of the right size amplicon in a gel should be enough for giving a positive diagnosis, this approach is a serious risk for specificity and therefore any of the additional tests previously described must be included. This can be avoided by the use of Rt-PCR with internal probes. However, even in the best conditions, failure of any step, or even errors, can occur. Therefore, the use of correct controls is crucial to detect false results. For PCR or RT-PCR positive and negative controls, corresponding to nucleic acid from strains of homologous and heterologous viruses, should be subjected to the same protocol. However, this is not enough because failure could occur during the extraction step. Therefore, infected and uninfected tissues should be employed as positive and negative control, respectively. The use of positive controls can compromise the result of simultaneously processed samples. Therefore, the ideal positive control would be that producing a PCR product amplified with the same set of primers, but giving a different size. This has been the approach by Cunningham and Hoffs [201], who designed a plasmid with an insert to be used for positive control, yielding an amplified fragment of 200 bp, easily differentiated from the 155 bp specific amplicon obtained from infected material. However, this control does not detect failures in the extraction step. Therefore, an additional approach is the amplification of a gene from the fish [202]. To avoid false results due to carryover of contaminants, a correct organization of the working areas is also important, separating the different steps and tools [82,154,203]. Limit of detection of molecular techniques: Table 32.3 gives a summary of the DLs reported for different fish and shellfish viruses. Comparing with other techniques, the DL values obtained by HAN
15 ng [27]
1 pg [55]
—
—
—
—
—
50 ng [72]
—
—
—
—
—
IPNV
IHNV
VHSV
SVCV
ISAV
VERV
SAV
TRV
EHNV
OMV
RSIV
GIV
LDV
nad
vte
—
—
—
10 copies [232]
—
—
—
—
—
105 TCID50/g [151]
—
—
104 TCID50 [27]
NAHa
2.5 ng [161]
50 fg [156]
10 fg [227]
—
—
0.1 pg [231]
—
25 fg [207]
37 fg [202]
—
32 TCID50 [205]
10 fg (1 fg w/Nt)f [204]
10
3.5
—
—
pfu [227]
—
1–10 pfu [162]
—
—
—
—
1 pg [182]
—
—
—
—
—
—
0.01–0.1 TCID50 [229] 100 copies [159,230]
—
—
—
—
na
10−1 TCID50 [167]
1–100 inf. Unitsg [228]
1 TCID50 [205]
100 TCID50/mL [205]
vt
1 pg [26]
15 fg [206]
na
PCR/RT-PCRb
—
vt
(continued)
—
—
—
—
—
—
0.1 TCID50 [177]
10 copies [176]
10 TCID50 [178]
—
—
0.5 ffui [171]
100 copiesh [172]
Rt-PCRc
Table 32.3 Detection Limits of Fish and Shellfish Viruses by Different Molecular Techniques of Diagnosis
Techniques of Diagnosis of Fish and Shellfish Virus and Viral Diseases ◾ 629
—
—
—
—
KHV
CCV
WSSV
IHHNV
—
—
—
—
—
vte
99 fg [212]
100 g [212]
1 fg [155]
1 pg [209]
—
na
vt
—
—
—
—
2.7 pfu [233]
PCR/RT-PCRb
—
—
—
—
—
na —
vt
—
2 copies [169]
—
10 copies [179]
Rt-PCRc
i
h
g
f
e
d
c
b
a
Nucleic acid hybridization (NAH). Polymerase chain reaction or reverse transcription-PCR. Real-time PCR. Extracted nucleic acid. Viral titer (or na copies). With nested PCR. Infectious units. Copies of plasmid or of in vitro synthetized RNA. Fluorescent foci units.
CCV, Channel catfish virus; EHNV, Epizootic hematopoietic necrosis virus; GIV, Grouper iridovirus; IHNV, Infectious hematopoietic necrosis virus; IHHNV, Infectious hypodermal and hematopoietic necrosis virus; IPNV, Infectious pancreatic necrosis virus; ISAV, Infectious salmon anemia virus; KHV, Koi herpesvirus; LDV, Lymphocystis disease virus; LMBIV, Largemouth bass iridovirus; OMV, Oncorhynchus masou virus; RSIV, Red sea bream iridovirus; SAV, Salmonid alphavirus; SVCV, Spring viremia of carp virus; TRV, Turbot aquareovirus; VERV, Viral encephalopathy and retinopathy virus; VHSV, Viral hemorrhagic septicemia virus; WSSV, White spot syndrome virus.
—
LMBIV
NAHa
Detection Limits of Fish and Shellfish Viruses by Different Molecular Techniques of
◾
nad
Table 32.3 (continued) Diagnosis
630 Handbook of Seafood and Seafood Products Analysis
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631
are relatively high, perhaps due to the absolute limit of sensitivity of 104 –105 molecules by direct hybridization reported by Desselberger [203]. The values given in the table are the lowest ones; other authors have reported different values, which demonstrate that the performance of a technique depends on many factors and therefore standardization is a requirement before being able to compare protocols. For instance, from the data shown by Arakawa et al. [26] and López-Vázquez et al. [204], it seems that the base line for RT-PCR detection of IHNV (1 pg) is 104 times over that for VHSV (0.1 fg) in terms of the minimum detectable quantity of purified genome. However, the value of 0.1 fg reported by López-Vázquez et al. [206] was obtained after re-amplification by nested PCR. In this same study, DL observed by plain RT-PCR was 10 fg, which still is 100 times lower than that from Arakawa et al. [26]. On the other hand, in a study by Williams et al. [205] the DL for IHNV was 30 times lower. Similar levels to those for IHNV were reported by López-Lastra et al. [198] and Rimstad et al. [148] for IPNV, though Wang et al. [206] obtained 1000 times more sensitivity. Similar DL values have been reported for other RNA viruses such as ISAV (37 fg [202]) or betanodavirus (25 fg [207] or 100 fg [208]). For DNA viruses, low DL have also been reported (Table 32.3). Except for two cases of koi herpesvirus (KHV), with a DL of 1 fg by PCR [209] or, even more remarkable, the DL reported by PCR for lymphocystis disease virus (LDV) (2.5 ng [161]). For channel catfish virus (CCV), first reports gave DL values in the level of pg (0.1 pg [210]), though it has been lowered to 10 fg by RT-PCR plus SB [157], and even to 1 fg by Nt-PCR [155]. Finally, for shellfish viruses the limits seem to be between 0.1 and 1 pg [150,211–213]. In terms of minimum detectable viral titers, the best result was reported for SVCV by RT-PCR [168] and SRV by Rt-PCR [175], with a DL of 0.1 TCID50, or WSSV [169] with a DL of two genome copies.
32.9.3 Molecular Methods of Diagnosis: Brief Description of Protocols The aim of this chapter is not to constitute a manual or a list of protocols for each technique, but to show a general view of the procedure. For each step, different materials (enzymes, buffers, primes…) and different conditions (time and temperature of incubation) have been reported by many authors. As shown in the previous section, it is not easy to decide which protocol (if any) is actually the best one. Even, it might depend on so many factors, including the type of virus, the best advice to the diagnostician is to test several options before using one in routine diagnosis. In addition, only the techniques most frequently employed will be approached, NAH, PCR/ RT-PCR, and Rt/Qt-PCR. Those interested in other techniques described earlier may consult the specific references. Nucleic acid hybridization: Table 32.4 gives a summary of the steps of a basic protocol for dot blot hybridization. The first step is necessarily the preparation of the probe. For this purpose, two alternatives were originally available. The most common is the use of oligonucleotides [55,147,148], which have the convenience that they are easily selected with available software, and can be purchased already labeled. However as already cited, some authors believe they can be occasionally responsible of false positives [82]. The second alternative was the cloning technology [27,72] to obtain larger fragments of 200–600 bp. Fortunately, this cumbersome procedure to obtain this type of probe has been substituted by PCR, which allows the simultaneous labeling of the specific fragment, producing large quantities of probe in a short time. The most convenient choices of reporters for probe labeling are biotin and digoxigenin, though better sensitivities are obtained with chemiluminescence. Any method for target extraction can be chosen. The unique condition is the purity of the extracted nucleic acid, which must be free of
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Handbook of Seafood and Seafood Products Analysis Table 32.4
Steps in the Protocol of Nucleic Acid Hybridization
I. Probe preparation I.1. Designing of the probe I.2. Labeling II. Sample preparation II.1. The extraction of target nucleic acid II.2. Blotting onto membrane, and denaturing III. Hybridization III.1. Prehybridization solution and selection of stringency III.2. Prehybridization III.3. Hybridization III.4. Posthybridization washes IV. Development of results
contaminated salts. Then, the sample nucleic acid must be blotted onto a nylon membrane previously soaked in 2× SSC (1× SSC: 0.15 M NaCl, 0.015 M sodium citrate). If the target is dsRNA, it must be pre-denatured by treatment at 100°C for 5 min, immediately transferred to an ice bath, and methylmercury (10 mM) added [72,82]. In the case of ssRNA, denaturing is not needed [55]. DNA targets may be pre-denatured by boiling, though it can be denatured by NaOH treatment after blotting [215]. Finally, the samples are blotted onto the membrane using a 96 wells dotblotting minifold. The nucleic acid can be fi xed by baking 1–2 h at 80°C under vacuum, or by UV light using a crosslinker. For hybridization, the important concerns are the stringency conditions [214] and the prehybridization solution. Stringence must be selected based on previous tests but in their absence medium level stringency can be chosen. For the prehybridization solution, Denhart’s [55,71] and Hybrisol (Oncor), in combination with sonicated salmon sperm DNA or thyme DNA demonstrated good results. The membrane is subjected to prehybridization for 1–2 h at the temperature corresponding to the chosen stringency (40°C–55°C). The probe is then denatured by boiling and added to the prehybridization solution. After incubating from 2 to 24 h (usually o/n is enough) at the same temperature, the membrane is rinsed and subjected to several washes in salt concentrations specific for the selected stringency, using SSC buffer supplemented with 0.1% sodium dodecyl sulfate. Finally, the membrane is developed following the instruction of the manufacturer of the labeling kit employed. PCR and RT-PCR: Table 32.5 enumerates the steps and parameters to consider in the designing of a PCR protocol for the diagnosis of DNA viruses. The first parameter is the quantity of DNA subjected to amplification, which normally must be around 100 ng to 1 mg, though much lower quantities (2.5 ng) have been reported [156,209]. The primers, usually designed to amplify fragments of 200–600 bp, must be supplied in a final concentration of 0.2–0.5 mM. Higher concentrations are usually responsible for unspecific bands at the bottom of the lane, which can be confusing if small
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Table 32.5 Steps and Parameters to Consider in DNA Amplification I. PCR Mix I.1. Target DNA I.2. Primers set I.3. Enzyme I.4. Buffer I.5. MgCl2/MgSO4 I.6. dNTP II. Initial denaturation III. Cycle III.1. Number of cycles III.2. Denaturation III.3. Annealing III.4. Extension IV. Final extension V. The development of results
specific fragments have been chosen. Lower concentration, on the other hand, may get exhausted during the cycles and cause poor amplifications. Taq DNA polymerase is added to a final concentration of 0.04 U/mL in a buffer (supplied by the manufacturer) supplemented with 1.5 M MgCl2 (previous tests are advised to optimize the concentration of Mg2+), and dNTPs to a final concentration normally between 0.4 and 0.6 mM, although lower values have been published. The amplification normally starts with a previous denaturation step at 95°C for 5 min, though some laboratories skip this step [156,215], followed by a number in between 30 and 40 cycles consisting of denaturation (normally 94°C, 30 s), annealing, and extension (normally 72°C, 30 s). The annealing temperature depends on the characteristics of primers and target DNA and, therefore must be assayed for each case. In some cases, low (46°C [166]) or high (68°C–72°C [209,216]) temperatures have been employed, but most frequently annealing is performed at 56°C–63°C for 30 s. After the cycles, a final extension period (72°C, 7 min) is advised to end incomplete synthesis. For RNA viruses, a previous step of cDNA synthesis is required. In the case of ssRNA viruses, as for the amplification of viral mRNA, all buffers must be nuclease-free, and a supplement of RNase inhibitors is recommended [161,208,215,217]. An important parameter is the quantity of template RNA to use in the RT reaction. It is usually set at 0.5–1 mg, but 2 mg [218] or even higher values (5 mg [219]) have been reported. The author’s laboratory has performed a validation of the optimum quantity of RNA (extracted by resin-based methods) for RT synthesis of IPNV, VHSV, and betanodavirus RNA, and observed that 100–200 ng of RNA (or around 10 ng/mL of reaction) is enough to obtain reliable results in the PCR amplification [23].
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There is a variety of commercial reverse transcriptases available, any of them with good performance. Previous test are advised to determine the best conditions for the enzyme, including Mg2+ and dNTP concentration. Regarding the primers, the procedure for the RT step is slightly different depending on the chosen version. In this sense, if the 1-step RT-PCR system has been selected, the reaction mixture must be supplemented with both sense and antisense primers, to a final concentration of around 0.5–1 mM, or 10–15 pmol per reaction. For the 2-step version only one primer (sense or antisense) is needed in this first step but, some authors reported better performance by using random primers [202,219–221]. Although many authors have reported an initial denaturation step, previous to addition of the enzyme, at 90°C–98°C [23,208,215] or 65°C–70°C [203,214,222] for 5 min, others skip this step obtaining good results [161,207,218]. Finally, the enzyme is inactivated by treatment at 95°C–99°C for 2–10 min. If using the 2-step method, the PCR mixture must be added to the RT product (or vice versa). If the 1-step version has been selected, the DNA polymerase must be thermostable to avoid inactivation during the last step. However, this is not really a concern of the user since all one-step systems provide the ready-to-use mixture of both enzymes. PCR protocols are quite similar to that cited earlier, but the diagnostician must use specific references for the virus under assay. An important subject is the type or types of primers used for the specific amplification. As already indicated, normally sets of primers are selected by specific software on known viral sequence, but must be tested in previous assays to ensure their reliability. Special care must be taken when variants of a virus with large differences can be present. For those cases, some authors prefer the design of primers specific for each type [219], but others prefer the use of degenerate primers to ensure that mismatches will not be responsible of false negatives [115,120,222]. An alternative to ensure the detection (and/or differential diagnosis) of different viruses is the multiplex PCR /RT-PCR, using mixtures of different (normally 2) pairs of primers [207,213,217,221]. However, special care must be taken to optimize the conditions of multiplex amplification, as specificity and sensitivity can be compromised [213]. Finally, for analysis and confirmation of the diagnostic results, different approaches, other than simple gel electrophoresis, can be performed, as already indicated. Real-time PCR: The protocol to apply this technique for diagnosis is quite similar to that of PCR except for the addition of an extra component constituted by the intercalating dye (SYBR-Green) or the internal labeled probe (TagMan or Molecular Beacon). Therefore, for precise description of the procedure we redirect the readers to those protocols reported elsewhere in the literature for fish and shellfish viruses of both, DNA [170,182,183,224] or RNA [143,171–173,175–178,183,184].
32.10 Nonlethal Methods of Diagnosis The official directives published by the OIE and other organizations for the surveillance of diseased and healthy populations [3,7,29] are mainly based on the sacrifice of the animals for sampling of internal organisms or tissues. This can be admissible for regular surveillance and monitoring, but not when the populations under study are in-danger species or broodstocks [224]. For some scientists, the available methods of diagnosis are supposed to yield more reliable results if applied onto internal organs. However, this assumption is not necessary true. For nonlethal sampling and diagnosis there have been different approaches. The detection of antibodies in fish serum has been used (and still is) by many authors, which for some authors might be helpful for the selection of breeders, in some cases even with better detection capacities than PCR [129]. However, the OIE does not accept this as an official method of diagnosis of fish
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◾ 635
viruses due to the partial lack of knowledge on the immune response of fish to viral infection under certain conditions [7]. Serum has also been employed for viral detection by PCR [80] or by isolation in cell culture. However, the level of virus in serum can be, in some cases, much lower than in other tissues and organs. For breeders selection, sex products such as sperm or ovarian fluids have been used for diagnosis by means of PCR [129,160], CC isolation, or even flow cytometry [72]. However, the best results have been obtained by the detection of the virus in leukocytes by both, isolation of the virus by cocultivation of the leukocytes in cell monolayers, or by the inoculation of lysed leukocytes in CC [21,23,24,60,137], or by PCR, or RT-PCR detection [23,26,155,158,160,204,225–227]. However, although the best results of viral detection in carrier fish seem to be obtained by PCR-based technologies applied on blood samples [23,204,225], recent studies reported the lower performance of the nonlethal methods, compared with the traditional lethal diagnosis [226]. Therefore, much effort is needed to standardize and validate the methods of diagnosis to be applied on nonlethal sampling.
References 1. Anon 2000. Aquaplan zoning policy guidelines. Agriculture, Fisheries, Forestry. Canberra, Australia, p. 41. 2. EC Council Directive 91/67/EEC [and further amends] concerning the animal health conditions governing the placing on the market of aquatic animals and products. 3. EC Commission Decision 92/532/EEC [and further amends] laying down the sampling plans and diagnostic methods for the detection and confirmation of certain fish diseases. 4. FAO 2004. Surveillance and zoning for aquatic animal diseases. FAO Fisheries Technical paper 451. 5. OIE 2006. Aquatic animal health code. OIE, Paris. 6. Cameron, A. 2004. Principles for the design and conduct of surveys to show presence or absence of infectious diseases in aquatic animals. Nat. Aquat. Anim. Health-Tech. Work G.- Policy Doc. Aus. Vet. Animal Health Services. p. 37. 7. OIE 2000b. Manual of diagnostic tests for aquatic animals. OIE, Paris. 8. Dopazo, C.P. and Barja, J.L. 2001. A comparison between polymerase chain reaction and serological techniques for detection of fish viruses. In: Risk Analysis in Aquatic Animal Health Rodgers, C.J. (Ed.), World Org. An. Health (OIE), Paris. pp. 271–275. 9. Dohoo, I., Martin, W., and Stryhn, H. 2003. Veterinary epidemiology research. Prince Edward Island. AVC Ic. 10. Cameron, A.R. 2002. Survey toolbox for aquatic diseases—A practical manual and software package. Australian Center for International Agriculture Research (ACIAR). Monograph no. 94, Canberra, Australia. 11. Mulcahy, D. and Batts, W.N. 1987. Infectious hematopoietic necrosis virus detection by separation and incubation of cells from salmonid cavity fluid. Can. J. Fish. Aquat. Sci. 44: 1071–1075. 12. McAllister, P.E., Schill, W.B., Owens, W.J., and Hodge, D.L. 1993. Determining the prevalence of infectious pancreatic necrosis virus in asymptomatic brook trout Salvenilus fontinalis: A study of clinical samples and processing methods. Dis. Aquat. Org. 15: 157–162. 13. Hostnik, P., Barlic-Maganja, D., Strancar, M., Jencic, V., Toplak, I., and Grom, J. 2002. Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods. Dis. Aquat. Org. 52: 179–184. 14. Agius, C., Richardson, A., and Walker, W. 1983. Further observations on the co-cultivation method for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri, Richardson. J. Fish Dis. 6: 477–480.
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15. Hedrick, R.P., McDowell, T., Rosemark, R., Aronstein, D., and Chan, L. 1986. A comparison of four apparatuses for recovering infectious pancreatic necrosis virus from rainbow trout. Prog. Fish-Cultur. 48: 47–51. 16. Smail, D.A., Burnside, K., Watt, A., and Munro, E.S. 2003. Enhanced cell culture isolation of infectious pancreatic necrosis virus from kidney tissue of carrier Atlantic salmon (Salmo salar L.) using sonication of the cell harvest. Bull. Eur. Ass. Fish Pathol. 23: 250–254. 17. Grant, R. and Smail, D.A. 2003. Comparative isolation of infectious salmon anaemia virus (ISAV) from Scotland on TO, SHK-1 and CHSE-214 cells. Bull. Eur. Ass. Fish Pathol. 23: 80–85. 18. Agius, C., Mangunwiryo, H., Johnson, R.H., and Smail, D.A. 1982. A more sensitive technique for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri Richardson. J. Fish Dis. 5: 285–292. 19. Griffiths, S. and Melville, K. 2000. Non-lethal detection of ISAV in Atlantic salmon by RT-PCR using serum an mucus samples. Bull. Eur. Ass. Fish Pathol. 20: 157–162. 20. LaPatra, S.E. 1996. The use of serological techniques for virus surveillance and certification of finfish. Ann. Rev. Fish Dis. 6: 15–28. 21. Munro, E.S., Gahlawat, S.K., and Ellis, A.E. 2004. A sensitive non-destructive method for detecting IPNV carrier Atlantic salmon, Salmo salar L., by culture of virus from plastic adherent blood leucocytes. J. Fish Dis. 27: 129–134. 22. St-Hilaire, S., Ribble, C., Traxler, G., Davies, T., and Kent, M.L. 2001. Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org. 46: 173–179. 23. Cutrin, J.M., Lopez-Vazquez, C., Olveira, J.G., Castro, S., Dopazo, C.P., and Bandin, I. 2005. Isolation in cell culture and detection by PCR-based technology of IPNV-like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.). J. Fish Dis. 28: 713–722. 24. Gahlawat, S.K., Munro, E.S., and Ellis, A.E. 2004. A nondestructive test for detection of IPNVcarriers in Atlantic halibut, Hippoglossus hippoglossus (L.). J. Fish Dis. 27: 233–239. 25. Gray, W.L., Williams, R.J., Jordan, R.L., and Griffin, B.R. 1999. Detection of channel catfish virus DNA in latently infected catfish. J. Gen. Virol. 80: 1817–1822. 26. Arakawa, C.K., Deering, R.E., Higman, K.H., Oshima, K.H., O’Hara, P.J., and Winton, J.R. 1990. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus. Dis. Aquat. Org. 8: 165–170. 27. Dopazo, C.P., Hetrick, F.M., and Samal, S.K. 1994. Use of cloned cDNA for diagnosis of infectious pancreatic necrosis virus infections. J. Fish Dis. 17: 1–16. 28. Wang, W.-S., Wi, Y.-L., and Lee, J.-S. 1997. Single-tube, non-interrupted reverse transcription PCR for detection of infectious pancreatic necrosis virus. Dis. Aquat. Org. 28: 229–233. 29. Thoesen, J. (Ed.). 1994. Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens. 4th edn. Fish Health Section, American Fisheries Society. Bethesda, MD. 30. Humphrey J.D. 1995. Australian quarantine policies and practices for aquatic animals and their products: A review for the scientific working party on aquatic animal quarantine. Bureau of Resource Sciences, Canberra, Australia. 31. Aguirre Guzman, G. and Ascendio Valle, F. 2000. Infectious disease in shrimp species with aquaculture potential. Recent Res. Dev. Microbiol. 4: 333–348. 32. Brock, J.A. and Main, K. 1994. A guide to the common problems and diseases of cultured Penaeus vannamei. Oceanic Inst., Makapuu Point, Honolulu, Hamaii, p. 241. 33. Nylund, A., Krossoy, B., Devold, M., Asphaug, V., Steine, N.O., and Hovland, T. 1998. Outbreak of ISA during first feeding of salmon fry (Salmo salar). Bull. Eur. Assoc. Fish Pathol. 19: 71–74. 34. Wolf, K. 1988. Fish Viruses and Fish Viral diseases. Cornell University Press, Ithaca, NY. 35. Woo, P.T.K. and Bruno, D.W. (Eds). 1999. Fish Diseases and Disorders, Vol 3: Viral, Bacterial and Fungal Infections. CABI Publishing, U.K.
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36. Groocock, G.H., Getchell, R.G., Wooster, G.A., Britt, K.L., Batts, W.N., Winton, J.R., Casey, R.N., Casey, J.W., and Bowser, P.R. 2007. Detection of viral hemorrhagic septicemia in round gobies in New York. Dis. Aquat. Org. 76: 187–192. 37. Kwak, K.T., Gardner, I.A., Farver, T.B., and Hedrick, R.P. 2006. Rapid detection of white sturgeon iridovirus (WSIV) using a polymerase chain reaction (PCR) assay. Aquaculture 254: 92–101. 38. McClure, C.A., Hammell, K.L., Stryhn, H., Dohoo, I.R., and Hawkins, L.J. 2005. Application of surveillance data in evaluation of diagnostic tests for infectious salmon anemia. Dis. Aquat. Org. 63: 119–127. 39. Opitz, H.M., Bouchard, D., Anderson, E., Blake, S., Nicholson, B., and Keleher, W. 2000. A comparison of methods for the detection of experimentally induced subclinical infectious salmon anaemia in Atlantic salmon. Bull. Eur. Assoc. Fish Pathol. 20: 12–22. 40. Wilson, L., McBeath, S.J., Adamson, K.L., Cook, P.F., Ellis, L.M., and Bricknell, I.R. 2002. An alkaline phosphatase-based method for the detection of infectious salmon anaemia virus (ISAV) in tissue culture and tissue imprints. J. Fish Dis. 25: 615–619. 41. Evensen, O. and Lorenzen, E. 1997. Simultaneous demonstration of infectious pancreatic necrosis virus (IPNV) and Flavobacterium psychrophilum in paraffin-embedded specimens of rainbow trout Oncorhynchus mykiss fry by use of paired immunohistochemistry. Dis. Aquat. Org. 29: 227–232. 42. Lai, Y.-S., Chiu, H.-C., Murali, S., Guo, I.-C., Chen, S.-C., Fang, K., and Chang, C.-Y. 2001. In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel). J. Fish Dis. 24: 237–244. 43. Faisal, M. and Ahne, W. 1984. Spring viremia of carp virus (SVCV): Comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen. J. Fish Dis. 7: 57–64. 44. Dannevig, B.H., Nilsen, R., Modahl, I., Jankowska, M., Taksdal, T., and Press, C. McL. 2000. Isolation in cell culture of nodavirus from farmed Atlantic halibut Hippoglossus hippoglossus in Norway. Dis. Aquat. Org. 43: 183–189. 45. Drolet, B.S., Rohovec, J.S., and Leong, J.C. 1993. Serological identification of infectious hematopoietic necrosis virus in fi xed tissue culture cells by alkaline phosphatase immunocytochemistry. J. Aquat. Anim. Health. 5: 265–269. 46. Hyatt, A.D., Eaton, B.T., Hengstberger, S., and Russel, G. 1991. Epizootic haematopoietic necrosis virus detection by ELISA, immunohistochemistry and immunoelectron-microscopy. J. Fish Dis. 14: 605–617. 47. Dannevig, B.H., Olesen, N.J., Jentoft, S., Kvellestad, A., Taksdal, T., and Haastein, T. 2001. The first isolation of a rhabdovirus from perch (Perca fluviatilis) in Norway. Bull. Eur. Ass. Fish Pathol. 21: 145–153. 48. Dixon, P.F., Hattenberger-Baudouy, A-M., and Way, K. 1994. Detection of carp antibodies to spring viraemia of carp virus by a competitive immunoassay. Dis. Aquat. Org. 19: 181–186. 49. Wang, C.S., Tang, K.F.J., Kou, G.H., and Chen, S.N. 1997. Light and electron microscopic evidence of white spot disease in the giant tiger shrimp, Penaeus monodon (Fabricius), and the kuruma shrimp, Penaeus japonicus (Bate), cultured in Taiwan. J. Fish Dis. 20: 323–331. 50. Choi, D.L., Sohn, S.G., Bang, J.D., Do, J.W., and Park, M.S. 2004. Ultrastructural identification of a herpes-like virus infection in common carp Cyprinus carpio in Korea. Dis. Aquat. Org. 61: 165–168. 51. Hyatt, A.D., Hine, P.M., Jones, J.B., Whittington, R.J., Kearns, C., Wise, T.G., Crane, M.S., and Williams, L.M. 1997. Epizootic mortality in the pilchard Sardinops sagax neopilchardus in Australia and New Zealand in 1995. 2. Identification of a herpesvirus within the gill epithelium. Dis. Aquat. Org. 28: 17–29.
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164. Mjaaland, S., Rimstad, E., Falk, K., and Dannevig, B.H. 1997. Genome characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): An orthomyxo-like virus in a teleost. J. Virol. 71: 7681–7686. 165. Quere, R., Commes, T., Marti, J., Bonami, J.R., and Piquemal, D. 2002. White spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus simultaneous diagnosis by miniarray system with colorimetry detection. J. Virol. Methods 105: 189–196. 166. Iida, Y. and Nagai, T. 2004. Detection of flounder herpesvirus (FHV) by polymerase chain reaction. Fish Pathol. 39: 209–212. 167. Koutna, M., Vesely, T., Psikal, I., and Hulova, J. 2003. Identification of spring viraemia of carp virus (SVCV) by combined RT-PCR and nested PCR. Dis. Aquat. Org. 55: 229–235. 168. Dhar, A.K., Roux, M.M., and Klimpel, K.K. 2002. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry. J. Clin. Microbiol. 39: 2835–2845. 169. Durand, S.V. and Lightner, D.V. 2002. Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis. 25: 381–400. 170. Tang, F.F.J. and Lightner, D.V. 2001. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR. Dis. Aquat. Org. 44: 79–85. 171. Chico, V., Gomez, N., Estepa, A., and Perez, L. 2006. Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J. Virol. Methods 132: 154–159. 172. Overturf, K., LaPatra, S., and Powell, M. 2001. Real-time PCR for the detection and quantitative analysis of IHNV in salmonids. J. Fish Dis. 24: 325–333. 173. Purcell, M.K., Hart, S.A., Kurath, G., and Winton, J.R. 2006. Strand-specific, real time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, infectious hematopoietic necrosis virus. J. Virol. Methods 132, 18–24. 174. Graham, D.A., Taylor, C., Rodgers, D., Weston, J., Khalili, M., Ball, N., Christie, K.E., and Todd, D. 2006. Development and evaluation of a one-step real-time reverse transcription polymerase chain reaction assay for the detection of salmonid alphaviruses in serum and tissúes. Dis. Aquat. Org. 70: 47–54. 175. Hodneland, K. and Endresen, C. 2006. Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan). J. Virol. Methods 131: 184–192. 176. Zhang, H., Wang, J., Yuan, J., Li, L., Zhang, J., Bonami, J.-R., and Shi, Z. 2006. Quantitative relationship of two viruses (MrNV and XSV) in white-tail disease of Macrobrachium rosenbergii. Dis. Aquat. Org. 71: 11–17. 177. Grove, S., Faller, R., Soleim, K. B., and Dannevig, B. H. 2006. Absolute quantitation of RNA by a competitive real-time RT-PCR method using piscine nodavirus as a model. J. Virol. Methods 132, 104–112. 178. Nerland, A.H., Skaar, C., Eriksen, T.B., and Bleie, H. 2006. Detection of nodavirus in seawater from rearing facilities for Atlantic halibut Hippoglossus hippoglossus larvae. Dis. Aquat. Org. 73: 201–205. 179. Chi, S.C., Shieh, J.R., and Lin, S.-J. 2003. Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan. Dis. Aquat. Org. 55: 221–228. 180. Caipang, C.M.A., Haraguchi, I., Ohira, T., Hirono, I., and Aoki, T. 2004. Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP). J. Virol. Methods 121: 155–161. 181. Wang, X.W., Ao, J.Q., Li, Q.G., and Chen, X.H. 2006. Quantitative detection of a marine fi sh iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon. J. Virol. Methods 133: 76–81. 182. Caipang, C.M., Hirono, I., and Aoki, T. 2003. Development of a real-time PCR assay for the detection and quantification of red reabream iridovirus (RSIV). Fish Pathol. 38: 1–8. 183. Dalla-Valle, L., Toffolo, V., Lamprecht, M., Maltese, C., Bovo, G., Belvedere, P., and Colombo, L. 2005. Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR. Vet. Microbiol. 110: 167–179.
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184. Graham, D.A., Jewhurst, H., McLoughlin, M.F., Sourd, P., Rowley, H.M., Taylor, C., and Todd, D. 2006. Sub-clinical infection of farmed Atlantic salmon Salmo salar with salmonid alphavirus—a prospective longitudinal study. Dis. Aquat. Org. 72: 193–199. 185. Compton, J. 1991. Nucleic acid sequence based amplification. Nature 350: 91–92. 186. Starkey, W.G., Millar, R.M., Jenkins, M.E., Ireland, J.H., Muir, K.F., and Richards, R.H. 2004. Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA). Dis. Aquat. Org. 59: 93–100. 187. Starkey, W.G., Smail, D.A., Bleie, H., Muir, K., Ireland, J.H., and Richards, R.H. 2006. Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification. Dis. Aquat. Org. 72: 107–113. 188. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acid Res. 28: E63. 189. Savan, R., Kono, T., Itami, T., and Sakai, M. 2005. Loop-mediated isothermal amplification: An emerging technology for detection of fish and shellfish pathogens. J. Fish Dis. 28: 573–581. 190. Gunimaladevi, I., Kono, T., Venugopal, M.N., and Sakai, M. 2004. Detection of koi herpesvirus in common carp, Cyprinus carpio L., by loop-mediated isothermal amplification. J. Fish Dis. 27: 583–589. 191. Gunimaladevi, I., Kono, T., LaPatra, S.E., and Sakai, M. 2005. A loop mediated isothermal amplification (LAMP) method for detection of infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Onchorhynchus mykiss). Ach. Virol. 150: 899–909. 192. Shivappa, R.B., Savan, R., Kono, T., Sakai, M., Emmenegger, E., Kurath, G., and Levine, J.F. 2008. Detection of spring virevia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L. J. Fish Dis. 31: 249–258. 193. Soliman, H. and El-Matbouli, M. 2006. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet. Microbiol. 114: 205–213. 194. Kono, T., Savan, R., Sakai, M., and Irame, T. 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 115: 59–65. 195. Pillai, D., Bonami, J.-R., and Sri Widada, J. 2006. Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification. J. Fish Dis. 29: 275–283. 196. Nerette, P., Dohoo, I., and Hammell, L. 2005. Estimation of specificity and sensitivity of three diagnostic tests for infectious salmon anaemia virus in the absence of a gold standard. J. Fish Dis. Vol: 89–99. 197. Chomczynski, P. and Sachi, N. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal. Biochem. 162: 156–159. 198. Lopez-Lastra, M., Gonzalez, M., Jashes, M., and Sandino, A.M. 1994. A detection method for infectious pancreatic necrosis virus (IPNV) based on reverse transcription (RT)-polymerase chain reaction (PCR). J. Fish Dis. 17: 269–282. 199. Rimstad, E., Hornes, E., Olsvik, O., and Hyllseth, B. 1990. Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments. J. Clin. Microbiol. 28: 2275–2278. 200. Dopazo, C.P., De Blas, I., Miossec, L., Cameron, A.R., Vallejo, A., and Dalsgaard, I. 2006. Common errors in surveillance and monitoring programs on fish populations. In XI International Symposium on Veterinary Epidemiology and Economics. 6–11 August 2006, Cairns, Queensland, Australia. 201. Cunningham, C.O. and Hoffs, M.S. 2002. Development of a positive control for detection of infectious salmon anaemia virus (ISAV) by PCR. Bull. Eur. Assoc. Fish Pathol. 22: 212–217. 202. Devold, M., Krossoy, B., Aspehaug, V., and Nylund, A. 2000. Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection. Dis. Aquat. Org. 40: 9–18.
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203. Desselberger, U. 1995. Medical Virology: A Practical Approach. IRL Press, Oxford, U.K., p. 214. 204. López-Vázquez, C., Dopazo, C.P., Olveira, J.G., Barja, J.L., and Bandín, I. 2006. Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus. J. Virol. Methods 133: 167–17. 205. Williams, K., Blake, S., Sweeney, A., Singer, J.T., and Nicholson, B.L. 1999. Multiplex reverse transcriptase PCR assay for simultaneous detection of three fish viruses. J. Clin. Microbiol. 37: 4139–4141. 206. Wang, W.S., Lee, J.S., Blake, S.L., and Nicholson, B.L. 1995. Developing the polymerase chain reaction technique to detect aquatic birnaviruses. Taiwan J. Vet. Med. Anim. Husb. 65: 167–180. 207. Yoganandhan, K., Sri Widada, J., Bonami, J.R., and Sahul Hameed, A.S. 2005. Simultaneous detection of Macrobrachium rosenbergii nodavirus and extra small virus by a single tube, one-step multiplex RT-PCR assay. J. Fish Dis. 28: 65–69. 208. Nishizawa, T., Mori, K., Nakai, T., Furusawa, I., and Muroga, K. 1994. Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Dis. Aquat. Org. 18: 103–107. 209. Gilad, O., Yun, S., Andree, K.B., Adkison, M.A., Zlotkin, A., Bercovier, H., Eldar, A., and Hedrick, R.P. 2002. Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi. Dis. Aquat. Org. 48: 101–108. 210. Boyle, J. and Blackwell, J. 1991. Use of polymerase chain reaction to detect latent channel catfish virus. Am. J. Vet. Res. 52: 1965–1968. 211. Kim, C.K., Kim, P.K., Sohn, S.G., Sim, D.S., Park, M.A., Heo, M.S., Lee, T.H., Lee, J.D., Jun, H.K., and Jang, K.L. 1998. Development of a polymerase chain reaction (PCR) procedure for the detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimp. J. Fish Dis. 21: 11–17. 212. Thakur, P.C., Corsin, F., Turnbull, J.F., Shankar, K.M., Hao, N.V., Padiyar, P.A., Madhusudhan, M., Morgan, K.L., and Mohan, C.V. 2002. Estimation of prevalence of white spot syndrome virus (WSSV) by polymerase chain reaction in Penaeus monodon postlarvae at time of stocking in shrimp farms of Karnataka, India: A population-based study. Dis. Aquat. Org. 49: 235–243. 213. Yang, B., Song, X.-L., Huang, J., Shi, C.-Y., Liu, Q.-H., and Liu, L. 2006. A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp. J. Fish Dis. 29: 301–305. 214. Sambrook, J., Fritsch, E.F., and Maniatis, T. 2001. Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring, NY. 215. Seng, E.K., Fang, Q., Lam, T.J., and Sin, Y.M. 2004. Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR. J. Virol. Methods 118: 111–122. 216. Yuasa, K., Sano, M., Kurita, J., Ito, T., and Iida, T. 2005. Improvement of a PCR Method with the Sph I-5 primer set for the detection of koi herpesvirus (KHV). Fish Pathol. 40: 37–40. 217. Barlic-Maganja, D., Strancar, M., Hostnik, P., Jencic, V., and Grom, J. 2002. Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virus. J. Fish Dis. 25: 73–80. 218. Taksdal, T., Dannevig, B.H., and Rimstad, E. 2001. Detection of infectious pancreatic necrosis (IPN)-virus in experimentally infected Atlantic salmon parr by RT-PCR and cell culture isolation. Bull. Eur. Assoc. Fish Pathol. 21: 214–219. 219. Gagne, N., Johnson, S.C., Cook-Versloot, M., MacKinnon, A.M., and Olivier, G. 2004. Molecular detection and characterization of nodavirus in several marine fish species from the northeastern Atlantic. Dis. Aquat. Org. 62: 181–189. 220. Nylund, A., Devold, M., Mullins, J., and Plarre, H. 2002. Herring (Clupea harengus): A host for infectious salmon anemia virus. Bull. Eur. Assoc. Fish Pathol. 22: 311–318. 221. Miller, T.A., Rapp, J., Wastlhuber, U., Hoff mann, R.W., and Enzmann, P.-J. 1998. Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells. Dis. Aquat. Org. 34: 13–20.
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222. Goodwin, A.E., Khoo, L., LaPatra, S.E., Bonar, C., Key, D.W., Garner, M., Lee, M.V., and Hanson, L. 2006. Goldfish hematopoietic necrosis herpesvirus (Cyprinid Herpesvirus 2) in the USA: Molecular confirmation of isolates from diseased fish. J. Aquat. Anim. Health 18: 11–18. 223. Gilad, O., Yun, S., Zagmutt-Vergara, F.J., Leutenegger, C.M., Bercovier, H., and Hedrick, R.P. 2004. Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis. Aquat. Org. 60: 179–187. 224. Bandín, I. and Dopazo, C.P. 2006. Restocking of salmon in Galician rivers: A health management program to reduce risk of introduction of certain fish viruses. DIPNET Newslett 35. 225. Olveira, J.G., Soares, F., Engrola, L., Dopazo, C.P., and Bandín, I. 2008. Antemorten versus postmortem methods for detection of betanodavirus in Senegalese sole (Solea senegalensis). J. Vet. Diag. Invest. 20: 215–219. 226. Munro, E.S. and Ellis, A.E. 2008. A comparison between non-destructive and destructive testing of Atlantic salmon, Salmo salar L., broodfish for IPNV – destructive testing is still the best at time of maturation. J. Fish Dis. 31: 187–195. 227. Oshima, S., Hata, J.-I., Hirasawa, N., Ohtaka, T., Hirono, I., Aoki, T., and Yamashita, S. 1998. Rapid diagnosis of red sea bream iridovirus infection using the polymerase chain reaction. Dis. Aquat. Org. 32: 87–90. 228. Strommer, H.K. and Stone, D.M. 1998. Detection of viral hemorrhagic septicaemia (VHS) virus in fish tissues by semi-nested polymerase chain reaction (PCR). In: Barnes, A.C., Davidson, G.A., Hiney, M.P., and McIntosh, I. (Eds.), Methodology in Fish. Disease Research. Fisheries Research Services, Aberdeen. 229. Loevdal, T. and Enger, O. 2002. Detection of infectious salmon anemia virus in sea water by nested RT-PCR. Dis. Aquat. Org. 49: 123–128. 230. Grotmol, S., Nerland, A.H., Biering, E., Totland, G.K., and Nishizawa, T. 2000. Characterisation of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. Dis. Aquat. Org. 39: 79–88. 231. Juan, L., Tiehui, W., Yonglan, Y., Hanqin, L., Renhou, L., and Hongxi, C. 1997. A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction. Dis. Aquat. Org. 29: 7–12. 232. Gou, D.F., Kubota, H., Onuma, M., and Kodama, H. 1991. Detection of salmonid herpesvirus (Oncorhynchus masou virus) in fish by Southern-blot technique. J. Vet. Med. Sci. 53: 43–48. 233. Grizzle, J.M., Altinok, I., and Noyes, A.D. 2003. PCR method for detection of largemouth bass virus. Dis. Aquat. Org. 54: 29–33.
Chapter 33
Marine Toxins Cara Empey Campora and Yoshitsugi Hokama Contents 33.1 Introduction ..................................................................................................................650 33.2 Common Ichthyosarcotoxins ........................................................................................650 33.2.1 Ciguatoxin .......................................................................................................650 33.2.1.1 Overview .........................................................................................650 33.2.1.2 Clinical Symptoms...........................................................................651 33.2.1.3 Detection Methods ..........................................................................651 33.2.2 Tetrodotoxin ....................................................................................................653 33.2.2.1 Overview .........................................................................................653 33.2.2.2 Clinical Symptoms...........................................................................653 33.2.2.3 Detection Methods ..........................................................................654 33.3 Common Shellfish Toxins .............................................................................................655 33.3.1 Saxitoxin—Paralytic Shellfish Poisoning .........................................................655 33.3.1.1 Overview .........................................................................................655 33.3.1.2 Clinical Symptoms...........................................................................656 33.3.1.3 Detection Methods ..........................................................................656 33.3.2 Okadaic Acid—Diarrhetic Shellfish Poisoning ................................................658 33.3.2.1 Overview .........................................................................................658 33.3.2.2 Clinical Symptoms...........................................................................658 33.3.2.3 Detection Methods ..........................................................................658 33.3.3 Domoic Acid—Amnesic Shellfish Poisoning .................................................. 660 33.3.3.1 Overview ........................................................................................ 660 33.3.3.2 Clinical Symptoms.......................................................................... 660 33.3.3.3 Detection Methods ..........................................................................661
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33.3.4 Brevetoxin—Neurotoxic Shellfish Poisoning .................................................. 662 33.3.4.1 Overview ........................................................................................ 662 33.3.4.2 Clinical Symptoms.......................................................................... 662 33.3.4.3 Detection Methods ......................................................................... 663 33.4 Other Toxins ................................................................................................................ 664 33.4.1 Hepatotoxins—Microcystins .......................................................................... 664 33.4.1.1 Overview ........................................................................................ 664 33.4.1.2 Clinical Symptoms.......................................................................... 664 33.4.1.3 Detection Methods ..........................................................................665 33.4.2 New and Emerging Toxins ............................................................................. 666 33.4.2.1 Pinnatoxins ..................................................................................... 666 33.4.2.2 Azaspiracids .................................................................................... 666 33.4.2.3 Gymnodimine ................................................................................ 666 33.4.2.4 Spirolides ........................................................................................ 666 33.5 Conclusion ................................................................................................................... 666 References ............................................................................................................................... 667
33.1
Introduction
Fish poisoning dates back to antiquity. It was cited in Homer’s Odyssey in 800 BC and was observed during the time of Alexander the Great (356–323 BC) when armies were forbidden to eat fish in order to avoid the accompanying sickness and malaise that could threaten his conquests [1]. Marine toxins, often the cause of various seafood poisonings, arise naturally from marine algal sources and accumulate through the food chain, ultimately depositing in predator fish- or filter-feeding bivalves destined for mammalian consumption. Such seafood-borne diseases account for a large and growing proportion of all food poisoning incidents, and are associated with several acute and chronic diseases in humans worldwide, which are characterized by gastrointestinal, neurological, and/or cardiovascular disturbances that can persist or recur for many months. In this chapter, selected marine toxins originating from phytoplankton or their associated bacteria will be discussed in detail with respect to their general mechanisms of action, clinical symptoms, and available bioassay, immunoassay, and analytical detection methods.
33.2
Common Ichthyosarcotoxins
33.2.1 Ciguatoxin 33.2.1.1
Overview
Ciguatera fish poisoning was described as early as 1606 in the South Pacific island chain of New Hebrides [2]. A similar outbreak there and in nearby New Caledonia was reported by the famous English navigator Captain James Cook in 1774 [3], who described the clinical symptoms of his sick crew—symptoms that coincide with the clinical manifestations described today for ciguatera fish poisoning [4,5]. Representing a crude bioassay, viscera from the same fishes given to Cook’s crew were also given to pigs, causing their deaths [3].
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The term “ciguatera” originated in the Caribbean area to designate intoxication induced by the ingestion of the marine snail, Turbo livona pica (called cigua), as described by a Cuban ichthyologist. Today, it is widely used to denote the most commonly reported marine toxin disease in the world resulting from the ingestion of certain fishes, primarily reef fish, encountered in the islands of the Caribbean Sea, the Pacific and Indian Oceans, and other tropical and subtropical regions circumglobally between the Tropic of Cancer and the Tropic of Capricorn. Ciguatera fish poisoning affects between 50,000 and 500,000 people annually, and stems from the consumption of fish containing high levels of ciguatoxins (CTXs), a family of complex, lipid-soluble, highly oxygenated cyclic polyether compounds produced by the benthic marine dinoflagellate, Gambierdiscus toxicus. CTXs are small molecular weight toxins (∼1111 Da) with some 21 Pacific congeners varying in toxicity elucidated thus far [6–9]. They are biomagnified through the food chain, ultimately causing human and mammalian illness, as they are heat stable, colorless, odorless, and cannot be inactivated through cooking or freezing [7]. The CTXs are the most potent sodium channel toxins known, with the Pacific CTX-1 congener in mice having an intraperitoneal (IP) LD50 of 0.25 mg/kg [10]. CTXs and the closely related brevetoxins (a family of lipid-soluble polyether toxins produced by the marine dinoflagellate Karenia brevis, detailed in Section 33.3.4) are characterized by their ability to cause the persistent activation of voltage-sensitive sodium channels, leading to increased cell Na+ permeability. As a consequence, Na+-dependent mechanisms in numerous cell types are modified, leading to increased neuronal excitability and neurotransmitter release, impairment of synaptic vesicle recycling, and induced cell swelling [10].
33.2.1.2
Clinical Symptoms
CTXs cause gastrointestinal and neurological symptoms that typically persist for days to weeks, with common symptoms such as vomiting, diarrhea, nausea, abdominal pain, dysesthesia, pruritus, and myalgia. Severe cases of ciguatera may involve hypotension and bradycardia, although fatalities are rare. Neurological signs may persist for several months or even years [11]. Remarkably, the diagnosis of ciguatera is still largely dependent on the astuteness of the clinician. A history of recent consumption of potentially toxic fish, and at least one neurological sign and one other typical symptom are required to establish the clinical diagnosis. In the absence of a confirmatory laboratory test, a sizable proportion of cases still go undiagnosed and unreported. Treatment is largely empiric and symptomatic. In severe cases, supportive care, particularly monitoring fluid and electrolyte balance, is paramount, and local anesthetics and antidepressants may also be useful in some instances. Following its somewhat serendipitous use for a coma victim in the Marshall Islands who was later diagnosed with severe ciguatera, intravenous mannitol is now the mainstay of therapy [12]. Mannitol, however, is not universally beneficial, and is best when used during the acute phase of severe intoxications.
33.2.1.3
Detection Methods
33.2.1.3.1 Bioassays A commonly used method to detect CTXs involves the IP injection of mice with the crude extracts of fish [13,14]. Using estimates from known cases of ciguatera fishes obtained by the Hawaii Department of Health and other laboratories, it has been found that 1 mouse unit (MU) = 7–8 ng
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of CTX [15], which is equivalent to the concentration of toxic extract injected IP that kills a 20 g mouse within 24 h. The general protocol for testing crude fish extract is as follows: Swiss-Webster mice weighing 20–25 g are injected IP with 100 mg of crude fish extract resuspended in 1 mL of 1% Tween 60 in saline. Symptoms displayed by the mouse are observed from 0.5 to 48 h after injection and rated on a scale of 0–5 according to toxicity. Characteristic ionotropic responses to various toxin extracts including CTX have also been established using the guinea pig atrial assay, which involves specialized dissection techniques, requires a small amount of test material, and gives some measure of specificity, as the actions are at the sites of the sodium channel [16]. Other organisms, such as brine shrimp [17], mosquitoes [18,19], chickens [20], and dipteral larvae [21] have been used to screen for CTX, however, most have been found to be nonspecific, nonquantitative, and generally unreliable for routine screening. Directed cytotoxicity to the sodium channels of neuroblastoma cells has been established for purified CTXs, brevetoxins, saxitoxin (STX), and crude seafood extracts [22]. Using a microplate high-throughput format, this assay takes several days to complete and serves as a valuable tool for marine toxin studies, detecting CTX at subpicogram levels. A fluorescent-based assay detecting sodium channel activators has also been useful in the nonspecific analysis of crude extract [23], and recently, a rapid hemolysis assay based on the neuroblastoma cell bioassay using red cells from the red tilapia (Sarotherodon mossambicus) has been developed for the detection of sodium channel-specific marine toxins, including CTX [24].
33.2.1.3.2 Immunoassays The radioimmunoassay (RIA) [25] and membrane immunobead assay (MIA) [26] are advances in simple, rapid, sensitive, and specific qualitative detection methods for CTX. The MIA is a field usable assay that employs a monoclonal antibody to purified moray eel CTX-1 coated with polystyrene microbeads and a hydrophobic membrane laminated onto a solid plastic support. The membrane binds polyether lipids such as CTX and specifically detects the toxin using the monoclonal antibody to CTX coated with microbeads. The intensity of the color on the membrane correlates to the concentration of toxin on the solid support. This assay has a limit of detection at ∼0.032 ng CTX/g fish tissue and has a sensitivity of 91% and specificity of 87%. However, immunochemical methods are subject to cross-reactivity issues with other polyether compounds, and often there is a limited supply of antibody for use.
33.2.1.3.3 Chemical Methods Because CTX and brevetoxin share a common receptor at the sodium channel receptor site 5, the use of labeled brevetoxin (3H-PbTx-B) allows CTX to be quantified by competitive-binding assay with sodium channel containing proteins using isolated rat brain synaptosomes [27]. This method requires a small amount of fish extract, is rapid and simple, and has a high sensitivity. Best suited for research purposes, this method is likely impractical for large-scale fish screening because of specialized equipment and the use of radiolabeled compounds. Gradient reverse phase high-performance liquid chromatography/mass spectroscopy (HPLC/ MS), fast-atom bombardment tandem mass spectroscopy (MS/MS), and other chemical methods have recently been used to elucidate CTXs and their structures. While CTXs do not possess a useful chromophore for selective spectroscopic detection, they do contain a reactive primary hydroxyl group that can be labeled after a clean-up step. HPLC coupled to fluorescence detection
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has proven effective when screening for CTX in crude fish extracts [28,29]. HPLC with ionspray MS has shown promise as a confirmatory analytical assay for CTXs in fish flesh [30]. Nuclear magnetic resonance (NMR) has been used to characterize CTXs in fish flesh [31] and wild and cultured G. toxicus extracts [7,32], and Lewis and Jones [33] used gradient reverse phase LC/ MS methods to identify 11 new P-CTX congeners in a partially purified sample of toxic moray eel viscera. Similarly, LC–ESI-MS/MS (ESI = electrospray ionization) was reported to detect the levels of CTX equivalent to 40 ng/g P-CTX-1 and 100 ng/kg C-CTX-1 in fish flesh [34].
33.2.2
Tetrodotoxin
33.2.2.1
Overview
Tetrodotoxin (TTX), often referred to as puffer fish poisoning, is one of the most potent and common lethal marine poisonings. It occurs primarily in Southeast Asia where fugu (puffer fish fi llet) in Japan is considered a delicacy. It is one of the oldest known natural toxins, recorded as early as 2700 BC in Chinese literature describing the toxicity of the puffer [35]. Because there is no cure or antidote, the mortality rate is relatively high, although incidence is steadily declining due in part to increased government regulations and legislation regarding preparation and marketing of aquacultured nontoxic fish. According to the Japanese Ministry of Health and Welfare, there were ∼88 deaths due to TTX poisoning in 1965 compared to five deaths in 2001 [35]. TTX concentrates in the liver of bony fish in the order Tetraodontiformes, mainly from the family Tetraodontidae, which includes the puffer fish and toadfish. However, TTX has also been found in xanthid crabs, horse-shoe crabs and their eggs, the blue-ringed octopus, newts, and several other fish species such as marine gobies [36]. TTX has also been found in several bacterial species, including Shewanella sp. and Vibrio sp., and is believed to be bacterial in origin [35]. TTX is a water-soluble heterocyclic guanidine that blocks Na+ conductance over the single nanomolar range by binding extracellularly to receptor site 1 of voltage-gated sodium channels. This mechanism of action prevents the access of monovalent cations to the outer pore of the channel and primarily affects the control of peripheral nerve excitability by influencing the generation of action potentials and impulse conduction [37].
33.2.2.2
Clinical Symptoms
The type, the severity, and the range of symptoms of TTX poisoning are dependent on the amount of toxin ingested, and the age and the preexisting health of the victim. The minimum dose for developing TTX poisoning symptoms in humans is ∼2 mg of TTX. Early symptoms are sensory, including perioral and distal limb numbness and paresthesia, taste disturbances, dizziness, headache, diaphoresis, and other symptoms such as salivation, nausea, vomiting, diarrhea, and abdominal pain [35]. Mild poisoning cases might include several sensory features and minor gastrointestinal effects. Patients with moderate poisoning may develop distal muscle weakness, weakness of the bulbar and facial muscles, and ataxia and incoordination with normal reflexes [36]. Severe poisoning causes generalized flaccid paralysis, respiratory distress with possible eventual respiratory failure, extreme hypotension, seizures, and loss of deep tendon and spinal reflexes. Although some patients may exhibit impaired mental capabilities, most remain fully conscious for 6–24 h,
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after which the prognosis for recovery is good. Otherwise, death is caused by cardiovascular effects and ascending paralysis involving the respiratory muscles. The diagnosis of TTX poisoning is based on the clinical examination and the history of the consumption of toxic organisms. Because TTX may remain detectable and quantifiable in urine using HPLC up to 5 days following exposure [38], testing for exposure immediately after a suspected poisoning is likely to be the most sensitive method of determination. There are no known antidotes or antitoxins to TTX and therefore treatment involves careful observation and supportive care, including serial neurological assessments, and admission to intensive care units so that respiratory failure or cardiac effects are appropriately anticipated and treated. The case reports have suggested the use of neostigmine in an effort to reduce paresthesia and numbness [39,40], although other reports indicate that it has no effect on symptom improvement [41]. The prevention of TTX poisoning, with an emphasis on public education, is the primary method of avoiding illness.
33.2.2.3
Detection Methods
33.2.2.3.1 Bioassays The mouse bioassay is commonly used to determine the toxicity of TTX in a given sample, as well as the identification of unknown toxin extract when compared to a TTX-specific dose death time relationship curve. Although the mouse bioassay is the animal of choice for such determinations, drawbacks to the method include low accuracy due to inherent individual variation in a biological system, lack of specificity, and the inconvenience and controversy that often accompany the use of live animals for experimentation. Cell-based bioassays have been employed for the quantitative measurement of the sodium channel blocker TTX, even at low levels (∼3 nmol/L). This assay is based on the ability of sodium channel-blocking toxins to antagonize the combined effects of the chemicals veratridine and ouabain on neuroblastoma cell lines. While veratridine at 0.075 mmol/L and ouabain at 1.0 mmol/L cause the cells to round up and die, the presence of TTX counters this effect and the cells exhibit growth. The amount of toxin can then be estimated from the linear relationship of the relative abundance of living cells and the concentration of toxin in the samples. A modified assay that employs a water-soluble tetrazolium salt to quantitate the assay using a microplate reader streamlines the process [42,43]. The sensitivity of this method is much higher than that of the mouse bioassay, however, it is time consuming, requires laboratory expertise, and is not suitable for routine screening.
33.2.2.3.2 Immunoassays Several attempts to develop immunoassay techniques to detect TTX have been made in recent years with limited success [44–47]. However, recently a monoclonal antibody against TTX has been developed from Balb/c mice immunized with TTX-bovine serum albumin conjugate by which a rapid and highly sensitive enzyme immunoassay capable of monitoring seafood has been established for the quantitative analysis of TTX. It detects concentrations as low as 2–100 ng/mL in 30 min [48]. Using this highly specific monoclonal antibody, immunoaffinity column chromatography methods have also been developed for identification of TTX from the urine of poisoned patients, detecting as low as 2 ng/mL [49].
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33.2.2.3.3 Chemical Methods HPLC methods have been examined for both the qualitative and the quantitative analyses of TTX and its derivatives, including a fluorometric HPLC continuous analyzer first constructed in 1982 [50] and reconfigured in 1989 to improve the detection and the separation of TTX and TTX analogues including 6-epiTTX [51]. Reversed-phase HPLC is a fast and efficient method used by many researchers for analyzing TTX and its analogues by using heptanesulfonic acid as a counterion [52,53]. Methods such as thin layer chromatography (TLC) and electrophoresis are useful techniques for detecting the levels down to 2 mg of TTX in laboratories where HPLC and other costly analytical systems are not available [35]. Capillary isotachophoresis is also a rapid, accurate, and potential detection method for TTX, with a quantitative detection limit of ∼0.25 mg of TTX [54]. LC–MS is considered an accurate method of detecting TTX [55], combining an HPLC–MS equipped with a 1.5 × 150 mm column coupled to a mass spectrometer, using acetonitrile (50%, flow rate 70 mL/min) as the mobile solvent. This method has also shown promise in screening biological samples such as blood and urine at a detection limit of 12.5 nM, equivalent to about 3.9 ng/mL [56]. Several other methods including UV spectroscopy [57], gas chromatography–MS [58], infrared spectrometry [59], fast-atom bombardment MS [60], and ESI-time of flight/MS [61] have all been used in the determination of TTX and its derivatives, as well as 1H NMR spectrometry for determining absolute configurations [62].
33.3
Common Shellfish Toxins
Microscopic planktonic algae are critical food sources for filter-feeding bivalve shellfish such as oysters, mussels, scallops, and clams. It is not clear why some microalgal species produce toxins; however, during the past few decades the frequency, intensity, and geographic distribution of toxic compounds produced by marine algae have increased, contributing to the awareness of poisoning events from the ingestion of contaminated shellfish products. The four groups of shellfish toxins and their associated poisonings will be reviewed, namely: STX (paralytic shellfish poisoning [PSP]), okadaic acid (OA) (diarrhetic shellfish poisoning [DSP]), domoic acid (DA) (amnesic shellfish poisoning [ASP]), and brevetoxin (neurotoxic shellfish poisoning [NSP]).
33.3.1
Saxitoxin—Paralytic Shellfish Poisoning
33.3.1.1
Overview
The water-soluble STX and its derivatives, including the gonyautoxins (GNTXs), are responsible for PSP, and are accumulated from dinoflagellates from the genus Alexandrium as well as Pyrodinium bahamense and Gymnodinium catenatum by shellfish filter feeders, primarily mussels, oysters, and clams, and passed through the food chain to humans in tropical and moderate climate zones. The link between shellfish toxicity and dinoflagellates was first identified in the San Francisco Bay in 1927 [63,64] following an outbreak of PSP in the region. The PSP toxins behave pharmacologically similar to TTX in that they bind with nanomolar affinity to receptor site 1 on the sodium channel and are the reversible blockers of voltage-gated sodium channels. Structural differences between the various congeners of STX alters the rates at which they bind and release from the binding site on the sodium channel, and the lifetime of the open channel is reversibly correlated with toxin concentrations and association constants [65].
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Clinical Symptoms
The outbreaks of PSP occur periodically, attributed in part to poorly understand environmental changes that may be related to “red tides.” Individual sensitivity to the toxins determines the level at which PSP toxins cause illness; for example, oral intake causing mild symptoms ranges from 144 to 1660 mg of STX equivalents/person, and fatal intoxications were calculated ranging from 456 to 12,400 mg STX equivalents/person [66]. The fluctuation in the methods of determination and the reconstruction of STX values based on the remaining toxic food sources may contribute to the variations in toxicity reported. In cases of mild poisoning, clinical symptoms may include a tingling sensation or numbness around the lips within 30 min of ingestion, due to localized absorption of the toxins through the buccal mucous membranes. Gradually these symptoms spread to the face and neck, and prickly sensations in the fingertips and toes as well as headaches, dizziness, nausea, vomiting, and diarrhea are commonly observed. In cases of moderately severe poisoning, paresthesia progresses to the arms and legs, and incoherent speech, motor incoordination, and ataxia are frequent. In severe cases, respiratory difficulties including muscular paralysis are pronounced and death through respiratory paralysis may occur within 2–24 h of ingestion [65]. The overall mortality is reportedly between 1% and 10%, and appears to depend to some degree on medical care, age, and previous health status of the patient [67]. Supportive treatment generally resolves the symptoms, although several weeks or months may pass before the fatigue, tingling, or memory loss is completely resolved. Initial treatments may include gastric lavage to remove unabsorbed toxin, maintenance of adequate ventilation, and fluid therapy to correct acidosis and facilitate renal excretion of the water-soluble toxins. Animal studies have shown that 4-aminopyridine may be useful as a therapeutic antidote for STX intoxication by markedly improving the cardiorespiratory performances in rats and guinea pigs exposed to STX [68,69].
33.3.1.3
Detection Methods
33.3.1.3.1 Bioassays The detection of STXs is challenging because there are a large number of different but related causative compounds that can be encountered at low levels. The original bioassay for the STXs was a mouse bioassay based on the IP injection of mice [70] and is still in use as the current benchmark technique in food safety, although it cannot distinguish STX from TTX. The refined procedure, standardized by the Association of Official Analytical Chemists (AOAC), produces a rapid and reasonably accurate measurement of total PSP toxins [71]. In most countries, the action level for the closure of a fishery is 400 MU/100 g of shellfish, where 1 MU is defined as the amount of toxin that kills a 20 g mouse in 15 min by IP injection, equivalent to 0.18 mg of STX [72]. The limit of the detection of the assay is ∼40 mg STX/100 g shellfish tissue with a precision of ±15%–20% [66]. While alternative organisms have been sought, including houseflies and other insects, they have yet to show the same precision and efficiency as mammalian-based assays, and may be less accurate as the predictors of human oral potency. The drawbacks to the mouse bioassay include maintenance of mice colonies at specific weights, strains, and sizes, lack of linearity between the time of death and the toxin levels, time and labor-intensive procedures, and the use and the sacrifice of animals during the process. To reduce the number of mouse tests in several European countries, a qualitative technique that involves the direct monitoring of toxic algal cells in seawater is often used [73].
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A cell bioassay modified by Jellet et al. [74] incorporates the use of an automated microplate reader using mouse neuroblastoma cells, which swell and lyse in the presence of ouabain and veratridine by enhancing sodium ion influx. The addition of STX will block the sodium channel and the cells will remain morphologically normal, and changes can be detected using the absorption of stained cells. This method has a detection limit of about 10 ng STX equivalents/mL of extract, or 2.0 mg STX equivalents/100 g shellfish tissue. This method is a promising screening tool; however, it is recommended that any results measuring close to regulatory limits be reevaluated using another method to confirm. By sequencing, the addition of veratridine, ouabain, and extracted samples to neuroblastoma cells, a hemolysis assay [24] recently developed reportedly detects STXs in concentrations at 0.3 mg/mL, although its value as a practical shellfish-screening tool has not yet been evaluated.
33.3.1.3.2 Immunoassays Indirect enzyme-linked immunoassays (ELISAs) have been developed and are commercially available for the detection of STX [75] and more recently adapted to detect STX derivatives including neoSTX, GNTX1, and GNTX3 [76]. Such methods appear to be more sensitive than LC and more specific than the mouse bioassay. In addition to indirect ELISAs, direct competitive ELISAs have also been available for the detection of STX and derivatives [77,78] and show excellent correlation between the ELISA data and the mouse assay results, often detectable at concentrations lower than the regulatory limits. However, ELISAs are prone to cross-reactivity, and the difficulty of adequately detecting STX derivatives at low levels limits the use of ELISA as a means of regulating shellfish for PSP toxins.
33.3.1.3.3 Chemical Methods The alkaline oxidation of PSP toxins yields fluorescent products, allowing determination using fluorometric techniques [79,80] and has given way to the development of a fluorescent sensor that is reportedly selective for STX and not TTX using acridinyl crowns [81]. LC techniques are the most widely used nonbioassay methods for PSP compound determination and are generally based on the separation of toxins by ion-interaction chromatography and use of a postcolumn reactor that oxidizes the column effluent to produce readily detectable derivatives. The methodology developed by the United States Food and Drug Administration was reported to resolve 12 carbamate and sulfocarbamoyl PSP toxins at detection limits with an order of magnitude lower than that of the mouse bioassay, and validation against the mouse bioassay showed good correlation between the two methods at r > 0.9 [82]. However, in practice, this method has shown some difficulties in separating STX from the derivative dcSTX and has gone out of use in many European laboratories screening for PSP toxins [83]. Though LC methods are promising, operating such a system requires a considerable amount of skill and time, and may not be robust enough to handle the large numbers of samples that are necessary for screening during a bloom event [84]. MS, specifically LC–MS, has been used for qualitative determination of STX with detection limits five times lower than that of the mouse bioassay [85–87] and variations in the methods have shown promise in confirming accumulation of PSP toxins in mussel and shellfish samples.
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33.3.2
Okadaic Acid—Diarrhetic Shellfish Poisoning
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33.3.2.1
Overview
DSP is a toxic syndrome that is caused by the consumption of shellfish that has been contaminated with algal toxins produced by marine dinoflagellates belonging to the generas Dinophysis spp. and Prorocentrum spp. The DSP toxins, which are heat stable polyether lipophilic compounds, can be grouped into three categories based on their unique chemical structures. The first group are acidic toxins including OA and related dinophysistoxin (DTX) derivatives, and are potent phosphate inhibitors, which can cause inflammation of the intestinal tract and diarrhea in humans [88]. The second group is neutral polyether–lactones of the pectenotoxins (PTXs), 10 of which have been isolated. The third group are sulfated polyether compounds called yessotoxins (YTXs), and their derivative 45-hydroxyyessotoxin (45-OH-YTX) [89,90]. Interestingly, the YTXs do not cause diarrhea, but rather attack the cardiac muscle in mice after IP injection, while the desulfated YTX damages the liver [90]. The reevaluation of their toxicities may lead to the removal of these toxins from classification as a DSP toxin, although they currently remain as such [91]. OA is a potent inhibitor of the serine/threonine phosphatases PP1 and PP2A. Because these phosphatases are enzymes responsible for phosphorylation and dephosphorylation of proteins associated with critical metabolic processes within a cell, their dysregulation leads to the specific symptoms associated with DSP. It is suggested that diarrhea in humans is caused by the hyperphosphorylation of proteins that control sodium secretion by intestinal cells or by the increased phosphorylation of junctional moieties that regulate solute permeability, resulting in the passive loss of fluids [90,92].
33.3.2.2
Clinical Symptoms
The clinical symptoms vary depending on the DSP toxin and the intensity depends on the amount of toxin ingested. While rarely fatal, the predominant symptoms from OA and DTX include diarrhea, nausea, vomiting, and abdominal pain within 30 min to several hours after ingestion, and complete recovery is expected within 3 days generally without hospitalization. Intravenous injection of an electrolyte can assist in ameliorating the symptoms. The data indicates that the minimum dose to induce toxic effects in humans is 48 mg of OA and 38.4 mg of DTX1 [70]. The primary clinical result from the ingestion of PTXs is liver necrosis [93], while YTXs can cause cardiac muscle damage when administered intraperitoneally in mice [94]. The prevention of exposure is enforced in many countries including the frequent inspection of seawater around aquaculture facilities and monitoring programs that keep records on the occurrence of toxic phytoplankton and the closures of harvesting areas when toxic algae levels are high. In Europe, the maximum level of OA, DTXs, and PTXs together in edible tissues of molluscs, echinoderms, tunicates, and marine gastropods are 160 mg OA equivalents/kg of shellfish meat, while YTX levels are 1 mg YTX equivalents/kg of meat [95]. Shellfish containing more than 2 mg OA/g hepatopancreas and/or more than 1.8 mg DTX/g hepatopancreas are considered unsafe for human consumption [92].
33.3.2.3
Detection Methods
33.3.2.3.1 Bioassays The mouse bioassay is a preferred method of analysis for DSP toxins in Europe and Japan, although complementary chemical or immunological analyses may accompany the evaluation [66], and is
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the officially recognized regulatory method for detection in the European Union (EU) [96]. The mouse bioassay, first developed by Yasumoto et al. [97], involves the extraction of shellfish tissues using acetone, followed by IP injection into a 20 g mouse and survival monitoring for 24–48 h. The toxicity of the sample, expressed in MU/g of whole tissue, is determined as the minimum quantity of toxin capable of killing a 20 g mouse within 24 h after IP injection. In many countries, the regulatory level is set at 0.05 MU/g whole tissue. The disadvantages to this assay include: lack of specificity in that there is no differentiation between the various components of DSP toxins or unknown toxic groups exhibiting ichthyotoxic and hemolytic properties, subjectivity to the time of death in the animals, and the need for routine maintenance of laboratory animals. In addition, the selectivity, specificity, and toxin recovery depend greatly on the selection, the purity, and the ratios of the organic solvents used in the extraction and the clean up. A semiquantitative method for OA and DTX toxin evaluation is a rat bioassay in which animals are starved, then fed suspect shellfish tissue and observed for signs of diarrhea, fecal consistency, and food refusal. However, this method, officially allowed in the EU, does not detect PTXs and YTXs. An inexpensive, sensitive method for screening OA and some coextracting toxins is a bioassay using small planktonic crustaceans, Daphnia magna, and has been reported to measure OA levels 10 times below the threshold of the mouse bioassay method [98]. Cytotoxicity assays using rat hepatocytes and KB cells (a human cell line derived from epidermoid carcinoma) have shown promise for detecting some DSP toxins. The hepatocyte assay is based on morphological changes in the cell, and can differentiate between the diarrhetic DSP toxins and the nondiarrhetic toxins [99]. OA and PTX appear to have a high toxicity on KB cell lines, and thus several different assays using the cell line have been developed using various methods [100].
33.3.2.3.2 Immunoassays A variety of ELISA kits are commercially available to detect DSP toxins, including the DSPCheck® ELISA test kit (UBE Industries, Japan), used to screen OA and DTX1 at a claimed detection limit of 20 ng/g. While reports about its performance vary, it appears to be more sensitive and specific than LC. The Rougier Bio-Tech® ELISA kit (Montreal, Canada) has undergone extensive comparisons using analytic methods for DSP toxin detection and has been found to be reliable for OA quantification in both mussel and phytoplankton extracts [92]. A direct ELISA developed by Biosense® (Bergen, Norway) for YTX is still being evaluated for efficacy in detecting YTX and its analogues. Immuno biosensors, which are defined as “a self-consistent bioanalytical device incorporating a biologically active material, either connected to, or integrated within, an appropriate physicochemical transducer, for the purpose of detecting-reversibly and selectively—the concentration or activity of chemical species in any type of sample [101],” have been applied in the development of sensors for DSP toxins. A semiautomated chemiluminescent immunosensor for OA in mussels has already been described [102], and it is expected that such technology will further advance in the coming years. The phosphate inhibition bioassays using colorimetric or fluorometric detection are capable of the quantitative measurement of OA and have been shown to be rapid, accurate, specific, and simple procedures for detecting OA in buffered or complex solutions [103–105].
33.3.2.3.3 Chemical Methods Chromatography methods are often used to assess DSP toxins. TLC offers a fairly simple method of assessing the acidic DSP toxins at levels of ∼1–3 mg of toxin [92], however, these high
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detection limits can be a limiting factor in the use of TLC. LC methods are commonly used for the determination of OA and DTX1. The original method involves sequential extraction of shellfish tissue with methanol, ether, and chloroform, derivatization with 9-anthryldiazomethane, silica Sep-pak clean up, and determination by HPLC with fluorescence detection [106]. Permutations to the original method have been made to streamline the analysis, including use of various solvents [107], changes to the derivatization reagents including coumarin, luminarine-3, and 9-chloromethylanthracene [108], and adapting the analysis to include the determination of YTXs and PTXs using fluorescent labeling [109,110]. LC combined with ESI-MS can achieve a detection limit of 1 ng/g shellfish tissue, resulting in a fast, sensitive technique for determination of DSP toxins even when analytical standards are not readily available. The interlaboratory studies of a new LC–MS method for the determination of ASP and DSP toxins have obtained consistent results and represents an encouraging alternative to the mouse bioassay [111].
33.3.3 Domoic Acid—Amnesic Shellfish Poisoning 33.3.3.1
Overview
First discovered in Prince Edward Island, Canada in 1987, amnesic or encephalopathic shellfish poisoning (ASP) primarily affects the central nervous system, leading to severe memory loss and confusion. The causative toxin, DA, is a heat stable, water soluble, neuroexcitatory amino acid that acts like the neurotransmitter glutamic acid and is produced by diatoms from the genus Pseudonitzschia. Specifically, Pseudonitzschia pungens f. multiseries, P. australis, and P. pseudodelicataissima have been implicated in human and bird intoxications [112,113]. Until the toxic event in Canada, it was thought that phycotoxins were only produced by dinoflagellates, and diatoms were not considered a potential source of toxins. Cultured blue mussels, soft-shelled clams, razor clams, and some species of scallops have all been shown to potentially contain DA in Canada and from the California coast up to Washington in the United States. In addition, DA was found in the viscera of Dungeness crabs from Oregon and Washington, and some species of anchovies and mackerel have been found to be contaminated with DA after sea lions and water birds died in Central and Northern California and Baja Mexico after ingestion of these fish. DA is an agonist of the glutamate receptor [114], and binds with high affinity to the glutamate receptors of the quisqualate type, which are targets for neurotransmitters. The receptor serves to conduct Na+ ion channels in the postsynaptic membrane; DA acts to open these Na+ channels, leading to Na+ influx which induces depolarization, with the resulting increased influx of Ca 2+ ions leading to cell death. DA is about 100 times more potent than glutamate [66].
33.3.3.2
Clinical Symptoms
The diagnosis of ASP is difficult because there have only been a few outbreaks reported, however, a combination of gastrointestinal and neurological features, particularly memory loss and confusion after ingestion of shellfish appear to be common. In the Canadian outbreak, 107 patients were reported to have an acute illness after the ingestion of mussels contaminated with DA [115,116]. Patients presented with gastrointestinal symptoms ∼5.5 h after ingestion, including vomiting, abdominal cramps, and diarrhea. Unusual neurological features developed after 48 h including headache, confusion, disorientation, and short-term memory loss correlated with age, mutism,
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seizures, disordered eye movements, myoclonus, and coma. Hemodynamic instability, cardiac arrhythmias, and respiratory secretions were also noted [116]. Four patients died and 14 were severely affected with ongoing neurological abnormalities, while the remaining patients recovered fully. Treatment is supportive, and symptomatic and/or neurological dysfunction should be carefully monitored and treated accordingly.
33.3.3.3
Detection Methods
33.3.3.3.1 Bioassays The AOAC approved mouse bioassay for PSP toxins [117] can also be used to detect DA at the concentrations of ∼40 mg/g of tissue because the symptoms of ASP in mice are distinguishable from the classic PSP symptoms. The typical sign of DA presence in an extract is a unique scratching of the mouse shoulder by the hind leg, followed by convulsions over an observation period of 4 h. The common regulatory limit for DA is 20 mg DA/g of mussel tissue, and as such the mouse bioassay is not sensitive enough to quantify the toxin for routine screenings. In vitro assays for detecting the toxin include a competitive receptor-binding assay in which frog (Rana pipiens) brain synaptosomes are used and assayed based on binding competition with radiolabeled kainic acid for the kainite/quisqualate glutamate receptor. This assay was further optimized [118] to use a cloned rat GLUR6 glutamate receptor and is suitable for the analysis of DA in seawater extracts from algae and shellfish tissue.
33.3.3.3.2 Immunoassays An ELISA for DA determination in mussel extracts measuring total DA content including a diastereoisomer and at least two cis–trans isomers was developed in 1995 [119] using a polyclonal antiserum raised in mice against an ovalbumin–DA conjugate. A limit of detection was found to be 0.25 mg/mL of extract, representing 0.5 mg DA/g of extracted mussel tissue [119]. The routine monitoring of DA levels in cultured bivalve molluscs can be accomplished through a commercial indirect ELISA originally developed in 1998 [120] where the limit of quantitation is 10 mg/ kg shellfish. According to the manufacturer (Biosense®, Bergen, Norway), method validation between reference laboratories in Scotland, Chile, and New Zealand yielded excellent results. New antibody-based approaches involve the use of biosensors [121] wherein DA is bound to the surface of a sensor and detected with polyclonal antibodies raised to DA–human serum albumin conjugates with the promising limits of detection. It is expected that biosensor technology will become more refined and effective for use in regulatory situations in the near future.
33.3.3.3.3 Chemical Methods DA can be determined by TLC as a weak UV-quenching spot that stains yellow following treatment with 1% ninhydrin [122], although normal amino acids present in crude extracts have the potential to interfere, thus a separation step is required. A clean up procedure using strong anion change solid phase extraction, or SAX-SPE, yields fractions that can be used directly in onedimensional TLC. The detection limit of DA using TLC is ∼10 mg/g in shellfish tissues and is a useful tool, particularly as a secondary screen following immunoassay detection, or for laboratories that do not have LC available for use.
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The liquid and/or ion exchange chromatography can analyze and preparatively isolate DA. Reverse phased LC–UV gives the fastest and the most efficient separations, and has become a preferred analytical technique for the determination of DA in shellfish following an AOAC collaborative study [123]. The detection limit using this method is about 10–80 ng/mL, depending on the sensitivity of the UV detector used. When crude extracts are analyzed without clean-up, the practical limit of quantitation is ∼1 mg/g [124], which is suitable for regulatory laboratories concerned with detecting contamination at levels greater than 20 mg/g. The use of fluorescent derivatives can detect DA as low as 15 pg/mL in marine matrices such as seawater and phytoplankton as well as shellfish extracts [125,126]. In addition to other methods, capillary electrophoresis is a relatively simple method that allows for rapid, high-resolution separations and gives comparable precision and accuracy rates when compared with LC. Electrospray is a technique used to interface LC with MS [127–129]. The interlaboratory studies of the LC–MS method for the determination of ASP toxins in shellfish have been performed and yielded consistent sets of data, and were shown to be a viable alternative to mouse bioassay [111]. The certified materials including a DA calibration solution and a mussel tissue reference material have been developed for ASP to aid in analytical quality assurance through the Certified Reference Materials Programme of the National Research Council, Canada [130].
33.3.4 33.3.4.1
Brevetoxin—Neurotoxic Shellfish Poisoning Overview
The first documented event of a “red tide” dinoflagellate bloom of K. brevis (also known as Gymnodinium breve and Ptychodiscus breve) was over 100 years ago. Since that time, scientific interest in the mammalian intoxications, massive fish, and bird kills that result from such blooms along the Gulf coast of the United States and in other parts of the world has increased and resulted in advanced research. An unusual feature of K. brevis is the formation of toxic aerosols through wave action that can lead to asthma-like symptoms in humans. NSP is caused by brevetoxins (PbTxs), which are tasteless, odorless, heat and acid stable, lipid-soluble, and cyclic polyethers. The molecular structure of the brevetoxins consists of 10–11 transfused rings; their molecular weights are around 900 Da, and 10 brevetoxins have been isolated and identified from field blooms and K. brevis cultures [131]. The two major brevetoxins, PbTx-2 and PbTx-3, have been shown to act on receptor site 5 of the voltage-sensitive sodium channel where they bind and cause persistent activation, increased sodium flux, and subsequent depolarization of excitable cells at resting potential.
33.3.4.2
Clinical Symptoms
The toxic effects of brevetoxin can be passed through inhalation and the dermal exposure of aerosolized dinoflagellate particles, and the oral ingestion of raw or cooked shellfish contaminated with brevetoxins. Dermal exposure occurs when the fragile K. brevis is broken open during rough surf, releasing the toxins that can cause irritation of the eye and the nasal membranes of the swimmers or those in direct contact with toxic blooms [132,133]. In addition to skin irritations, inhalation of aerosolized red tide brevetoxins may cause respiratory distress, conjunctival irritations, rhinorrhea, nonproductive cough, and bronchoconstriction. Other symptoms such as dizziness, tunnel vision, and skin rashes are also common. The condition is readily reversible in most individuals once they leave the affected area, however, those with asthma or chronic lung conditions have reported more difficulties including prolonged lung disease as a result of exposure
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[133,134]. Brevetoxin is thought to cause chronic immunosuppression, possibly mediated through interactions with cysteine cathepsins that are naturally present in immune cells and involved in antigen presentation [135]. The oral ingestion of contaminated shellfish induces a toxic syndrome similar to PSP and ciguatera fish poisoning, although with a lesser degree of severity. The symptoms of brevetoxin through ingestion generally appear within 30 min to 3 h of exposure and may include nausea, vomiting, diarrhea, chills, sweats, reversal of temperature sensation, hypotension, numbness, tingling, paresthesias of lips, face, and extremities, bronchoconstriction, paralysis, and even coma. Fatalities are extremely rare, chronic symptoms as a result of ingestion have not been reported, and treatment is primarily supportive.
33.3.4.3
Detection Methods
33.3.4.3.1 Bioassays The mouse bioassay involves the IP injection of a crude lipid obtained from a diethylether extraction of shellfish into mice weighing 20 g where 1 MU is defined as the amount of crude toxic residue that on an average will kill 50% of the test animals in 930 min. In practice, a residue toxicity of 20 MU per 100 g shellfish tissue was adopted, and remains as the guidance level for the prohibition of shellfish harvesting [136]. The drawbacks to the mouse assay are that it requires large numbers of animals, uses relatively large amounts of tissue extracts, the results are interpreted subjectively, and it lacks specificity [137]. Mosquito fish (Gambusia affinis) bioassays can be conducted in 20 mL seawater (3.5% salinity) using one fish per vessel with toxin added in 0.01 mL ethanol and median lethal doses determined using the tables in Weil from 1952 [138]. The fish bioassay is generally used to determine the potency of either the contaminated seawater or crude and purified toxin extracts [139]. A neuroblastoma cell assay takes advantage of the toxic effects of NSPs and their affinity for voltage-sensitive Na+ channels. Using this method, the detection limit for PbTxs is 0.25 ng/10 mL tissue extract and can be detected within 4–6 h, though the detection limit can be decreased with an incubation time of 22 h [140]. The detection is based on functional activity rather than on the recognition of a structural component, as is the case of an antibody-based assay and the affinity of a toxin for its receptor is directly proportional to its toxic potency, which can affect the specificity and the sensitivity of this assay. Fairey et al. [141] reported a further modification of the receptor-binding assay in neuroblastoma cells to a reporter gene assay that utilizes luciferase-catalyzed light generation as an endpoint and a microplate luminometer for quantification. The results indicated that the assay was capable of meeting or exceeding the sensitivity of bioassays for sodium channel active algal toxins. Van Dolah et al. [142] developed a high-throughput synaptosome-binding assay for brevetoxins using microplate scintillation technology. The microplate assay can be completed within 3 h, has a detection limit of less than 1 ng and can analyze dozens of samples simultaneously. The assay has been demonstrated to be useful for assessing algal toxicity, for purification of brevetoxins, and for the detection of brevetoxins in seafood.
33.3.4.3.2 Immunoassays A competitive RIA was developed for the detection of PbTx-2 and PbTx-3 at 1 nM concentrations [143], and ELISA methods for brevetoxin detection have since ensued. The modifications to early
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ELISA methods have resulted in improved detection and specificity to where the method can be used to screen for brevetoxins in dinoflagellate cells, in shellfish and fish seafood samples, in seawater and culture media, and in human serum samples [144–147] with detection limits ranging from 0.33 pmol for PbTx-3 to 2.5 mg/100 g shellfish meat in spiked oysters.
33.3.4.3.3 Chemical Methods Using micellar electrokinetic capillary chromatography, brevetoxins were isolated from cell cultures and fish tissue and the method detection limit in fish tissue was ∼4 pg/g [148]. The reversedphase LC–ESI-MS was successfully applied to the separation and the identification of brevetoxins associated with red tide algae [149], and an ionspray LC–MS method was shown to have mass detection limits as low as 10 pg (10 fmol) when using the selected ion monitoring of the (M + H)+ ions. The analyses by LC–MS can be very rapid (as low as 2 min in some cases) and can be completely automated [150]. A fish tissue procedure based on gradient reversed-phase LC/MS/MS was used for the detection of PbTx-2 in fish tissue, and the detection limit in fish flesh using this method was at least 0.2 ng/g [34].
33.4
Other Toxins
33.4.1 Hepatotoxins—Microcystins 33.4.1.1
Overview
Cyanobacteria, also known as blue-green algae, are Gram-negative photosynthetic prokaryotes that can be found in both terrestrial and aquatic habitats, generally preferring temperatures between 20°C and 25°C [151]. Toxins produced by cyanobacteria differ according to their toxicological properties and chemical structures, which include hepatotoxic cyclic peptides such as microcystins and nodularins, neurotoxic alkaloids, and lipopolysaccharides. Cyanobacterial genera that produce microcystins include Microcystis, Planktothrix (Oscillatoria), Anabaena, Nostoc, Anabaenopsis, and Hapalosiphon [152], while nodularins are produced by Nodularia spumigena, a brackish water cyanobacterium [153]. Currently there are more than 60 variants of microcystin, which differ in toxicity [154,155], however, microcystin-LR is considered the most common in cyanobacteria. Nodularins are structurally similar to microcystins and exert similar toxicities. Microcystins contain five invariant amino acids, namely, d-alanine, d-methylaspartic acid, adda, d-glutamic acid, and N-methyldehydroalanine, and two variant L-amino acids. The “adda” amino acid (3-amino-9-methoxy-2,6,8-trimethyl-10phenyl-4,6-dienoic acid) contributes to the toxicity of the microcystins and the nodularins by inhibiting several eukaryotic processes such as growth, protein synthesis, glycogen metabolism, and muscle contraction, and provides the microcystins with a characteristic absorption wavelength at 238 nm due to the presence of a conjugated diene group in the long carbon chain. This absorption provides a means of analysis after separation using reverse phase chromatography [155].
33.4.1.2
Clinical Symptoms
Human exposure to cyanobacterial toxins is mainly through ingestion and direct contact with contaminated waters. In the case of ingestion, drinking water contaminated with toxic blue-green
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algae has been reported, as has the consumption of fish and blue-green algal products used as food supplements. Swimming in waters where toxic blooms are occurring can lead to dermatitis and gastrointestinal symptoms such as vomiting and diarrhea [154]. Hepatotoxins induce massive hemorrhages, hepatocyte necrosis, disruption of mammalian liver systems, tumor promotion, and adverse kidney effects. Apoptotic and morphological changes have been observed at the cellular level including cell shrinkages, chromosomal breakage, and organelle redistribution.
33.4.1.3
Detection Methods
33.4.1.3.1 Bioassays Bioassays involving mice, Artemia salina, Sinapis alba seedlings, and animal cell lines offer simple and rapid screening for microcystins [156], however these methods often lack the specificity necessary for adequate detection and validation. Protein phosphatase inhibition assays (PPIAs) can be radioisotopic and colorimetric, and have been developed based on the ability of microcystins to inhibit serine–threonine protein phosphatase enzymes [157–159]. The detection has also been reported using bioluminescence and fluorogenic substrates [160,161].
33.4.1.3.2 Immunoassays A specific, sensitive ELISA has been developed using either polyclonal [162] or monoclonal antibodies [163,164] and while this and the PPIA detect microcystins that are below the guideline levels of the World Health Organization (WHO), which recommends that drinking water should have less than 1 mg/L, there are compatibility issues including cross-reactivity of the antibodies with variants, and underlying phosphatase activity in the sample preparation that masks the effects of the toxin.
33.4.1.3.3 Chemical Methods HPLC retrofitted with UV detection or MS is a powerful tool for the identification of microcystins, capable of providing both quantitative and qualitative data [165]. In general, microcystins are separated on C18 silica column using a gradient of water and acetonitrile, acidified with trifluoroacetic acid or formic acid. Microcystins have characteristic spectra with absorption maxima at either 238 nm due to the “adda” residue, or at 222 nm for microcystins containing tryptophan [166]. One issue in the quantitative analysis of microcystins is the lack of suitable standards, as there are some 60 microcystin variants. In the absence of such standards, variants are often expressed as equivalents of microcystin-LR [167–169]. The HPLC–MS is widely accepted for the qualitative analysis of microcystins of interest. In this method, molecules are converted to desolvated ions, which are resolved based on mass and charge [170]. Other related methods including ESI and MS/MS have been utilized with success. More time-consuming, less specific methods include TLC, gas chromatography–MS, and capillary zone electrophoresis. Novel approaches for the environmental monitoring of cyanobacterial blooms are developing with the advent of DNA sequencing and polymerase chain reaction. Such sequencing has led to the coding of microcystin genes in several major producers and has enabled the design of primers and probes to specifically detect and identify toxin-producing species in natural samples with low
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quantities [171,172]. Obstacles to water resource management include the inability to differentiate between toxic and nontoxic cyanobacterial blooms without isolation and testing, as neither strain shows a measurable difference in appearance.
33.4.2 New and Emerging Toxins 33.4.2.1
Pinnatoxins
Pinnatoxins are potent marine toxins common to the bivalve from genus Pinna, common in China and Japan where human intoxication is a regular occurrence [173]. Symptoms include diarrhea and neurological disturbances, and the toxin is thought to be a Ca 2+ channel activator.
33.4.2.2
Azaspiracids
Azaspiracids, first found in mussels after a toxic incident in the Netherlands, exhibit the symptoms typical of DSP, including nausea, vomiting, diarrhea, and abdominal cramps. However, structural and toxicological studies show that the target organs and the mode of action are distinctly different from those of DSP, PSP, and ASP toxins [174,175].
33.4.2.3
Gymnodimine
Oysters from South Island, New Zealand in 1994 were found to contain a potent compound whose causative organism is Gymnodinium sp. This toxin exhibits potent mouse and ichthyotoxicity, with mice dying within 5–15 min following a minimum lethal dose of 450 mg/kg and fish at levels of 250–500 ppb. The structure of the toxin has been resolved through NMR [176].
33.4.2.4
Spirolides
Spirolides were isolated from the digestive glands of shellfish collected near Nova Scotia, Canada and possess an unusual seven-membered cyclic imine moiety that is spirolinked to a cyclohexane ring. The macrocyclic toxins may activate Ca 2+ channels [176].
33.5
Conclusion
Many varied dynamics characterize the field of algal toxins, posing a challenge for biologists, toxicologists, biochemists, and pharmacologists interested in elucidating the molecular mechanisms and developing more sophisticated detection methods for such toxins. While there have been major advancements in this field in recent years, the increasing incidence of marine toxin poisonings worldwide as well as the continual discovery of new toxins demonstrate the need for the development of additional tools for biotoxin monitoring in seafood intended for mammalian consumption. Analytical methods that could allow for the accurate estimates of the toxicity of the multiple classes of toxins using a single procedure would be ideal in managing the risks posed by phycotoxins. While such a global approach does not appear likely in the near future, continued efforts toward more rapid, sensitive, specific, and accurate testing methodologies will be encouraged in an effort to monitor marine toxins in the environment.
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96. Mouratidou, T. et al., Detection of marine toxin okadaic acid in mussels during a diarrhetic shellfish poisoning (DSP) episode in Thermaikos Gulf, Greece, using biological, chemical and immunological methods, Sci. Tot. Environ., 366, 894, 2006. 97. Yasumoto, T., Oshima, Y., and Yamaguchi, M., Occurrence of a new type of shellfish poisoning in the Tokohu District, Bull. Jpn. Soc. Sci. Fish., 44, 1249, 1978. 98. Vernoux, J.P. et al., The use of Daphnia magna for detection of okadaic acid in mussel extracts, Food Add. Contam., 10, 603, 1993. 99. Aune, T., Yasumoto, T., and Engeland, E., Light and scanning electron microscopic studies on effects of marine algal toxins toward freshly prepared hepatocytes, J. Toxicol. Environ. Health, 34, 1, 1991. 100. Amzil, Z. et al., Short-time cytotoxicity of mussel extracts: A new bioassay for okadaic acid detection, Toxicon, 30, 1419, 1992. 101. Botrè, F. and Mazzei, F., Inhibition enzymic biosensors: An alternative to global toxicity bioassays for the rapid determination of phycotoxins, Int. J. Environ. Pollut., 13, 173, 2000. 102. Marquette, C.A., Coulet, P.R., and Blum, L.J., Semi-automated membrane based chemiluminiscent immunosensor for flow injection analysis of okadaic acid in mussels, Anal. Chim. Acta, 398, 173, 1999. 103. Simon, J.F. and Vernoux, J.P., Highly sensitive assay of okadaic acid using protein phosphatase and paranitrophenyl phosphate, Nat. Toxins, 2, 293, 1994. 104. Tubaro, A. et al., A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels, Toxicon, 34, 743, 1996. 105. Vieytes, M.R. et al., A fluorescent microplate assay for diarrheic shellfish toxins, Anal. Biochem., 248, 258, 1997. 106. Lee, J.S. et al., Fluorimetric determination of diarrhetic shellfish toxins by high-performance liquid chromatography, Agric. Biol. Chem., 51, 877, 1987. 107. Aase, B. and Rogstad, A., Optimization of sample clean-up procedure for determination of diarrhetic shellfish poisoning toxins by use of experimental design, J. Chromatogr. A., 764, 223, 1997. 108. GFL, Determination of Okadasäure in mussels with HPLC (L 12.03/04-2), in Official Collection of Methods under Article 35 of the German Federal Act; Methods of Sampling and Analysis of Foods, Tobacco Products, Cosmetics and Commodity Goods/BgVV, vol. 1., Köln, Beuth Verlag GmbH, Berlin, 2001. 109. Yasumoto, T. and Takizawa, A., Fluorimetric measurement of Yessotoxins in shellfish by highpressure liquid chromatography, Biosci. Biotech. Biochem., 61, 1775, 1997. 110. Sasaki, K. et al., Fluorometric analysis of Pectenotoxin-2 in microalgal samples by high performance liquid chromatography, Nat. Toxins, 7, 241, 1999. 111. Holland, P. and McNabb, P., Inter-Laboratory Study of an LC-MS Method for ASP and DSP Toxins in Shellfish, Cawthron Report No. 790, Cawthron Institute, Nelson, New Zealand, April 2003. 112. Subba Rao, D.V., Quilliam, M.A., and Pocklington, R., Domoic acid—A neurotoxic amino acid produced by the marine diatom Nitzscia pungens in culture, Can. J. Fish. Aquat. Sci., 45, 2076, 1988. 113. Fritz, L. et al., An outbreak of domoic acid and poisoning attributed to the pinnate diatom Pseudonitzschia australis, J. Phycol., 28, 439, 1992. 114. Takemoto, T., Isolation and structural identification of naturally occurring excitatory amino acids, in Kainic Acid as a Tool in Neurobiology, McGeer, E.G., Olney, J.W., and McGeer, P.L., Eds., Raven Press, New York, 1978, p. 1. 115. Perl, T.M. et al., An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid, N. Engl. J. Med., 322, 1775, 1990. 116. Teitelbaum, J.S. et al., Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels, N. Engl. J. Med., 322, 1781, 1990. 117. AOAC, Paralytic Shellfish Poison. Biological method. Final action, in Official Method of Analysis, 15th edn., Sec 959.08., Hellrich, K., Ed., Association of Official Analytical Chemists (AOAC), Richmond, VA, 1990, p. 881.
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118. Van Dolah, F.M. et al., A microplate receptor assay for the amnesic shellfish poisoning toxin, domoic acid, utilizing a cloned glutamate receptor, Anal. Biochem., 245, 102, 1997. 119. Smith, D.S. and Kitts, D.D., Enzyme immunoassay for the determination of domoic acid in mussel extracts, J. Agric. Food. Chem., 43, 367, 1995. 120. Garthwaite, I. et al., Polyclonal antibodies to domoic acid and their use in immunoassays for domoic acid in seawater and shellfish, Nat. Toxins, 6, 93, 1998. 121. Traynor, I.M. et al., Detection of the marine toxin domoic acid in bivalve molluscs by immunobiosensor, Poster presented at the 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerpen, Belgium, 2002. 122. Quilliam, M.A., Thomas, K., and Wright, J.L.C., Analysis of domoic acid in shellfish by thin-layer chromatography, Nat. Toxins, 6, 147, 1998. 123. Lawrence, J.F., Charbonneau, C.F., and Ménard, C., Liquid chromatographic determination of domoic acid in mussels, using AOAC paralytic shellfish poison extraction procedure: Collaborative study, J. AOAC Int., 74, 68, 1991. 124. Lawrence, J.F. et al., Liquid chromatographic determination of domoic acid in shellfish products using the paralytic shellfish poison extraction procedure of the association of official analytical chemists, J. Chromatogr. 462, 349, 1998. 125. Pocklington, R. et al., Trace determination of domoic acid in seawater and phytoplankton by highperformance liquid chromatography of the fluorenylmethoxycarbonyl (FMOC) derivative, Intern. J. Environ. Anal. Chem., 38, 351, 1990. 126. Wright, J.L.C. and Quilliam, M.A., Methods for domoic acid, the amnesic shellfish poisons, in Manual on Harmful Marine Microalgae, IOC Manuals and Guides, No. 33, Hallegraeff, G.M. et al., Eds., UNESCO, Paris, 1995, p. 113. 127. Hess, P. et al., Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid in shellfish from Scotland by liquid chromatography and mass spectrometry, J. AOAC Int., 84, 1657, 2001. 128. Powell, C.L. et al., Development of a protocol for determination of domoic acid in the sand crab (Emerita analoga): A possible new indicator species, Toxicon, 40, 485, 2002. 129 Furey, A. et al., Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry, Rapid Commun. Mass Spectrom., 16, 238, 2002. 130. National Research Council Canada (NRC) Institute for Marine Biosciences, 2003. Available at http:// www.nrc.ca/imb. 131. Benson, J.M., Thischler, D.L., and Baden, D.G., Uptake, distribution, and excretion of brevetoxin 3 administered to rats by intratracheal instillation, J. Toxicol. Environ. Health Part A, 56, 345, 1999. 132. Cembella, A.D. et al., In vitro biochemical and cellular assays, in Manual on Harmful Marine Microalgae, IOC Manuals and Guides, No. 33, Hallegraeff, G.M., Anderson, D.M., and Cembella, A.D., Eds., UNESCO, Paris, 1995. 133. Fleming, L.E. and Baden, D.G., Florida Red Tide and Human Health: Background, 1999. Available at http://www.redtide.whoi.edu/hab/illness/floridaredtide.html. 134. Watters, M.R., Organic neurotoxins in seafoods, Clin. Neurol. Neurosurg., 97, 119, 1995. 135. Van Dolah, F.M., Roelke, D., and Greene, R.M., Health and ecological impacts of harmful algal blooms: Risk assessment needs. Hum. Ecol. Risk Assess., 7, 1329, 2001. 136. Dickey, R. et al., Monitoring brevetoxins during a Gymnodinium Breve red tide: Comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts, Nat. Toxins, 7, 157, 1999. 137. Hokama, Y., Recent methods for detection of seafood toxins: Recent immunological methods for ciguatoxin and related polyethers, Food Addit Contam., 10, 71, 1993. 138. Weil, C.S., Tables for convenient calculation of median effective dose (LD50 or ED50) and instruction in their use, Biometrics, 8, 249, 1952. 139. Viviani, R., Eutrophication, marine biotoxins, human health, Sci. Total Environ. Suppl., 631, 631–662, 1992.
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140. Manger, R.L. et al., Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: Semiautomated assay for saxitoxin, brevetoxin and ciguatoxins, Anal. Biochem., 214, 190, 1993. 141. Fairey, E.R., Edmunds, J.S.G., and Ramsdell J.S., A cell-based assay for brevetoxins, saxitoxins, and ciguatoxins using a stably expressed c-fos-luciferase reporter gene, Anal. Biochem., 251, 129, 1997. 142. Van Dolah, F.M. et al., Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins, Nat. Toxins, 2,189, 1994. 143. Trainer, V.L. and Baden, D.G., An enzyme immunoassay for the detection of Florida red tide brevetoxins, Toxicon, 29, 1387, 1991. 144. Baden, D.G. et al., Modified immunoassays for polyether toxins: Implications of biological matrixes, metabolic states, and epitope recognition, J. AOAC Int., 78, 499, 1995. 145. Naar, J. et al., Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium, Toxicon, 39, 869, 2001. 146. Naar, J. et al., A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish and mammalian body fluid, Environ. Health Perspect., 10, 179, 2002. 147. Garthwaite, I. et al., Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand, J. AOAC Int., 84, 1643, 2002. 148. Shea, D., Analysis of brevetoxins by micellar electrokinetic capillary chromatography and laserinduced fluorescence detection, Electrophoresis, 18, 277, 2002. 149. Hua, Y. et al., On-line high-performance liquid chromatography-electrospray ionization mass spectrometry for the determination of brevetoxins in “red tide” algae, Anal. Chem., 67, 1815, 1995. 150. Quilliam, M.A., Liquid chromatography-mass spectrometry: A universal method for analysis of toxins? In Harmful Algae, Proceedings of the VIII International Conference on Harmful Algae, Reguera, B., Blanco, J., Fernandez, M., and Wyatt, T., Eds., UNESCO, Paris, 1998, p. 509. 151. Msagati, T.A.M., Siame, B.A., and Shushu, D.D., Evaluation of methods for the isolation, detection and quantification of cyanobacterial hepatoxins, Aquat. Toxicol., 78, 382, 2006. 152. Carmichael, W.W., The cyanotoxins, in Advances in Botanical Research, vol. 27, Callow, J.A., Ed., Academic Press Inc., San Diego, CA, 1997, p. 211. 153. Mankiewicz, J. et al., Natural toxins from cyanobacteria, Acta Biol., Cracov. Bot., 45, 9, 2003. 154. Falconer, I.R., Cyanobacterial toxins of drinking water supplies: Cylindrospermopsins and microcystins, in Cyanobacterial Poisoning of Livestock and People, CRC Press, Boca Raton, FL, 2005, ch. 5. 155. Falconer, I.R., Is there a human health hazard from microcystins in the drinking water supply?, Acta Hydrochim. Hydrobiol., 33, 64, 2005. 156. Rapala, J. and K. Lahti, Methods for detection of cyanobacterial toxins, in Detection Methods for Algae, Protozoa and Helminthes in Fresh and Drinking Water, Water Quality Measurement Series, Palumbo, F., Ziglip, G., Van der Beken, A., Eds., Wiley, New York, 2002, ch. 7. 157. Lambert, T.W. et al., Quantitation of the microcystin hepatotoxins in water at environmentally relevant concentrations with the protein phosphatase bioassay, Environ. Sci. Technol., 28, 753, 1994. 158. Wong, B.S.F. et al., A colorimetric assay for screening microcystin class compounds in aquatic systems, Chemosphere, 38, 1113, 1999. 159. Almeida, V.P.S. et al., Colorimetric test for the monitoring of microcystins in cyanobacterial culture and environmental samples from southeast Brazil, Braz. J. Microbiol., 37, 192, 2006. 160. Sugiyama, Y. et al., Sensitive analysis of protein phosphatase inhibitors by the firefly bioluminescence system; application to PP1, Biosci. Biotechnol. Biochem., 60, 1260, 1996. 161. Bouaicha, N. et al., A colorimetric and fluorometric microplate assay for the detection of microcystinLR in drinking water without preconcentration, Food Chem. Toxicol., 40, 1677, 2002. 162. Metcalf, J.S., Bell, S.G., and Codd, G.A., Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin, Water Res., 34, 2761, 2000.
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163. Mikhailov, A. et al., Production and specificity of mono and polyclonal antibodies against microcystins conjugated through N-methyldehydroalanine, Toxicon, 39, 477, 2001. 164. Zeck, A. et al., Highly sensitive immunoassay based on a monoclonal antibody specific for [4-arginine] microcystins, Anal. Chim. Acta, 441, 1, 2001. 165. Merliuoto, J.A.O., Chromatography of microcystins, Anal. Chim. Acta, 352, 277, 1997. 166. Moollan, R.W., Rae, B., and Verbeek, A., Some comments on the determination of microcystin toxins in waters by high performance liquid chromatography, Analyst, 121, 233, 1996. 167. Barco, M., Rivera, J., and Caixach, J., Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed-phase liquid chromatography-electrospray ionisation mass spectrometry, J. Chromatogr. A., 959, 103, 2002. 168. Barco, M. et al., Determination of microcystin variants and related peptides present in a water bloom of Planktothrix (Oscillatoria) rubescens in a Spanish drinking water reservoir by LC/ESI-MS, Toxicon, 44, 881, 2004. 169. McElhiney, J. and Lawton, L.A., Detection of the cyanobacterial hepatotoxins microcystins, Toxicol. Appl. Pharmacol., 203, 219, 2005. 170. Graves, P.R. and Haystead, T.A.J., Molecular biologist’s guide to proteomics, Microb. Mol. Biol. Rev., 66, 39, 2002. 171. Rouhiainen, L. et al., Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90, Appl. Environ. Microb., 70, 686, 2004. 172. Ouellette, A.J., Handy, S.M., and Wilhelm, S.W., Toxic Microcystis is widespread in Lake Erie: PCR detection of toxin genes and molecular characterization of associated cyanobacterial communities, Microb. Ecol., 51, 154, 2006. 173. Twohig, M., New analytical methods for the determination of acidic polyether toxins in shellfish and marine phytoplankton, MSc thesis, Cork Institute of Technology, Cork, Ireland, 2001. 174. Satake, M. et al., Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis, J. Am. Chem. Soc., 120, 9967, 1998. 175. Ito, E. et al., Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland, Toxicon, 38, 917, 2000. 176. Gago Martinez, A. and Lawrence, J.F., Shellfish toxins, in Food Safety, D’Mello, J.P.F., Ed., CAB International, Wellingford, U.K., 2003, p. 47.
Chapter 34
Detection of Adulterations: Addition of Foreign Proteins Véronique Verrez-Bagnis Contents 34.1 34.2 34.3 34.4 34.5 34.6 34.7
Introduction ..................................................................................................................675 Electrophoresis ..............................................................................................................676 Immunological Techniques .......................................................................................... 677 Visible and Near-Infrared Spectrometry ........................................................................678 Microscopic Methods ....................................................................................................678 Chromatographic Techniques .......................................................................................679 DNA Methods ..............................................................................................................679 34.7.1 PCR-Sequencing ............................................................................................. 680 34.7.2 Species-Specific PCR or Multiplex PCR.......................................................... 680 34.7.3 Amplified Fragment Length Polymorphism .....................................................681 34.7.4 PCR-Restriction Fragment Length Polymorphism ...........................................681 34.7.5 Real-Time PCR ................................................................................................681 34.7.6 PCR Lab-on-a-Chip........................................................................................ 682 34.7.7 Commercial PCR Kits for Fish Species Differentiation ................................... 682 34.8 Conclusion ................................................................................................................... 683 References ............................................................................................................................... 683
34.1 Introduction Recent food scares such as bovine spongiform encephalopathy (BSE), malpractices of some food producers, religious reasons, and food allergies have tremendously reinforced public awareness in 675
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the composition of food products [1]. Therefore, the description and/or labeling of food must be honest and accurate, particularly if the food has been processed removing the ability to distinguish one ingredient from another [2]. There are several ways in which food can be misdescribed: (1) the nondeclaration of processes (e.g., previous freezing or irradiation), (2) substitution of high-quality materials with ones of lower value, (3) overdeclaring a quantitative ingredient declaration, and (4) extending or adulteration of food with a base ingredient, such as water [2]. However, because labels do not provide sufficient guarantee about the true contents of a product, it is mandatory to identify and/or authenticate the components of processed food, thus protecting both consumers and producers from illegal substitutions [3]. Woolfe and Primrose [2] wrote on the needs of methods for detecting misdescription and fraud, that detecting the total substitution of one ingredient is easier than investigating partial substitution or adulteration. In many cases, it is necessary to know the possible adulterant before it can be detected. To decide whether it is adventitious mixing or deliberate substitution, the amount of adulterant present is usually to be confirmed. Many different chemical and biochemical techniques have been developed for determining the authenticity of food. However, some techniques work well with raw products but lose their discrimination when applied to cooked or highly processed foods [2]. Molecular authentification or molecular traceability, based on the polymerase chain reaction (PCR) amplification of DNA, which has been developed in recent years, offers promising solutions for these issues [1]. In this chapter on seafood product adulteration by addition of foreign proteins, the different techniques used to identify such adulterations will be summarized. In fact, there are, strangely, few studies on seafood adulteration even if a considerable number of fish products may contain muscle or other tissue from one or more fish species. Examples are cooked and sterilized fish commodities such as cakes, pies, pastries, soups, patés, and industrial products such as fish meals. As Ascensio Gil [4] reported there are many forms of adulteration for economic gain such as the addition of undeclared cheaper fish in fish products that are labeled using the names of higher price and quality fish species. This chapter gives the reader, through results of research studies, an idea of the main methods used to detect seafood adulteration by substitution or addition of unlabeled component (foreign proteins).
34.2
Electrophoresis
The identification of fish can be problematic when morphological characteristics, such as the head, the skin, and the fins are removed. A well-used method for identifying raw fish is the characterization of muscle proteins using electrophoresis [5–11]. The methods used depend upon the separation of the muscle proteins (sarcoplasmic proteins and/or myofibrillar proteins) into speciesspecific profiles which, when compared with those of authentic species obtained under the same electrophoretic conditions, enable the species to be established unequivocally [12]. The identity of raw fish or shellfish is generally determined from the muscular water-soluble or sarcoplasmic proteins obtained by isoelectric focusing (IEF: separation of proteins according to their pI) [5], while cooked fish is analyzed by sodium dodecyl sulfate (SDS) electrophoresis of SDS protein extracts (myofibrillar, connective, and sarcoplasmic proteins) [6,8,10,11]. When such procedures are applied to detect adulteration rather than substitution of one species by another (i.e., mixed species products), their success depend upon the characteristic zones of the component species being identifiable in the profile of mixture. For most species of fish, IEF of the sarcoplasmic proteins is the preferred analytical system as the profiles generally have more species-specific components, with differences between species being much greater than SDS electrophoresis. A report of
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the European Commission (EC) project “Identification and Quantification of species in marine products” [13] noted that it is possible to identify and to evaluate the concentration of each species in a binary mixture (in the study: saithe and ling, respectively) by IEF if each species contained distinguishing protein bands with measurable intensity. In the same way, Podeszewski and Zarzycki [14] have previously applied the starch-gel electrophoresis on sarcoplasmic protein fractions of fish and demonstrated that this made it possible to demonstrate the presence of another fish species in minced fish meat stated to contain only one species. Under favorable circumstances, it is also possible to detect and identify foreign additions in a mixture of more than two fish species [14]. The IEF method, however, is not suitable for identifying mixtures of crustacean species, as the profiles have few zones and most of them are focused within the same narrow range of pH [12]. However, Craig et al. [12] in a study to detect adulteration of raw reformed breaded scampi (Nephrops norvegicus) demonstrated the successful application of SDS acrylamide gel electrophoresis to the identification of scampi and other crustacean species such as tropical shrimp (Penaeus indicus) and Pacific scampi (Metanephrops andamanicus) when present in reformed scampi products. Martinez and co-workers [10,15,16] thought that two-dimensional gel electrophoresis (2-DE) could have a major application within food authentification to characterize the species and tissue. They noted that obviously, the first studies carried out on given species will have to deal with 2-DE, sequencing marker proteins, and identification with reference organisms. However, as the databases for food and feed material increase, it is possible that, in the future, the procedure will be made easier, faster, and perhaps cheaper by using, for example, tailor-made peptide chips for each product type.
34.3 Immunological Techniques Blot hybridization, enzyme-linked immunosorbent assay (ELISA), immunodot, and immunodiffusion tests are immunological techniques which could be used for the detection of adulteration as these techniques are specific and sensitive analytical methods. Immunoassays using antigenspecific antibodies offer a powerful tool for the detection of the added proteins in a complex food protein mixture. Some research studies have been realized on the implementation of immunological techniques for the detection of seafood product adulteration by the addition of foreign proteins. Verrez et al. [17,18] have focused on immunological methods such as immunoblots and ELISA to detect the addition of crab meat in surimi (washed fish mince)-based products. Indeed, the label of surimi-based crabsticks sometimes indicates the addition of crab flesh in these products; analytical methods should be implemented to check this assertion. The authors have shown that using antiarginine kinase antibodies (arginine kinase is a cytoplasmic protein present in many invertebrates and absent from vertebrates), crustacean flesh could easily be detected in surimi crab supplemented preparations with a correlation between the level of crab added to surimi and the level of immunological response. Taylor and Leighton Jones [19] have developed an immunoassay based on a noncompetitive indirect ELISA using antibodies directed to albacore, bonito, skipjack, and yellowfin to detect the adulteration of high-value crustacean tail meat products with lower value white fish. Another study was done on the development of a dipstick immunoassay for the detection of trace amounts of egg proteins in food [20]. Actually, allergy against egg, for example, can be caused by relatively small amounts of egg proteins and can exhibit typical symptoms, however lifethreatening anaphylactic reactions to egg are very rare. In this study, the authors have developed
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tests with antibodies against egg white and ovalbumin and they have tested different food samples. In nearly all analyzed foods where egg proteins have been declared, they were detected by the dipstick method. On the contrary, seafood product could also be added to meat products and adulterate them. Indeed, surimi, which is a source of high content in myofibrillar proteins, was expected to become an additive to meat products. In order to distinguish the addition of Alaska pollock surimi to meat products, Dreyfuss et al. [21] have developed a test for rapid identification of pollock surimi in raw meat products. Their test based on the detection by antibodies directed against proteins of Alaska pollock surimi gave positive results with all the finfish species tested (14 species). The test was specific for Alaska pollock surimi at 2% concentration and showed detectable sensitivity to surimi from other finfish at concentration between 2% and 4%, and was 100% accurate in the laboratory trials.
34.4
Visible and Near-Infrared Spectrometry
The origin of the near-infrared (NIR) spectra of agro-food products is the absorption of the NIR light by chemical bounds of organic molecules. Spectra of food products include mainly absorption bands characteristic of the main constituents of the materials (i.e., water, proteins, fat, and carbohydrates) [22]. The main limitation of the NIR technique is its indirect nature as it measures no single target such as a specific molecule, DNA fragment, or proteins. However, NIR-based methods were proposed for the detection of meat and bone meal (MBM) in compound feeds [23,24]. To prevent the transmission to BSE between animals and humans, European authorities [25] prohibited the use of animal meat for feeding to ruminants. This measure includes fish meals, although this disease does not affect fish. The objective of this measure is to prevent adulteration and cross-contamination between fish and land animal meals. That is why analytical methods have been proposed for the determination of animal origin of feeding stuffs. Murray et al. [24] have developed a method based on a partial least squared (PLS) discriminant analysis, using visible and NIR reflectance spectra. From their results, it seems that visible–NIR reflectance spectroscopy could routinely provide the first line of defense of the food chain against accidental contamination or fraudulent adulteration of fish meal with MBM. In their article, van Raamsdonk et al. [22] noted that a NIR spectrometer coupled to a microscope (NIRM) could also be proposed to tackle the problem of detection of MBM in compound feed; the method can also be used to detect fish meal ingredients. When using a NIR microscope the subjective judgment of the microscopist is replaced by the spectra that can be subjected to statistical analysis. In another possible application field, Gayo et al. [26,27] have successfully used visible and NIR spectroscopy (Vis/NIR) to detect and quantify species authenticity and adulteration in crabmeat samples. In their studies, visible and NIR spectroscopy have been successfully used to detect the adulteration in crab meat samples adulterated with surimi-based imitation crabmeat and to detect the adulteration of Atlantic blue crab (Callinectes sapidus) meat with blue swimmer crab (Portunus pelagicus) meat in 10% increments.
34.5
Microscopic Methods
Following the measure to prohibit the use of animal proteins for feeding ruminant including also fish meals, EC directive 2003/126/EC [28] indicates the analytical method to be applied for
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the detection and characterization of processed animal proteins (PAPs) in feeds. This analytical method is based on a microscopic technique. However, this method is not applicable to fish meals, because some typical structures detected are common to both fish and land animals, and it only gives useful results when bones are present in the sample [29]. Whereas microscopic techniques could be used to check if fish meals are not adulterated by fraudulent addition of terrestrial animal meal. van Raamsdonk et al. [22] have reviewed different proficiency studies and ring trials organized since 2003 for the detection of mammalian PAP in fish meal. The first proficiency study, allowing the participants to apply their own protocol, revealed that microscopic detection of 1 g/kg of mammalian PAP in the presence of 50 g fish meal/ kg was realized in 44% of the cases. However, a microscopic detection of 98% can be reached by providing the application of an optimal protocol and a sufficient level of expertise. Recent studies showed that training, application of a decision support system, and use of an improved microscopy protocol resulted in a higher sensitivity. As van Raamsdonk et al. [22] noted, an attractive approach to detect fish meal adulteration by meat meal is the combination of the very low detection level of microscopy with identification by other methods (PCR and immunoassays). Microscopical techniques could also be used to detect other types of seafood product adulteration than adulteration of fish meals. Thus, Ebert and Islam [30] used histological examinations to identify, as caviar imitations; products labeled “special caviar product manufactured with sturgeonand salmon roe in the Russian way.” The caviar-like, spherical products appeared to be formed out of a homogeneous, unstructured mass and showed none of the fish roe specific biological structures.
34.6
Chromatographic Techniques
There is very little literature on the use of chromatographic techniques to detect adulteration. However, the study of Chou et al. [31] is based on an high-performance liquid chromatography (HPLC) method with electrochemical detection (HPLC-EC). They reported that a major advantage of EC detection is its ability to directly detect peptides and amino acids that exhibit little or no chromogenic or fluorescent properties. In addition, under appropriate chromatographic conditions and simultaneous use of a copper nanoparticle-plated electrode, reliable detection is feasible without sample pretreatment. In their study, Chou et al. [31] have tested the applicability of the method to detect the species in mixtures only on three land animals (beef, pork, and horse meats). However, as they could identify cod, crab, salmon, scallop, and shrimp with this method, it seems possible to apply such technique—that is fast, economic, and reliable for identification of meats from multiple species—to detect adulteration of seafood flesh by meat components.
34.7 DNA Methods Advances in DNA technologies have led to rapid development of genetic methods mainly based on PCR for fish species identification and for the detection of fish product adulteration as protein analysis are in general not suited to fish species identification in heat-processed matrices [32]. DNA offers advantages over proteins, including stability at high temperature, presence in all tissue types, and greater variation in genetic sequence [33]. Fish and fishery products authentification can be achieved by PCR-based methods (see reviews by Leighton Jones [34], Sotelo et al. [35], Mackie [36], Lockley and Bardsley [32], Asensio Gil [4]). Asensio Gil’s [4] work provided an extensive overview
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on various techniques such as PCR-sequencing, species-specific PCR primers or multiplex PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-single-stranded conformation polymorphism (PCR-SSCP), random amplification of polymorphic DNA (RAPD), real-time PCR, and PCR lab-on-a-chip. All these PCR-based techniques have high potential because of their rapidity, increased sensitivity, and specificity [32]. Nevertheless, some of these techniques such as RAPD analysis may not be suitable to identify adulterations. RAPD technique is not adapted to detect the species of origin in products containing mixtures of species containing 50%–50% mixtures of species that can interbreed [37]. On the other hand, quantitative PCR tests such as real-time PCR have been widely used for food authentification and quantification. In this section on DNA techniques, only those that could be used to detect adulteration are reviewed.
34.7.1
PCR-Sequencing
PCR-sequencing which is the most direct means of obtaining information from PCR products has been extensively used to identify various fish mainly based on mitochondrial genes such as cytochrome b or cytochrome oxidase I (COI) (e.g., see study of Jérôme et al. [38]). In addition fragments of nuclear genes such as a-actinine, 5S ribosomal DNA, rhodopsin, among others, have been sequenced for the discrimination of fish (e.g., see study of Sevilla et al. [39]). Dedicated Internet databases offer the possibility to rely on unknown sequences to reference fish species sequences (e.g., FishTrace database [40] and Fish-BOL [41]). Even if sequencing is time consuming, it produces large amounts of information that could be used in other PCR-based methods such as PCR-RFLP to fish species identification [42–44]. PCR-sequencing technique seems, therefore, difficult to adapt to check seafood adulteration by substituting partially foreign fish in labeled one fish species product.
34.7.2
Species-Specific PCR or Multiplex PCR
Because this method has the potential to detect qualitative admixture, it is a method adapted to adulteration analysis. Prior sequence knowledge is required in order to design primers and appropriate controls should be included to preclude the possibility of false positive or negative results being obtained [45]. In this idea, Colombo et al. [46] have developed a species-specific PCR to identify frozen and seasoned food labeled as pectinid scallop and suspected to be or to contain vertebrate (in particular teleostean fish). Multiplex PCR can also be used with the intention to examine fish meal for contamination with mammalian and poultry products. Bellagamba et al. [47] have seeked a method based on three species-specific primer pairs designed for the identification of ruminant, pig, and poultry DNA. The PCR specifically detected mammalian and poultry adulteration in fish meals containing 0.125% beef, 0.125% sheep, 0.125% pig, 0.125% chicken, and 0.5% goat. The multiplex PCR assay for ruminant and pig adulteration in fish meals had a detection limit of 0.25% after optimization. As different tuna species have different qualities and prices, a fraudulent replacement of valuable species by less valuable ones (e.g., Katsuwonus pelamis) may occur. Bottero et al. [48] have developed a multiplex primer-extension assay (PER) to discriminate four closely related species of Thunnus (T. alalunga, T. albacores, T. obesus, and T. thynnus) and one species of Euthynnus genus (K. pelamis) in raw and canned tuna. The technique enables the simultaneous and unambiguous identification of the five tuna species.
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Amplified Fragment Length Polymorphism
As Atlantic salmon (Salmo salar) is highly appreciated in the Chinese market, illegal practices can occur by adulterating or substituting rainbow trout products (Oncorhynchus mykiss) of much lower value in China for those of Atlantic salmon. Zhang and Cai [49] have developed a species-specific amplified fragment length polymorphism (AFLP) marker based on AFLP analysis and converted into reliable sequence-characterized amplified regions (SCARs) for constructing a direct and fast method to detect frauds in fresh and processed products of Atlantic salmon being adulterated and substituted by rainbow trout. The SCAR marker could be amplified and visualized in 1% agarose gel in all tested rainbow trout samples and was absent in all salmon samples. Using DNA admixtures, the detection of 1% (0.5 ng) and 10% (5 ng) rainbow trout DNA in Atlantic salmon DNA for fresh and processed samples, respectively, was readily achieved. In another study, Zhang et al. [50] have demonstrated that the detection sensitivity of AFLP-derived SCAR was higher than that of DNA amplicons separation by denaturing gradient gel electrophoresis (DDGE) when analyzing experimental mixtures of Atlantic salmon and rainbow trout. The AFLP-derived SCAR approach was sensitive and demonstrated to be a rapid and reliable method for identifying frauds in salmon products, and it could be extended for the applications of species identification in food industry.
34.7.4
PCR-Restriction Fragment Length Polymorphism
Only three examples of the use of PCR-RFLP in the detection of seafood product adulteration are detailed in this chapter. The first one is the study of Hold et al. [51] who have used this technique to develop a method to differentiate between several different fish species. The method was tested in a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. From a total of 120 tests performed, unknown samples identified by comparison with RFLP profiles of reference species were correctly identified in 96% of cases. They have also tested the ability of this method to analyze mixed and processed fish samples. In all cases, the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products. Horstkotte and Rehbein [52] have tested the usability of fish species identification with RFLP using HPLC for sturgeon, salmon, and tuna samples. Unequivocal species identification was achieved with HPLC, despite nucleotide sequence-depending separation. Separation of DNA fragments by HPLC could be demonstrated to be a fast and reliable alternative to electrophoresis. In the aim of guaranteeing the composition and security of fish meals, a method based on PCR and length polymorphism, followed by a RFLP was developed by Santaclara et al. [29]. Specific primers for every species were designed and calibrated to generate a PCR product with a specific size when DNA of each land species that can be used for elaboration of meat meals (cow, chicken, pig, horse, sheep, and goat) was present in the sample. This methodology allows verification of the adulteration and cross-contamination of fish meals with the six land species studied.
34.7.5 Real-Time PCR The specificity and sensitivity of this technique, combined with its high speed, robustness, reliability, and the possibility of automation contribute to the adequacy of the method for quantifying fish species in fishery products [53]. For instance, Asensio Gil [4] reported study of Sotelo et al. [54] who used TaqMan assay for the identification and the quantification of cod. In the same idea, Trotta et al. [55] used real-time PCR for the identification of fish fillets from grouper and
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common substitute species. They also used conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. These two approaches, real-time PCR and multiplex PCR made possible to discriminate grouper from substitute fish species. Hird et al. [56] have designed real-time PCR primer and probe set for the detection and quantification of haddock. The presence of this fish in concentrations of up to 7% in raw or slightly heat-treated products could be detected. While Lopez and Pardo [57] applied the realtime PCR technology for the identification and the quantification of albacore and yellowfin tuna, the real-time methodology described in their study was suitable to detect the fraudulent presence of yellowfin or even to identify the absence of albacore in cans labeled as white tuna. As Asensio Gil [4] reported, the accuracy of this technique could be affected by several factors, such as the DNA yield of the samples, which can be variable depending on the strength of the technique used to process the fish product, and by the fact that the sample material could be thermally processed in different ways. Owing to its cost, real-time PCR has only a remarkable interest in analyzing products with an important economic value. However, the enormous utility and possible applications of the real-time PCR will make it affordable for most laboratories in the near future.
34.7.6 PCR Lab-on-a-Chip This technology that uses microfluidic devices has been recently used for fish species authentification. Dooley et al. [58] used a chip-based capillary electrophoresis system to discriminate mixtures of salmon and trout. Experimental repeatability was less than 3%, allowing species identification without the need to run reference materials with every sample. Using DNA admixtures, the discrimination of 5% salmon DNA in trout DNA was readily achieved. This technology permitted an improvement of a published PCR-RFLP approach for fish species identification by the replacement of the gelelectrophoretic steps by capillary electrophoresis. Dooley et al. [59] used the same methodology for identification of 10 white fish species associated with the U.K. food products. The method was subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine identifications of admixture samples were achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials. Although this technology is relatively expensive, the cost of the instrumentation and disposable chips are relatively low when compared to that for real-time PCR analysis (cited by Ascensio Gil [4]).
34.7.7
Commercial PCR Kits for Fish Species Differentiation
In the recent years, advances in PCR-based methods have led to rapid development of different commercial kits for fish species identification. There is no literature on the use of these rapid diagnostic kits for the detection of substitution or adulteration in seafood products, but without doubt, these kits could be very useful for screening purposes in inspection programs. The present list below of those commercial kits or commercial proposals is not exhaustive and is only valuable for at present time. ◾ DNA kit for eight fish species identification provided by Tepnel Biosystems company ◾ Biofish kit for cod and gadiform species and Biofish kit for Atlantic salmon, sea trout, and rainbow trout commercialized by Biotools company
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◾ FishID kit for the identification of more than 200 fish species developed by Bionostra company ◾ GeneChip based on DNA microarray technology developed by Biomérieux ◾ Proposal of Eurofins/GeneScan company to develop methods and analysis kits based on PCR according to a specific request
34.8 Conclusion Of the wide range of analytical methods available, it is likely that DNA-based techniques will be the favorite approach for determining adulteration, because they are easy to use. However, now, they are generally more expensive than electrophoresis or chromatographic techniques and in some cases, the latter are sufficient to clearly demonstrate an adulteration in seafood products by addition of foreign proteins. Nevertheless, researches into relatively novel techniques such as PCR lab-on-chip and real-time PCR offer the greatest potential for the development on new fish discrimination applications and protocols (Asensio Gil [4]). Quantifying methods are, without doubt, the more adapted to analyze adulteration, and these methods are to be developed.
References 1. Teletchea, F., Maudet, C., and Hanni, C., Food and forensic molecular identification: Update and challenges, Trends Biotechnol., 23, 359, 2005. 2. Woolfe, M. and Primrose, S., Food forensics: Using DNA technology to combat misdescription and fraud, Trends Biotechnol., 22, 222, 2004. 3. Pascal, G. and Mahé, S., Identity, traceability, acceptability and substantial equivalence of food, Cell. Mol. Biol., 47, 1329, 2001. 4. Asensio Gil, L., PCR-based methods for fish and fishery products authentication, Trends Food Sci. Technol., 18, 558, 2007. 5. Mackie, I. M., Identifying species of fish, Anal. Proc., 27, 89, 1990. 6. Scobbie, A. E. and Mackie, I. M., The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis in fish species identification—A procedure suitable for cooked and raw fish, J Sci. Food Agric., 44, 343, 1988. 7. Rehbein, H., Etienne, M., Jerome, M., Hattula, T., Knudsen, L. B., Jessen, F., Luten, J. B. et al., Influence of variation in methodology on the reliability of the isoelectric focusing method of fish species identification, Food Chem., 52, 193, 1995. 8. Rehbein, H., Kundiger, R., Yman, I. M., Ferm, M., Etienne, M., Jerome, M., Craig, A. et al., Species identification of cooked fish by urea isoelectric focusing and sodium dodecylsulfate polyacrylamide gel electrophoresis: A collaborative study, Food Chem., 67, 333, 1999. 9. Etienne, M., Jerome, M., Fleurence, J., Rehbein, H., Kundiger, R., Mendes, R., Costa, H., and Martinez, I., Species identification of formed fishery products and high pressure-treated fish by electrophoresis: A collaborative study, Food Chem., 72, 105, 2001. 10. Piñeiro, C., Barros-Velázquez, J., Pérez-Martín, R. I., Martínez, I., Jacobsen, T., Rehbein, H., Kündiger, R. et al., Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: A collaborative study, Electrophoresis, 20, 1425, 1999. 11. Etienne, M., Jérôme, M., Fleurence, J., Rehbein, H., Kündiger, R., Mendes, R., Costa, H., Pérez-Martín, R., and Piñeiro-González, C., Identification of fish species after cooking by SDS-PAGE and urea IEF: A collaborative study, J. Agric. Food Chem., 48, 2653, 2000.
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12. Craig, A., Ritchie, A. H., and Mackie, I. M., Determining the authenticity of raw reformed breaded scampi (Nephrops norvegicus) by electrophoretic techniques, Food Chem., 52, 451, 1995. 13. European Commission, EU Project FAR UP-3-783. 1992–1995, Identification and quantitation of species in marine products, 1992–1995. 14. Podeszewski, Z. and Zarzycki, B., Identification of fish species by testing the minced-meat tissue, Food/Nahrung, 22, 377, 1978. 15. Martinez, I. and Friis, T. J., Application of proteome analysis to seafood authentication, Proteomics, 4, 347, 2004. 16. Martinez, I., James, D., and Loréal, H., Application of modern analytical techniques to ensure seafood safety and authenticity, FAO Fisheries Technical Paper No. 455, Ed., Food and Agriculture Organization of the United Nations, Rome, Italy, 2005, p. 73. 17. Verrez, V., Benyamin, Y., and Roustan, C., Detection of marine invertebrates in surimi-based products, in Quality Assurance in the Fish Industry, Huss, H. H., Jakobsen, M., and Liston, J. (Eds.), Elsevier Science Publisher B.V., Amsterdam, the Netherlands, 1992, p. 441. 18. Verrez-Bagnis, V. and Escriche Roberto, I., The performance of ELISA and dot-blot methods for the detection of crab flesh in heated and sterilized surimi-based products, J. Sci. Food Agricul., 63, 445, 1993. 19. Taylor, W. J. and Leighton Jones, J., An immunoassay for distinguishing between crustacean tailmeat and white fish, Food Agric. Immunol., 4, 177, 1992. 20. Baumgartner, S., Steiner, I., Kloiber, S., Hirmann, D., Krska, R., and Yeung, J., Towards the development of a dipstick immunoassay for the detection of trace amounts of egg proteins in food, Eur. Food Res. Technol., 214, 168, 2002. 21. Dreyfuss, M. S., Cutrufelli, M. E., Mageau, R. P., and McNamara, A. M., Agar-gel immunodiff usion test for rapid identification of pollock surimi in raw meat products, J. Food Sci., 62, 972, 1997. 22. van Raamsdonk, L. W. D., von Holst, C., Baeten, V., Berben, G., Boix, A., and de Jong, J., New developments in the detection and identification of processed animal proteins in feeds, Anim. Feed Sci. Technol., 133, 63, 2007. 23. Garrido-Varo, A., Pérez-Marín, M. D., Guerrero, J. E., Gómez-Cabrera, A., Haba, M. J. D. L., Bautista, J., Soldado, A. et al., Near infrared spectroscopy for enforcement of European legislation concerning the use of animal by-products in animal feeds, Biotechnologie, Agronomie, Société et Environnement, 9, 3, 2005. 24. Murray, I., Aucott, L. S., and Pike, I. H., Use of discriminant analysis on visible and near infrared reflectance spectra to detect adulteration of fishmeal with meat and bone meal, J. Near Infrared Spectrosc., 9, 297, 2001. 25. European Commission, Commission decision of 27 March 2002 (2002/248/EC) amending council decision 2000/766/EC and commission decision 2001/9/EC with regard to transmissible spongiform encephalopathies and the feeding of animal proteins, Off. J. Eur. Communities (L84 28/3/2002), 71, 2002. 26. Gayo, J. and Hale, S. A., Detection and quantification of species authenticity and adulteration in crabmeat using visible and near-infrared spectroscopy, J. Agric. Food Chem., 55, 585, 2007. 27. Gayo, J., Hale, S. A., and Blanchard, S. M., Quantitative analysis and detection of adulteration in crab meat using visible and near-infrared spectroscopy, J. Agric. Food Chem., 54, 1130, 2006. 28. European Commission, Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the official control of feeding stuffs, Off. J. Eur. Communities, L 339 24/12/2003, 0078, 2003. 29. Santaclara, F. J., Espiňeira, M., Cabado, A. G., and Vieites, J. M., Detection of land animal remains in fish meals by the polymerase chain reaction-restriction fragment length polymorphism technique, J. Agric. Food Chem., 55, 305, 2007. 30. Ebert, M. and Islam, R., Kaviar und Kaviarimitate: Verfälschungen des teuren Störrogens mit histologischer Methode nachweisbar (Caviar and caviar imitations adulterations of sturgeon roe provable by histological method), Fleischwirtschaft, 87, 124, 2007.
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31. Chou, C.-C., Lin, S.-P., Lee, K.-M., Hsu, C.-T., Vickroy, T. W., and Zen, J.-M., Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes, J. Chromatogr. B, 846, 230, 2007. 32. Lockley, A. K. and Bardsley, R. G., DNA-based methods for food authentication, Trends Food Sci. Technol., 11, 67, 2000. 33. Mackie, I. M., Authenticity of fish, in Food Authentification, Ashurt, P. R. and Dennis, M. J. (Eds.), Blackie Academic and Professional, London, U.K., 1996, p. 140. 34. Leighton Jones, J., DNA probes: Applications in the food industry, Trends Food Sci. Technol., 2, 28, 1991. 35. Sotelo, C. G., Pinciro, C., Gallardo, J. M., and Perez-Martin, R. I., Fish species identification in seafood products, Trends Food Sci. Technol., 4, 395, 1993. 36. Mackie, I. M., Fish speciation, Food Technol. Int. Eur., 1, 177, 1994. 37. Martinez, I. and Malmheden Yman, I., Species identification in meat products by RAPD analysis, Food Res. Int., 31, 459, 1998. 38. Jerome, M., Lemaire, C., Verrez-Bagnis, V., and Etienne, M., Direct sequencing method for species identification of canned sardine and sardine-type products, J. Agric. Food Chem., 51, 7326, 2003. 39. Sevilla, R. G., Diez, A., Noren, M., Mouchel, O., Jerome, M., Verrez-Bagnis, V., Van Pelt, H. et al., Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes, Mol. Ecol. Notes, 7, 730, 2007. 40. FishTrace: Genetic catalogue, www.fishtrace.org. 41. Fish Barcode of Life Initiative (FISH-BOL), www.fishbol.org. 42. Quinteiro, J., Sotelo, C. G., Rehbein, H., Pryde, S. E., Medina, I., Pérez-Martin, R. I., Rey-Méndez, M., and Mackie, I. M., Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna, J. Agric. Food Chem., 46, 1662, 1998. 43. Sebastio, P., Zanelli, P., and Neri, T. M., Identification of anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) by polymerase chain reaction, sequence of their mitochondrial cytochrome b gene, and restriction analysis of polymerase chain reaction products in semipreserves, J. Agric. Food Chem., 49, 1194, 2001. 44. Ram, J. L., Ram, M. L., and Baidoun, F. F., Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA, J. Agric. Food Chem., 44, 2460, 1996. 45. Edwards, M. C. and Gibbs, R. A., Multiplex PCR: Advantages, development, and applications, PCR Methods Appl., 3, S65, 1994. 46. Colombo, F., Trezzi, I., Bernardi, C., Cantoni, C., and Renon, P., A case of identification of pectinid scallop (Pecten jacobaeus, Pecten maximus) in a frozen and seasoned food product with PCR technique, Food Control, 15, 527, 2004. 47. Bellagamba, F., Valfre, F., Panseri, S., and Moretti, V. M., Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal, J. Food Prot., 66, 682, 2003. 48. Bottero, M. T., Dalmasso, A., Cappelletti, M., Secchi, C., and Civera, T., Differentiation of five tuna species by a multiplex primer-extension assay, J. Biotechnol., 129, 575, 2007. 49. Zhang, J. and Cai, Z., Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmon salar) by the AFLP-derived SCAR, Eur. Food Res. Technol., 223, 413, 2006. 50. Zhang, J., Wang, H., and Cai, Z., The application of DGGE and AFLP-derived SCAR for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), Food Control, 18, 672, 2007. 51. Hold, G. L., Russell, V. J., Pryde, S. E., Rehbein, H., Quinteiro, J., Vidal, R., Rey-Mendez, M. et al., Development of a DNA-based method aimed at identifying the fish species present in food products, J. Agric. Food Chem., 49, 1175, 2001.
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52. Horstkotte, B. and Rehbein, H., Fish species identification by means of restriction fragment length polymorphism and high-performance liquid chromatography, J. Food Sci., 68, 2658, 2003. 53. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M., Real time quantitative PCR, Genome Res., 6, 986, 1996. 54. Sotelo, C. G., Chapela, M. J., Rey, M., and Pérez-Martin, R. I., Development of an identification and quantitation system for cod (Gadus morhua) using Taqman assay, in First Joint Trans-Atlantic Fisheries Technology Conference, Reykjavik, Iceland, 2003, p. 195. 55. Trotta, M., Schonhuth, S., Pepe, T., Cortesi, M. L., Puyet, A., and Bautista, J. M., Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species, J. Agric. Food Chem., 53, 2039, 2005. 56. Hird, H. J., Hold, G. L., Chisholm, J., Reece, P., Russell, V. J., Brown, J., Goodier, R., and MacArthur, R., Development of a method for the quantification of haddock (Melanogrammus aeglefinus) in commercial products using real-time PCR, Eur. Food Res. Technol., 220, 633, 2005. 57. Lopez, I. and Pardo, M. A., Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares, J. Agric. Food Chem., 53, 4554, 2005. 58. Dooley, J. J., Sage, H. D., Brown, H. M., and Garrett, S. D., Improved fish species identification by use of lab-on-a-chip technology, Food Control, 16, 601, 2005. 59. Dooley, J. J., Sage, H. D., Clarke, M. A. L., Brown, H. M., and Garrett, S. D., Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: Application to detect white fish species in food products and an interlaboratory study, J. Agric. Food Chem., 53, 3348, 2005.
Chapter 35
Detection of Adulterations: Identification of Seafood Species Antonio Puyet and José M. Bautista Contents 35.1 Introduction ................................................................................................................. 688 35.2 Replacement Species and Adulterations ........................................................................ 688 35.2.1 Processed Products Adulteration ......................................................................691 35.3 Methods Based on Proteins ...........................................................................................691 35.4 Methods Based on DNA ...............................................................................................692 35.4.1 Sample Handling and DNA Extraction ...........................................................692 35.4.2 DNA Sequencing Methods ..............................................................................693 35.4.2.1 Standardized Fish Molecular Databases and Barcoding ...................693 35.4.2.2 Identification from General Databases ............................................ 697 35.4.3 Non-DNA Sequencing Methods ..................................................................... 697 35.4.3.1 RFLP .............................................................................................. 698 35.4.3.2 AFLP, RAPD, and Satellite DNA Analysis....................................... 698 35.4.3.3 SSCP and DGGE ........................................................................... 700 35.4.3.4 Selective Amplification .................................................................... 700 35.4.3.5 Quantitative Methods ..................................................................... 701 35.4.3.6 High-Throughput, Microarray Technologies and Bioinformatics .....702 35.5 Future Prospects ............................................................................................................703 References ............................................................................................................................... 704
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35.1 Introduction In the seafood market, substitution of valuable species for species of lower value is a common practice because it is uncomplicated and has immediate economic reward. In addition, it is favored by the depletion in some areas of highly appreciated species, the high variety of fish species, the global market, the difficult differential diagnosis, and the overall lack of taxonomical expertise. Studies around the world have shown that up to 75% of a given species from fish samples in the market can be mislabeled [1]. The need for fish species identification in seafood products rely on the consumer’s right to make informed choices and to guarantee consumer confidence. In a global trade, misleading or deceptive conduct in the commercialization of fisheries products should be always avoided and tracked if they happen, since the effectiveness of the seafood marketing and promotion could be, otherwise, depreciated. Also, at the level of public perception, whenever popular fish species are readily available in the marketplace, it supports the idea that there is abundant supply of them from fisheries, misleading the real condition of the stock. Thus, species mislabeling in the catch vessels could also negatively affect stock size assessment since incorrect data reported influences fisheries management. To this respect it should be mentioned that to identify fish correctly is not easy, and substitution or incorrect labeling can be unintended rather than deliberate, but always harms consumer perception and confidence in seafood products. Finally, it should be emphasized that fish species substitution is not only an economic or ecological fraud but also is a health threat since some seafood species from some areas may elicit maladies to susceptible populations, ranging from allergies [2] to serious illness [3]. For instance, the level of mercury or dioxins in fish species from some fishing grounds [4,5] might promote the medical or governmental advice to consumers, particularly vulnerable population, to limit their consumption, which, in turn, advocate for a correct fish labeling. With around 30,000 living fish species in the world, these can be only correctly identified by visual inspection when the specimens are undamaged. Nevertheless, even in these cases, professional education in fish identification (fishermen, fishmongers, and restaurateurs) may not be sufficient, particularly if there is a certain extent of overlapping features between taxa, as it frequently occurs in many fish species. Moreover, all processed fish products lose their morphological characteristics early in the processing food chain. Thus, potential misidentification or mislabeling of fish species can be considerable as it has been already reported in some market surveys [1,6], which would be specially significant in fish fillets, cooked food, or fish-transformed products [7]. Although not all fi sh species are subject to food trade, only in Europe, more than 500 species are currently in the market and 60% of them are caught outside controlled European waters. Similar trade values are found in the American, Australian, and Japanese markets, making the identification process as an essential tool for traceability of the transforming seafood chain.
35.2 Replacement Species and Adulterations Increasing vulnerability of fish species due to exploitation, climate change, overfishing, and byfishing [8,9] has augmented the commercial replacement of species everywhere. Although there are no wide screening studies on the identification of replacement species in global markets, the rising in the scientific publication of differential diagnostic systems for groups of fish species allows identifying major concerns at present in control laboratories and researchers as depicted in Table 35.1.
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Table 35.1 Potential Substitutions of Fish Species in Markets Worldwide According to the Main Differential Diagnostics Methodology Published Species
Substituted By
Market
Refs.
Cod (Gadus morhua)
Pacific cod (Gadus macrocephalus), Alaska Pollack (Theragra chalcogramma), Saffron cod (Eleginus gracilis), Arctic cod (Arctogadus glacialis), Southern blue whiting (Micromesistius australis), Chilean hake (Merluccius gayi), Southern hake (M. australis), Longfin codling (Laemonema longipes) or Blue grenadier (Macruronus novaezelandiae)
Worldwide (Japan, Europe, United States)
[10–12]
Grouper (Ephinephelus spp. and Mycteroperca spp.)
Nile perch (Lates niloticus) and wreck fish (Polyprion americanus)
Europe
[13]
Frigate tunas (Auxis thazard and Auxis rochei)
Skipjack (Katsuwonus pelamis), little tuny (Euthynnus alletteratus), or yellowfin tuna (Thunnus albacares)
Europe
[14]
Red snapper (Lutjanus campechanus)
Vermilion snapper (Rhomboplites aurorubens), crimson snapper (L. erythropterus), or Lane snapper (L. synagris)
United States
[15,16]
European anchovy (Engraulis encrasicolus)
Engraulis spp. (E. anchoita, E. ringens, E. japonicus, E. mordax), Coilia spp., Sardina spp., Sprattus spp., and Sardinella spp.
Europe
[17,18]
Atlantic salmon (S. salar)
Rainbow trout (Oncorhynchus mykiss)novaezelandiae
Worldwide
[19]
Pacific salmon (Oncorhyncus spp.)
Atlantic salmon (S. salar), Brown trout (Salmo trutta)
North America
[20]
European perch (Perca fluviatilis)
Nile perch (Lates niloticus), European pikeperch (Stizostedion lucioperca), Sunshine bass (Morone chrysops x saxatalis)
Europe
[21]
Albacore (Thunnus alalunga)
Skipjack tuna (Katsuwonus pelamis), yellowfin (T. albacares)
Europe
[22,23]
Mediterranean horse mackerel (Trachurus mediterraneus)
Blue jack mackerel (T. picturatus)
Europe
[24]
Japanese mackerel (Scomber japonicus)
Atlantic mackerel (Scomber scombrus)
Japan
[25] (continued)
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Table 35.1 (continued) Potential Substitutions of Fish Species in Markets Worldwide According to the Main Differential Diagnostics Methodology Published Species
Substituted By
Market
Refs.
European pilchard (Sardina pilchardus)
Other pilchards and sardinellas (Sardinops, Sardinella spp.)
Europe
[18,26]
European hake (Merluccius merluccius)
Deep water hake (Merluccius paradoxus), Senegalese hake (Merluccius senegalensis), Silver hake (Merluccius bilinearis), Chilean hake (M. gayi), Argentine hake (Merluccius hubbsi), Patagonian grenadier (Macruronus magellanicus)
Europe
[27,28]
Sturgeon caviar (Acipenser sturio)
Siberian sturgeon eggs (Acipenser baerii)
Worldwide
[29]
Surimi (Theragra chalcograma)
Merluccius spp.
Europe
[6]
Blue mussel (Mytilus galloprovincialis)
Green mussel (Perna spp.) and others (Aulacomya, Semimytilus, Brachidontes and Choromytilus spp.)
Europe
[30]
Prawn (Fenneropenaeus, Penaeus, Parapenaeus, Marsupenaeus, Melicertus, Solenocera, Pleoticus, and Aristeomorpha spp.) and shrimp (Farfantepenaeus and, Litopenaeus spp.)
Mislabeling
Europe
[32]
Shortfin squids (Family Ommastrephidae: Genera Loligo, Loliolus, Uroteuthis, and Alloteuthis) and longfin squids (family Loliginidae: Genera Todarodes, Illex, Todaropsis, Nototodarus, Dosidicus, and Ommastrephes)
Mislabeling
Europe
[32]
[Billfish species: Makaira nigricans (blue marlin), Makaira indica (black marlin), Istiophorus platypterus (sailfish), and Tetrapturus audax (striped marlin)
Swordfish (Xiphias gladius)
Wordwide
[33]
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35.2.1 Processed Products Adulteration Trade for processed seafood is particularly complex given the large number of species traded, countries involved, and production processes [36]. Protein addition from nondeclared species can be a source of the adulterations in seafood as it also happens in the meat industry [35]. Identification of this manipulation from nonseafood species usually follow specific methodology for detecting mixed DNA from different origin [35–37] that can be applied to groups of species [13]. Nevertheless it should be stressed that addition of artificially synthesized DNA in caviar [29] can be theoretically used to manipulate the detection of the origin, and thus the use of several markers would be recommended to identify foreign DNA in complex food mixtures. Other processed products adulterations are related to the trade of unrecognized parts of fish. This is the case of shark fin where types of fin are mainly described with English common names for sharks [38] in contrast with Chinese market categories that do not correspond to the taxonomic names of shark species. Since shark policy resources lacks species-specific catch and trade data for most fisheries [39], DNA-based species identification techniques have been used to determine the relationship between market category and species, showing that only 14 species made up approximately 40% of the auctioned fin weight in Hong Kong [38]. To follow food labeling regulations, in some instances, quantification of a given fish species in complex food mixtures is required. This has been usually based on the relative content of nitrogen determination, but more recently a model real-time polymerase chain reaction (PCR) has been developed for haddock [40] and tuna species [22] to determine proportion of muscle tissue relative to the amount detected of a single copy gene. This type of methodology is also allowing enforcement of the legislation regarding complex food mixtures containing fish species.
35.3
Methods Based on Proteins
Although DNA technologies are the fi rst choice in the identification of fi sh species since it is not dependent on the specific condition of the fi shery product (e.g., processed or not), several techniques based on protein analysis have also been described, particularly in the most recent past. Protein identification, as a label for species diagnosis, is based on physical and chemical properties of the polypeptide chain: size and net charge of the amino acidic sequence, three-dimensional structure exposure, and immunoreactivity of specific epitopes. Therefore, protein denaturation by heat, chemical additives, or proteolysis can severely modify those native properties. Separation and characterization of soluble sarcoplasmic proteins by isoelectric focusing (IEF) has been the most frequently described protein method for fish species identification [41–43]. Nevertheless, application of IEF is considered mostly limited to raw fish fillet [46] due to the processing of the fish carcass (involving heating, salting, drying, or smoking) which leads to the loss or modification of species-specific protein fragments and yields indefinite IEF patterns, not allowing clear-cut identifications [45,46] and requiring a faithful set of reference samples [46]. Moreover the identification procedure is generally laborious and requires skilled human resources to strictly follow highly optimized standard operation procedures [46], since reproducibility between laboratories is not always achieved [45]. From an IEF survey of 14 species obtained in a fish market, intraspecific polymorphisms and discrepancies were detected, some of them due to unpredictable band distortions that required special computer-assisted comparison of the IEF gels [42]. Although there are not large standardized sets of IEF standard patterns
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for seafood species identification, the Regulatory Fish Encyclopedia (RFE: http://www.fda. gov/Food/FoodSafety/Product-SpecificInformation/Seafood/RegulatoryFishEncyclopediaRFE/ default.htm) has compiled up to 94 fish species (July 2009) with their respective IEF pattern, the largest deposit at present (see Section 35.4.2.1). Other protein analytical techniques have also been developed for fish species identification including some attempts based on immunological procedures [47–49]. These methods have a narrow covering to identify different fish species and thus they have a very limited impact on routine analysis. The large number of different fish species and cellular types that could be involved in generating specific antibodies, either polyclonal [49] or monoclonal [47], would also require to analyze in parallel reference samples to verify that the antibodies used in the identification do not crossreact with other or similar species which is not clearly demonstrated with the available antibodies. Thus, although most of the protein-based methods can be of certain value in some instances, they are not suitable for forensic or certified analysis given that conservation and treatments alter three-dimensional structure and physical characteristics of the proteins, and therefore losing and/ or modifying their identification features.
35.4
Methods Based on DNA
35.4.1 Sample Handling and DNA Extraction All methods described below make use of some kind of PCR amplification and, in consequence, require the extraction of certain amounts of DNA from the sample, either fresh or processed seafood, as template. Nevertheless, in spite of the powerful ability of PCR to detect minute amounts of DNA, several problems may appear associated to its application for species identification in foods. DNA quality in the sample rely upon physical (temperature, moisture, time, mechanic pressure), chemical (pH, oxidizing agents), and biological (endogen nucleases and proteases, manipulation) factors. Depurination, strand break, pyrimidine dimerization, and deoxyribose fragmentation are usual DNA alterations as a consequence of oxidative and hydrolytic damage in the DNA. Thus, in a DNA purification protocol for identification analysis, particular care should be taken to 1. 2. 3. 4.
Sample contamination Low DNA yields associated to some species Inhibition of the DNA polymerase activity DNA degradation in processed products
Standard methods of DNA extraction from tissues are usually adequate for application to seafood and seafood products. The sample to be analyzed should be carefully removed from the food product to avoid contamination with other ingredients or particles from other specimens, preferably after rinsing of the surface and separation of an inner portion. Typically, a 5–50 mg muscle tissue sample from fish or shellfish, or equivalent amount from prepared/processed seafood, is finely minced and immersed in an extraction buffer containing detergents and proteinases. Proteins are removed by phenol and/or chloroform extraction and the nucleic acids are subsequently precipitated with ethanol or isopropanol [50]. These methods, fully manual and time consuming have been progressively replaced in the last years by commercial kits that allow simplifying the purification of multiple samples in a short period and have been successfully applied to processed seafood including canned tuna [51]. These kits usually consist of a single DNA-binding chromatography step in spin columns
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replacing both the protein extraction and the DNA precipitation steps, and can also be applied to the whole process (from tissue to DNA) when including a tissue-extraction initial step. It should be noticed that the DNA extraction method may be critical for a successful identification of some species. For instance, several species of chondrichthyes, eels, shellfish, and decapods can yield either low amounts or hardly amplifiable DNA. DNA purification for PCR amplification from these samples may require the use of modified or specialized methods. Chelex-100 has been widely used in problematic DNA extractions since it is a styrene–divinylbenzene chelating resin with functional iminodiacetic acid groups that protect against DNA degradation and is recommended for samples where other methods fail [52]. Also the cetyl–trimethyl ammonium bromide (CTAB) method has shown to isolate good quality DNA from diverse and difficult samples and organisms, particularly if those contain carbohydrates. The method was originally devised for plant tissues [53] based on the CTAB properties as cationic detergent that bind to negatively charged molecules like DNA [54]. The procedure use buffered 1% CTAB/0.7 M NaCl/10 mM EDTA to homogenize the tissue (even dehydrated) to be followed by chloroform extraction. Subsequently an isopropanol precipitation step in 1% CTAB/Tris/EDTA without salt is performed [53] where the CTAB/nucleic acids complex precipitate [54] and leave solubilized proteins and polysaccharides in the solution. Above all, DNA quality may be affected in processed food, particularly during storage and heat treatments [55]. Thus, while high-molecular weight DNA of a length of 20–50 kbp can be isolated from fresh meats, the DNA length is shortened to 15–20 kbp upon storage for only several days. Heating of the sample before extraction leads to a further degradation down to 300 bp for a 10 min cooking at 121°C [56,57]. Low DNA quality can also drive to misidentification when it occurs together with DNA contamination from other species within the sample. In these cases, if only PCR amplification is achieved from the contaminant DNA then, the proband individual is erroneously identified as belonging to the contaminant DNA. In order to detect early the problems of DNA quality in sample preparation, it is highly advisable to use a negative control of reagents used in DNA isolation together with positive and negative controls of PCR amplification in every diagnostic setup. At present, there is not a given DNA isolation method that could be successfully used for any type of sample. The convenience of a given procedure depends upon the nature of the sample and the use that will be given to the isolated DNA, and it should be empirically tested.
35.4.2
DNA Sequencing Methods
Due to the intraspecies genetic variability among fish populations and the lack of complete genetic information for many commercial species, DNA sequencing can be considered as one of the most reliable tools for fish identification. Moreover, comparative genetics of fish species has notably improved due, to a great extent, to the easy PCR amplification of specific DNA sequences in the last decade [58,59] and the subsequent automated and cheap DNA sequencing. Thus, direct DNA sequencing and database search has proved to be a highly accurate method for the unequivocal identification of fish species, subspecies, and even populations.
35.4.2.1
Standardized Fish Molecular Databases and Barcoding
Although current taxonomy and systematics tools permit the classification of practically all fish species, its usefulness is hindered by the lack of efficient and fast reference tools [60,61]. Nevertheless,
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there are fish identification databases that mainly collect taxonomical and general biological information from worldwide distributed species (e.g., FishBase: www.fishbase.org; The Census of Marine Life: www.coml.org; The FAO Species Identification and Data Programme, SIDP, at www.fao.org, and independent Natural History museums databases) that can be used for taxonomical identification and geographical identification of fish. On the other hand, the utility of DNA sequences for taxonomical purposes is well established at present [62], and even a single specific short sequence has been proposed to be sufficient to differentiate the vast majority of animal species [63], since congeneric species of animals regularly possess enough divergence between nucleotide sequences to ensure easy specific diagnosis. Thus, molecular features to taxon discrimination take advantage of the DNA sequence specificity to identify organisms and therefore, DNA sequences can be taken as identification markers or barcodes in any organism, and particularly in fish. This concept forms the basis for the implementation of databases for biological identifications through the DNA analysis, and is aimed to develop molecular systems based on DNA species-specific profiles or DNA-barcodes, which can be used as unique genetic fingerprint for living beings, allowing further investigations of DNA variation among them. Current studies in this field support the barcoding concept [63–65]. This potential is of particular interest to fisheries products for human consumption as well as to issues related to fisheries management. For traceability of fish, species identification should be potentially feasible on processed food, including fillets, ready-to-eat dishes, canned fish, etc., and to this respect, DNA-based diagnostic analysis is the most appropriate. Molecular genetics methodologies have widely progressed over the last decade in the area of DNA identification, and specific systems have been developed to obtain DNA fragments of diagnostic significance from most organisms, including fish. To this respect it should be pointed out that advanced and affordable DNA-sequencing equipment as well as private or academic enterprises can generate DNA sequences within 24 h with a relatively low cost that has a downward tendency, making feasible the identification of fish at species level at low cost and in a short time. Thus, several initiatives have been specifically developed for the identification of fish species based on DNA sequences. The species-diagnostic DNA sequences are online, publicly available and therefore accessible to control laboratories in any part of the world where also sequencing of short DNA fragments can be rapidly obtained at low cost from fish tissue samples. The FishTrace database (www.fishtrace.org) covers most teleost fish species of commercial, ecological, and zoological interest for the European countries, paying particular emphasis to local data collected in Europe. In addition, FishTrace database provides molecular data, detailed protocols, and tools for the correct identification of fish species [66], standardized photographs taken from fish specimens, otoliths and fish products, and also, a large list of relevant technical publications on taxonomy, distribution, ecology, and biological parameters that have been ad hoc collected for the database. Moreover, the information collected in FishTrace is connected to a biological reference collection from cataloged fish specimens validated by taxonomists. This additional endeavor allows cross-referring analyses with available vouchers deposited in Natural History Museums around Europe. Thus, the FishTrace database has developed a specific infrastructure in Europe for referencing and comparison of teleost fish sequences, information, and materials. The Fish DNA Barcode of Life (FishBOL: www.fishbol.org) is a global database effort to collect a reference DNA sequence for all fish species. Also, identifying DNA sequences are derived from voucher specimens identified by taxonomists. At present DNA sequences from more than 6800 species have been deposited (July 2009). In the United States, the National Atmospheric and
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Oceanic Administration (NOAA) already makes use of the FishBOL database for species identification purposes into their support for fisheries inspection and control. FishTrace and FishBOL share common concepts regarding standardization of common sequence information but differ in the standardized sequences chosen to identify fish. It is well accepted that metazoan mitochondrial genomes (mtDNA) are more suitable for the implementation of a microgenomic identification system than nuclear genomes. The usual limits of intraspecific divergence in mitochondrial genes derived from phylogenetic analyses were established between 1% and 2% in general animal species [67]. Fish genomes undergo genetic changes rapidly, often due to polyploidiation, gain of spliceosomal introns, speciation, and gene duplication phenomenon [68–70]. FishBOL focuses on a DNA-based identification system using a relatively small sequence fragment (∼600 bp) from the mitochondrial cytochrome c oxidase subunit I (COI). This DNA sequence provides sufficient identification labels in terms of nucleotide positions [65] to discriminate even between congeneric fish species, where a 2% sequence divergence is found in 98% of them [65]. Another mitochondrial gene used as DNA label is cytochrome b (cytb) which also contains enough resolution to discriminate from the intraspecific to the intergeneric level [71], possesses a phylogenetic performance equivalent to that of COI [72], and has been widely used to identify and develop diagnostic systems for seafood species [10,18,32,33,73,74]. FishTrace database uses a mitochondrial (cytb) and a nuclear (rhodopsin: rhod) sequence to construct a hybrid DNA-barcode that is used to identify European fish species. The short fragment of the COI sequence proposed as a universal DNA-barcode [63] presents low interspecific divergences, or what is the same, low phylogenetic resolution in some fish families like tunas [65]. In a recent study, ∼1% average interspecific Kimura 2-parameter (K2P) distance was obtained from the phylogenetic analysis of 46 tuna COI barcodes [65], while the average interspecific K2P distance obtained from the analysis of FishTrace DNA-barcodes in 29 tuna increased to ∼1.7% [66]. These results strengthen the practical efficacy of a DNA-barcode with cytb and rhod to identify fish species. Thus, although it is clear that safer identification labels depend upon the length of the DNA-barcodes, the DNA-barcoding efficiency can also be further improved by the simultaneous use of two genes with different evolutionary rates and genomic locations. This latter approach is used in the barcoding proposed in FishTrace by the use of the complete mitochondrial cytb (1141 bp) and a nuclear fragment (460 bp) of the rhod gene, with independent genetic variation rate for each of them [75]. In fact, both cytb and rhod genes have been widely used as effective molecular markers for fish species identification and for the establishment of unresolved or unknown fish phylogenies [76–80]. The absence of introns in the fish rhodopsin [81] makes easy the PCR amplification of a representative coding sequence for a nuclear gene, and being also conserved in chondrichthyes and tetrapods [81] allows the discrimination of teleosteii from other organism taxa in processed seafood. Moreover, the use of this nuclear gene in parallel with cytb has the advantage of including an internal phylogenetic control for each other, with an increased resolution and guarantees for the identification of fishes to the species level. From the phylogenetic analyses performed within FishTrace, both mitochondrial and nuclear DNA sequence data produce similar phylogenetic tree topologies and congruency with other taxonomical-based phylogenies [82–84]. In addition, the use of two independent genes allows avoiding erroneous ascribing of DNA-barcodes and potential crossover contamination or other errors occurred during the PCR amplification. These errors can be detected since each gene sequence can be independently validated and phylogenetically analyzed to finally perform a morphological cross-checking for testing the reliability of the formed clades. Furthermore, phylogenetic analysis of both the assembled sequences (cytb + rhod) reveal that most recent evolutionary changes are
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better resolved by the cytb, whereas basal phylogenetic relationships are better defined by the rhod gene, since it shows higher conservation than cytb (less overall changes between taxa). Some other DNA sequences have also been proposed as identification labels for fish species. Among them ribosomal subunits are the most popular (16S-rRNA [13,85,86]; 12S-rRNA [14]; 18S-rRNA [28,87,90]; 5S-rRNA [27]), but nuclear genes have also been employed for fish species identification [12]. When standardized sequences (COI, cytb, rhod, etc.) from fish species are not available at the time of developing an identification method, the possibility of finding at the general databases of NCBI, EMBL, and CIB-DDBJ* DNA sequences that could be used as identification labels, still exists. With the Taxonomy Browser at the NCBI site it is possible to identify rare fish species from which DNA sequences are available. Moreover, the Tree Tool within the basic local alignment search tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi) has also the ability to display a given sequence within a dendogram of closely related sequences to that query sequence uploaded by the user. Although caution should be taken for the correct phylogenetic clustering using this Tree Tool,† an adequate identification of a fish species can be performed provided that a matched sequence is available in the database (see below Section 35.4.2.2 where the DNA identification procedures from general databases are widely covered). In addition, based on the genetic data entered into the FishTrace or FishBOL databases, tailored molecular identification systems for fish teleost species can be also specifically developed [13]. According to this approach, forensically informative nucleotide sequencing (FINS) [88] has also been adapted to design molecular diagnostic identification of fish [17] and squid [33] species, and thus simplifying the barcoding method and minimizing the potential misassignations by nonsequencing methods like PCR-restriction fragment length polymorphism (RFLP) [10] (see below: Section 35.4.3.1). The Regulatory Fish Encyclopedia (RFE: http://www.fda.gov/Food/FoodSafety/ProductSpecificInformation/Seafood/RegulatoryFishEncyclopediaRFE/default.htm) is a project launched by the Food and Drug Administration, (FDA) United States, Center for Food Safety and Applied Nutrition (CFSAN) which compile fish species data in several formats to assist accurate identification. This is basically an initiative from administrative bodies focused to identify species substitution and economic deception in the marketplace. At present (July 2009), the RFE contains data on 94 commercially relevant fish species sold in the U.S. market including “chemical taxonomic” information consisting of species-characteristic biochemical patterns for comparison to patterns obtained by an appropriate laboratory analysis of the fish query. These are mainly protein-IEF and DNA-RFLP banding gel patterns. In addition the RFE comprises anatomical information in the form of pictures of the whole fish and their marketed product forms (such as fillets) and unique taxonomic features in a “checklist” format, to aid in identification. Nevertheless, IEF and RFLP patterns from similar or taxonomically closed fish species are not available at present, making practical comparisons between potential substitution species and differential diagnosis not a straightforward analysis. It is expected that in the near future the RFE would be able to include the DNA-barcodes of those species already listed and other relevant for practical analytical purposes [89]. An accurate barcoding procedure would improve species identification, which is essential in determining associated hazards, addressing economic fraud issues, and aiding in foodborne illness outbreak investigations. * NCBI: http://www.ncbi.nlm.nih.gov/; EMBL: www.ebi.ac.uk/; CIB-DDBJ: www.ddbj.nig.ac.jp/. † The phylogenetic analyses routinely performed in the NCBI Tree Tool are fast but technically limited and do not employ the most advanced (and computer-time consuming) methodology for accurate phylogenetic performance.
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35.4.2.2
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697
Identification from General Databases
Most sequencing identification methods are based on the nonstringent PCR amplification of a gene DNA section (the mitochondrial cytochrome b, 12S-rRNA, and cytochrome oxidase are the most commonly used target genes) followed by sequencing of the PCR product. The sequence is then compared to the current nucleotide database, and the identity of the specimen is established when the nucleotide identity fulfills a minimum score with one or more known specimens in the database, usually calculated by using the algorithm utilized in the Basic Local Alignment Search Tool (BLAST) software [90]. This approach should be carefully supervised, as nonvalidated databases, like NCBI or EMBL, may lead to DNA identities with entries erroneously labeled for a given species. In addition, BLAST results are typically formatted as a list of database entries arranged by the Expectation value (E) calculated as the number of different alignments with scores equivalent to or better than the score (sum of substitution and gap scores assigned by the similarity matrix used) that are expected to occur in a database search by chance; the lower the E value, the more significant the alignment between two given sequences. However, the E value is affected by the length of the alignments. Short alignments with higher identity may yield higher E values than longer alignments displaying slightly lower identity. As some database entries contain incomplete gene sequences, identical sequence matches may be overlooked behind lower identity full-length alignments. A more structured approach, which overcomes this inconvenience, should involve a taxonomical study of the sequence, involving the generation of a tree which will fit the input sequence in the appropriate taxonomy clade [92]. The building of phylogenetic trees requires the use of identical length sequences. The method, named FINS, has been applied in a number of applications to fish and other seafood species: the identification of scombrids [91]; sardines, including canned products [18,26]; cephalopods [92]; grey mullet, including processed ovary products [93]; or fish species in surimi [6,73] are just a few examples for the applicability of direct sequencing methods. Advantages of full sequencing of relatively short DNA sequences (200–400 bp) include the taxonomical identification of the individual down to subspecies or population level, allowing also the ascription of specimens to taxonomy groups even when there is no previous sequence information on a given species. There are, however, several drawbacks for using DNA sequencing as routine food analysis: It is not a straightforward method requiring the use of sophisticated equipment (sequencers) or the use of external facilities providing sequencing services. It also requires some skills to analyze data (database comparison, alignments), and it cannot be directly used on samples containing mixtures of fish species, as mixed PCR products will deliver unreadable sequencing patterns. To facilitate the sequencing and further data analysis, attempts have been made to automate all sample processing, DNA sequencing, and data analysis, with special reference to the identification of fish larvae present in ichthyoplankton [94].
35.4.3 Non-DNA Sequencing Methods Nonsequencing methods may not always provide forensically reliable information due to the absence of all appropriate control samples from potential substitution species or even because of intraspecific variation in the species. Thus a nonsequencing method can only be forensically applied if all controls have been considered and experimentally evaluated for a given differential identification of a group of fish species and their substitutions [13]. Partial comparison of fish species groups, which in practice can comprise some more species [95,96], are not recommended for fish species identification in control laboratories.
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RFLP
RFLP is a widespread procedure for species identification in food analysis [104]. The set up of the method requires the previous collection of a significant number of specimens of the species to be identified, the sequencing of the selected target DNA region, and its comparison with the same region sequence obtained from other species, in particular close phylogenetic relatives. Thus, single nucleotide positions showing either low or no intraspecific variations, but which are polymorphic with respect to all other species, can be used as single nucleotide polymorphism (SNP) markers for species identification. To carry out an RFLP analysis, these polymorphisms should be located at a restriction site, allowing the identification of the SNP by variations in the restriction pattern of the PCR product. After a protocol is established and tested, the identification process can be performed routinely by any laboratory skilled in DNA extraction, PCR, and agarose-gel electrophoresis. These methods can be easily adapted to new technical developments, like capillary electrophoresis or Lab-on-a-Chip systems [98], which reduce laboratory manipulations and help to standardize results. The reliability of RFLP methods depends largely on its careful design and testing with target and nontarget species. RFLP methods have been used to ascertain the origin of mackerels, either by using nontranscribed regions of nuclear DNA [25,99] or a double test using both mitochondrial cytb and nuclear 5S rRNA genes [100]. Several cytb gene regions have been successfully used as target sequences in PCR-RFLP fish identification methods, including gadoids [101], salmonids [102], or flatfishes [103], whether the p53 gene has been used for salmon and trout [104] and the 16S-rRNA gene for hairtail species [105]. RFLP in short DNA fragments has also been proved useful for the analysis of highly processed food. A diagnostic system set up to identify five different species in canned tuna show that the amplifiable fragment length could not exceed 278 bp due to DNA fragmentation during the sterilization process [23]. Recently, a wider range of tuna species, Thunnus thynnus, T. alalunga, T. obesus, T. albacares, Euthynnus pelands (Katsuivonus pelamis), E. affinis, Auxis thazard, and Sarda orientalis species in canned tuna could be identified by a combination of five restriction enzymes on two cytochrome b fragments [106]. The mitochondrial control region [107] and the 5S rRNA gene [27] have been used to identify several species of hake (Merluccius sp.), both in fresh and heat-treated samples. The identification of anchovy species is a representative example for the application of FINS and PCR-RFLP methods to discriminate closely related species. Engraulis japonicus and E. encrasicolus (Japanese and European anchovies, respectively) display highly similar cytb sequences. In addition, both species showed a relatively high genetic diversity, and the existence of subspecies or cryptic species of E. encrasicolus has been proposed [17,108,109]. As PCR-RFLP methods may not be fully selective for discrimination between these two species, the additional use of FINS or alternative methods may be needed for identification. PCR-RFLP methods have been also useful for the authentication of nonfish seafood, like crustaceans [86,110] and mollusks [111,112].
35.4.3.2
AFLP, RAPD, and Satellite DNA Analysis
Amplified fragment length polymorphism (AFLP) is based on the PCR amplification of endonuclease-restricted fragments ligated to synthetic adapters and then amplified using primers which carry selective nucleotides at their 3′ ends [113,114]. After the selection of adequate restriction enzymes, usually applied by pairs, which will yield a wide assortment of genomic DNA fragments upon digestion, and the attachment of linkers to the protruding ends, several sets of selective
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primers are tested to obtain a pattern of DNA fragments after PCR amplification. Differences among species or populations are visualized by the modification of such amplification patterns, due to the presence of polymorphic sequences located next to the restriction site. The selectivity of the method can be adjusted to identify species, strains, populations, or even lineages in cultured specimens, both in fish and shellfish species, as reported for sturgeon commercial products and interspecific hybrids [115], Morone and Thunnus species [116], and a wide variety of fish, mollusks, and crustacean species with the purpose of developing an AFLP database [117]. The randomly amplified polymorphic DNA approach (RAPD) has also been widely tested for identification of fish species and populations. The RAPD method relies on the generation of a collection of DNA fragments or fingerprint by using a single or limited number of arbitrary oligonucleotides as primers. The pattern of amplification is expected to be consistent for the set of primers used, the DNA, and the conditions used. The RAPD approach has been reported to be suitable for the identification, among others, of grouper, Nile perch and wreck [118], salmonids [87], and whiting [119]. Some other examples of primers used in RAPD methods for fish authentication have been previously compiled [120]. Both AFLP and RAPD techniques share some advantages such as the relatively low cost, the requirement of only small amounts of DNA, and that they do not require previous knowledge of the species DNA sequences. However, both methods are strongly dependent on the integrity of the DNA. Thus, samples containing highly fragmented DNA, as in thermally treated food, may lead to altered banding in AFLP and RAPD patterns. In addition, samples containing two or more DNA species cannot be easily analyzed by these methods, as the banding would become too complex to discriminate each species. These two above-mentioned methods are, however, a powerful tool for the identification of sequence characterized amplified regions (SCAR). Species-specific bands identified by AFLP or RAPD are isolated from separating gels, reamplified, and sequenced. These regions can be used subsequently as targets for species identification by using a sequence-specific identification method, as DGGE, selective amplification, RFLP, or array technologies. This approach has been successfully used for the discrimination of trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) [19]. Tandemly arrayed, highly repetitive DNA sequences on eukaryotic genomes, known as satellite DNA, are highly informative to investigate inheritance patterns and for the identification of intraspecific populations. These sequences range from very short (2–4 bp) repeated sequences (microsatellites), medium sized (10–64 bp) minisatellites, or long (>64 bp) satellite sequences, extensively dispersed throughout eukaryotic chromosomes. Among these, microsatellite sequences have become the most used nuclear markers for genetic analysis in fishes, which are estimated to occur once every 10 kbp [121]. These repeats are inherited, with new variants arising in the population during recombination and segregation by changes in the copy number of the repeat unit. Genetic isolation leads to the fi xation of these variants (alleles) in the populations, in which the length of the repeat can be ascertained by PCR amplification with oligonucleotide primers encompassing the neighbor DNA region. Due to its high power of discrimination, microsatellite analysis has become the most powerful tool to study the geographical distribution of populations, as has been reported for sockeye salmon [122], horse mackerel [123], and others [124–126]. It is also one of the preferred methods in aquaculture allowing the genotyping, parentage, stock structure studies, and the traceability of the products (see Chistiakov et al. [127]). One useful application of this approach is the differentiation of wild and hatchery-produced variants of the same species, as are the cases for chinook salmon [142]. The experimental comparison of the performance of microsatellites and SNPs to differentiate wild and farmed Atlantic salmon shows that both approaches produce similar results, being SNPs more suitable for high-throughput applications [129].
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Although mainly used for stock and population studies, the technology based on DNA satellites can also be used for species identification: for example, six microsatellite loci allow the identification of up to eight different grouper species [130]. In another report, the feasibility to differentiate 3 species of North Atlantic wolffishes using 16 tetranucleotide and dinucleotide microsatellite markers has also been demonstrated [131]. The use of satellite analysis for routine species identification is however hampered due to the extensive genetic analysis required during the set up of the method. Each microsatellite locus has to be identified and its flanking region sequenced for primer design, and the validation process may become laborious and time consuming.
35.4.3.3
SSCP and DGGE
Although initially developed for the identification of SNPs, single-strand conformation polymorphisms (SSCP) [132] is a useful tool for fish product identification [133]. The method relies on the differences observed in the electrophoretic mobility of short single-stranded DNA which differ in one nucleotide position. After PCR amplification of the region of interest, the resulting doublestranded product is denatured followed by rapid chilling to prevent reannealing of the strands, and separated by electrophoresis under nondenaturing conditions. An alternate technology to visualize mobility changes based on DNA sequences is denaturing gradient gel electrophoresis (DGGE) [134], which separates single-strand DNA fragments using a gel containing chemicals which break apart the strands of the DNA molecule. Because the amplicon segments are the same length, separation must be made based on the genetic sequences rather than on size. Denaturing gels have an increasing concentration gradient (from top to bottom) of denaturing chemicals. Furthermore, as the identification is based on one or a few electrophoretic bands, these methods can be used on complex samples containing DNA from different species. Recent applications of DGGE methods to fish identification have been reported [19]. Moreover, the comparison of the efficiency obtained by RFLP, SSCP, and DGGE methods to differentiate eight cod-fish species demonstrated that RFLP and SSCP were not able to identify all species tested, but DGGE achieved the best performance [11]. In spite of some advantages, SSCP and DGGE methods are not extensively used due to their lack of robustness and requirements for a skilled interpretation of the band patterns. Thus, accurate experimental conditions have to be maintained to obtain repetitive results, as small changes in temperature, pH, and gel composition may affect the band migration pattern. In addition, these methods are rather time consuming and laborious, and therefore are not well suited for routine monitoring laboratories.
35.4.3.4
Selective Amplification
Under selective amplification we include all PCR methods which deliver a species-specific amplification product under stringent conditions. This may be achieved by selecting short oligonucleotides that match DNA sequences found exclusively in the target species, either as primers for the Taq DNA polymerase, as internal probes in the amplicons, or both. Compared to other PCR-based identification methods, selective amplification is faster and simpler, as it requires little processing after the PCR has been completed. Lockley and Bardsley (2000) [135] used a single-step PCR for discrimination of tuna (T. thynnus) and bonito (Sarda sarda). The identification may be based on the direct detection of the expected size amplicon in agarose gels after electrophoresis, monitorization of double-chain DNA products by using nonselective fluorochromes like SYBRgreen, or the detection of fluorescence along the PCR using sequence-specific fluorescently labeled DNA probes
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(see below). Regardless of the method for visualization, PCR-specific methods rely strongly on the adequate selection of target sequences for oligonucleotide hybridization. The exponential increase in sequence information in the last years has assisted greatly to the development of this approach for species identification. Usual targets for PCR amplification from closely related species, like the mitochondrial cytb, COX1, D-loop, 12S-rRNA genes, or nuclear 18S-rRNA gene, may differ in only a few nucleotide positions. In the less-favorable condition, the selective PCR may depend solely on melting temperature (Tm) differences of one oligonucleotide to hybridize with a singlebase mismatch in its target sequence. Alternatively, the selective base position can be located at the 3′ end of the primer, preventing the initiation of DNA polymerization after hybridization with the nontarget DNA [136,137]. The selective amplification approach can be expanded in several ways. By combination of several primer pairs, or species-specific primers with a nonselective counterpart, it is possible to identify several species in the same reaction [95,138,139]. Also, nested PCR-based methods have been developed, in which the first reaction is carried out using nonselective or family-specific primers, and the second reaction makes use of species selective primers, yielding PCR products that can be analyzed by RFLP [23,28]. This latter approach increases the discrimination power of the method. The technology developed for real-time PCR can also be used to increase specificity, reduce the labor required, or automate the analysis. The real-time approach has found two main applications in genetic studies: the detection of SNPs and the quantification of the number of copies of target DNA. The technique relies on the monitorization of the accumulation of products at every PCR cycle, which can be achieved by the addition of an oligonucleotide probe labeled with a reporter and a quencher dye that binds to a target DNA between the flanking primers. The 5′ to 3′ exonuclease activity of the Taq DNA polymerase cleaves the probe during PCR, allowing the emission of fluorescence by releasing the reporter from the quencher. In either case, the fluorescent emission would increase exponentially at every PCR cycle [140]. Methods using fluorescent species-specific oligonucleotides probes in real-time PCR have been tested for the identification of eel species based on SNPs [141]. Alternatively, a fluorescent dye (SYBRgreen) which preferentially binds to double-strand DNA can be added to the reaction replacing the oligonucleotide probe [142]. This approach lowers the assay cost and avoids the need to use a specific probe for each species analyzed. In turn, the assay specificity relies exclusively on the primer sequence, which may require confirmation of the PCR product identity after the amplification. A method for identification of grouper and other substitution species based on a multiplex PCR assay using SYBRgreen and a postreaction DNA dissociation analysis has been developed [13]. The amplicons of different size display different melting temperatures, which can be monitorized as a decrease in fluorescence due to the separation of the dye when double-strand DNA dissociates. The identification could be performed based on the appearance of PCR products displaying a melting temperature corresponding to the species analyzed, and can be improved to high throughput screening [13].
35.4.3.5
Quantitative Methods
The development of real-time PCR technology has facilitated significantly the development of techniques aiming to the quantification of animal and vegetal ingredients in processed food, where mixtures of materials from different species are easily found, and there is an increasing need to differentiate purposely added ingredients from trace contaminant materials which may derive from the manufacturing process. The limit of detection of DNA-based qualitative methods may be well below the legal limits for a component to be considered as ingredient, and can therefore lead to conflictive results in the detection of frauds.
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As the amount of product accumulated at each PCR cycle is proportional to the initial copy number of the target DNA, it can be quantified using fluorescent monitored real-time PCR by comparison of the cycle at which the unknown and a standard DNA of known concentration reach the same fluorescence [143]. Although most protocols of quantitative real-time PCR for food authentication have been devoted to meat products [37,144,145] or transgenic material (see [146] and references therein), a few examples on applications to fish products have been reported. Thus, the feasibility to quantify albacore and yellowfin in binary mixtures [22] and the detection and precise quantification of haddock in mixed samples [40] are first-rate models for potential development of similar diagnostic systems in other groups of fish species. So far, considering the need for validation tests required for fish species identification, and the fact that quantification of DNA is a valuable data (although may not fully correlate with the amount of raw material or protein in the sample), real-time PCR appears as the most versatile and effective method for quantification of components in food products and mixtures.
35.4.3.6 High-Throughput, Microarray Technologies and Bioinformatics While most DNA-based methods can be successfully used for the identification of farmed species used in food manufacturing, setting up methods for identification of species from extractive fishing may become extremely laborious due to the very high diversity of species and the presence of intraspecific genetic variations. Compared to specific-amplification, RFLP, and other speciesspecific closed methods, DNA sequencing is an open method which may provide information on almost any species or variant. However, faster methods suitable for high-throughput analysis would be desirable for routine labs. The application of array technologies, widely used in SNP identification and gene expression applications, can be particularly suited for fish species identification as they have the potential to handle and identify hundreds of species in parallel. In their standard format, DNA microarrays are microscope slides on which oligonucleotides are spotted, whose DNA sequence are complementary to the DNA target sequences. The sample DNA is amplified by PCR using fluorescent-labeled primers flanking the hybridization region. The labeled PCR product hybridizes with the immobilized oligonucleotide on the microarray, and can be detected after washing steps. Despite some inherent drawbacks, as the high cost, methodological difficulties for standardization, and relatively high interlaboratory variability, there are examples of arrays developed for the identification of marine organisms in plankton [147,148] and fish species [149]. In this last report, a prototype assay with 11 commercial fish species were targeted for identification by using a single oligonucleotide probe (23–27 nucleotides long) from the 16S-rRNA gene per species, and a 600 bp fluorescent-labeled PCR product for hybridization. True-positive signals could be differentiated from false-positive due to their higher fluorescent signal, although those signal intensities were heterogeneous. This heterogeneity is common in hybridization array methods, being likely caused by the dependence of hybridization efficiency on several complex parameters like the nucleotide sequence, steric hindrance, secondary structures, and the relative position of the label at the target. This variability often leads to problems of reproducibility both intra- and interlaboratorily, being the main drawback for utilization as a general identification tool. In turn, this method can be the only practical approach for the routine analysis of very complex samples (e.g., plankton), in which a wide variety of known and unknown species can be found. Future improvements on the microarray design, like the use of multiple probe sets for each species to be identified, redundant hybridization using both DNA strands, addition of multiple-labeled target PCR products, and others, may allow a broader utilization of this methodology.
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An interesting alternative to the hybridization array approach for high-throughput analysis is the use of primer extension technology. These systems are based on the identification of polymorphisms by using a primer that hybridizes immediately upstream of the SNP, and is extended by just one base in the presence of a fluorescent-labeled dideoxynucleotide. The polymorphisms are detected by the emission of fluorescent signal at located spots (array) [150,151], or at specific peaks after separation by capillary electrophoresis or combined primer extension-capillary electrophoresis (SnaPshot, Applied Biosystems, Foster City, California). Following this approach, the efficiency of a multiplex primer-extension assay for the identification of five tuna species has been recently demonstrated [74]. After amplification of a 132 bp region from the cytb gene, the PCR product is used as template for simultaneous single-nucleotide extension using four primers which hybridize next to diagnostic base positions and have different lengths by the inclusion of poly(T) tails at the 5′ end. All four A, G, T, C dideoxynucleotides, each labeled with a different fluorochrome, are used in the reaction, and the resulting products are separated by capillary electrophoresis using a standard sequencing equipment. The resulting pattern of labeled bands allowed the unambiguous identification of five Thunnus species. As primer extension protocols require only very short DNA fragments for PCR amplification [152], they appear as a promising alternative for the analysis of highly processed food products.
35.5 Future Prospects Legislation and law enforcement of fish labeling rely on fast, efficient, and accurate diagnostic analysis of large sets of samples. At present powerful molecular technologies are available, but mostly partial diagnostic systems have only been provided by the scientific community to the control laboratories. Thus in Table 35.2, a suggested practical use of the different methodologies presently available for the identification of seafood species is given. Meanwhile many described Table 35.2 Recommended Fish Species Identification Methodologies for Different Types of Seafood Samples and Processing
Method
Fresh Single Species
Low Processed (Freezing, Mincing)
Highly Processed (High Temperature, Additives)
Mixed Species
Sequencing
A
A
AR
NA
RFLP
A
A
AR
AR
AFLP, RAPD
A
A
NA
NA
Microsatellite
AR
AR
NA
NA
SSCP
AR
AR
AR
NA
Selective PCR
A
A
AR
A
Array
A
A
AR
A
Quantitative PCR
A
A
AR
A
A, Adequate; AR, adequate with restrictions; NA, not adequate.
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molecular methods that show laboratory viability have limited use for routine analysis. In the near future we envisage that high-throughput technologies for DNA analysis associated to the automation of sample handling and processing that is controlled with specialized software and database identification could pave the path for an effective species diagnosis in seafood to overcome adulteration, mislabeling, and realistic fisheries control.
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708 ◾ Handbook of Seafood and Seafood Products Analysis 72. Zardoya, R. and Meyer, A., Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates, Molecular Biology and Evolution 13 (7), 933–942, 1996. 73. Pepe, T., Trotta, M., Di Marco, I., Cennamo, P., Anastasio, A., and Cortesi, M. L., Mitochondrial cytochrome b DNA sequence variations: An approach to fish species identification in processed fish products, Journal of Food Protection 68 (2), 421–425, 2005. 74. Bottero, M. T., Dalmasso, A., Cappelletti, M., Secchi, C., and Civera, T., Differentiation of five tuna species by a multiplex primer-extension assay, Journal of Biotechnology 129 (3), 575–580, 2007. 75. Vawter, L. and Brown, W. M., Nuclear and mitochondrial DNA comparisons reveal extreme rate variation in the molecular clock, Science 234 (4773), 194–196, 1986. 76. Zardoya, R. and Doadrio, I., Molecular evidence on the evolutionary and biogeographical patterns of European cyprinids, Journal of Molecular Evolution 49 (2), 227–237, 1999. 77. Farias, I. P., Orti, G., Sampaio, I., Schneider, H., and Meyer, A., The cytochrome b gene as a phylogenetic marker: The limits of resolution for analyzing relationships among cichlid fishes, Journal of Molecular Evolution 53 (2), 89–103, 2001. 78. Chen, T. Y., Hsieh, Y. W., Tsai, Y. H., Shiau, C. Y., and Hwang, D. F., Identification of species and measurement of tetrodotoxin in dried dressed fillets of the puffer fish, Lagocephalus lunaris, Journal of Food Protection 65 (10), 1670–1673, 2002. 79. Dettai, A. and Lecointre, G., Further support for the clades obtained by multiple molecular phylogenies in the acanthomorph bush, Comptes Rendus Biologies 328 (7), 674–689, 2005. 80. Jimenez, J., Schonhuth, S., Lozano, I. J., González, J. A., Sevilla, R. G., Diez, A., and Bautista, J. M., Morphological, ecological, and molecular analyses separate Muraena augusti from Muraena helena as a valid species, Copeia, 1007 (1), 101–113, 2007. 81. Venkatesh, B., Ning, Y., and Brenner, S., Late changes in spliceosomal introns define clades in vertebrate evolution, Proceedings of National Academy of Sciences U S A 96 (18), 10267–10271, 1999. 82. Nelson, J. S., Fishes of the World, 4th edn., John Wiley and Sons, Inc., New York, 2006. 83. Stiassny, M. L. J. and Moore, J. A., A review of the pelvic girdle of acanthomorph fi shes, with comments on hypotheses of acanthomorph intrarelationships, Zoological Journal of the Linnean Society 104 (3), 209–242, 1992. 84. Inoue, J. G., Miya, M., Tsukamoto, K., and Nishida, M., Basal actinopterygian relationships: A mitogenomic perspective on the phylogeny of the “ancient fish,” Molecular Phylogenetics and Evolution 26 (1), 110–120, 2003. 85. Akasaki, T., Saruwatari, T., Tomonaga, H., Sato, S., and Watanabe, Y., Identification of imported Chirimen at the genus level by a direct sequencing method using mitochondrial partial 16S rDNA region, Fisheries Science 72 (3), 686–692, 2006. 86. Brzezinski, J. L., Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method, Journal of Food Protection 68 (9), 1866–1873, 2005. 87. Jin, L., Cho, j. G., Seong, K. B., Park, J. Y., Kong, I. S., and Hong, Y. K., 18 rRNA gene sequences and random amplified polymorphic DNA used in discriminating Manchurian trout from other freshwater salmonids, Fisheries Science 72 (4), 903–905, 2006. 88. Bartlett, S. E. and Davidson, W. S., FINS (Forensically informative nucleotide sequencing): A procedure for identifying the animal origin of biological specimens, BioTechniques 12 (3), 408–411, 1992. 89. Yancy, H. F., Zemlak, T. S., Mason, J. A., Washington, J. D., Tenge, B. J., Nguyen, N. L., Barnett, J. D. et al., Potential use of DNA barcodes in regulatory science: Applications of the Regulatory Fish Encyclopedia, Journal of Food Protection 71 (1), 210–217, 2008. 90. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J., Basic local alignment search tool, Journal of Molecular Biology 215 (3), 403–410, 1990. 91. Paine, M. A., McDowell, J. R., and Graves, J. E., Specific identification of western Atlantic Ocean scombrids using mitochondrial DNA cytochrome C oxidase subunit I (COI) gene region sequences, Bulletin of Marine Science 80 (2), 353–367, 2007.
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92. Chapela, M. J., Sotelo, C. G., Calo-Mata, P., Perez-Martin, R. I., Rehbein, H., Hold, G. L., Quinteiro, J., Rey-Mendez, M., Rosa, C., and Santos, A. T., Identification of cephalopod species (Ommastrephidae and Loliginidae) in seafood products by forensically informative nucleotide sequencing (FINS), Journal of Food Science 67 (5), 1672–1676, 2002. 93. Murgia, R., Tola, G., Archer, S. N., Vallerga, S., and Hirano, J., Genetic identification of grey mullet species (Mugilidae) by analysis of mitochondrial DNA sequence: Application to identify the origin of processed ovary products (bottarga), Marine Biotechnology 4 (2), 119–126, 2002. 94. Richardson, D. E., Vanwye, J. D., Exum, A. M., Cowen, R. K., and Crawford, D. L., Highthroughput species identification: From DNA isolation to bioinformatics, Molecular Ecology Notes 7 (2), 199–207, 2007. 95. Asensio, L., Gonzalez, I., Fernandez, A., Cespedes, A., Rodriguez, M. A., Hernandez, P. E., Garcia, T., and Martin, R., Identification of nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene, Journal of the AOAC International 84 (3), 777–781, 2001. 96. Aranishi, F., Okimoto, T., and Izumi, S., Identification of gadoid species (Pisces, Gadidae) by PCRRFLP analysis, Journal of Applied Genetics 46 (1), 69–73, 2005. 97. Meyer, R., Höfelein, C., Lüthy, J., and Candrian, U., Polymerase chain reaction-restriction fragment length polymorphism analysis: A simple method for species identification, Journal of the AOAC International 78, 1542–1551, 1995. 98. Dooley, J. J., Sage, H. D., Clarke, M. A. L., Brown, H. M., and Garrett, S. D., Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: Application to detect white fish species in food products and an interlaboratory study, Journal of Agricultural and Food Chemistry 53 (9), 3348–3357, 2005. 99. Aranishi, F., PCR-RFLP analysis of nuclear nontranscribed spacer for mackerel species identification, Journal of Agricultural and Food Chemistry 53 (3), 508–511, 2005. 100. Karaiskou, N., Triantafyllidis, A., and Triantaphyllidis, C., Discrimination of three Trachurus species using both mitochondrial- and nuclear-based DNA approaches, Journal of Agricultural and Food Chemistry 51 (17), 4935–4940, 2003. 101. Calo-Mata, P., Sotelo, C. G., Perez-Martin, P. I., Rehbein, H., Hold, G. L., Russell, V. J., Pryde, S. et al., Identification of gadoid fish species using DNA-based techniques, European Food Research and Technology 217 (3), 259–264, 2003. 102. Russell, V. J., Hold, G. L., Pryde, S. E., Rehbein, H., Quinteiro, J., Rey-Mendez, M., Sotelo, C. G., Perez-Martin, R. I., Santos, A. T., and Rosa, C., Use of restriction fragment length polymorphism to distinguish between salmon species, Journal of Agricultural and Food Chemistry 48 (6), 2184–2188, 2000. 103. Sotelo, C. G., Calo-Mata, P., Chapela, M. J., Perez-Martin, R. I., Rehbein, H., Hold, G. L., Russell, V. J. et al., Identification of flatfish (Pleuronectiforme) species using DNA-based techniques, Journal of Agricultural and Food Chemistry 49 (10), 4562–4569, 2001. 104. Carrera, E., García, T., Céspedes, A., González, I., Fernández, A., Asensio, L. M., Hernández, P. E., and Martín, R., Identification of smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using PCR-restriction fragment length polymorphism of the p53 gene, Journal of the AOAC International 83, 341346, 2000. 105. Chakraborty, A., Aranishi, F., and Iwatsuki, Y., Polymerase chain reaction-restriction fragment length polymorphism analysis for species identification of hairtail fish fillets from supermarkets in Japan, Fisheries Science 73 (1), 197–201, 2007. 106. Lin, W. F. and Hwang, D. F., Application of PCR-RFLP analysis on species identification of canned tuna, Food Control 18 (9), 1050–1057, 2007. 107. Quinteiro, J., Vidal, R., Izquierdo, V., Sotelo, C. G., Chapela, M. J., Perez-Martin, R. I., Rehbein, H. et al., Identification of hake species (Merluccius genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences, Journal of Agricultural and Food Chemistry 49 (11), 5108–5114, 2001.
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108. Bembo, D. G., Carvalho, G. R., Snow, M., Cingolani, N., and Pitcher, T., Stock discrimination among European anchovies Engraulis encrasicolus, by means of PCR-amplified mitochondrial DNA analysis., Fishery Bulletin 94, 31–40, 1995. 109. Magoulas, A., Castilho, R., Caetano, S., Marcato, S., and Patarnello, T., Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy (Engraulis encrasicolus), Molecular Phylogenetics and Evolution 39 (3), 734–746, 2006. 110. Bossier, P., Wang, X. M., Catania, F., Dooms, S., Van Stappen, G., Naessens, E., and Sorgeloos, P., An RFLP database for authentication of commercial cyst samples of the brine shrimp Artemia spp. (International Study on Artemia LXX), Aquaculture 231 (1–4), 93–112, 2004. 111. Chapela, M. J., Sotelo, C. G., and Perez-Martin, R. I., Molecular identification of cephalopod species by FINS and PCR-RFLP of a cytochrome b gene fragment, European Food Research and Technology 217 (6), 524–529, 2003. 112. Fernandez, A., Garcia, T., Gonzalez, I., Asensio, L., Rodriguez, M. A., Hernandez, P. E., and Martin, R., Polymerase chain reaction-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment for authentication of four clam species, Journal of Food Protection 65 (4), 692–695, 2002. 113. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., and Kuiper, M., AFLP: A new technique for DNA fingerprinting, Nucleic Acids Research 23, 4407–4414, 1995. 114. Papa, R., Troggio, M., Ajmone-Marsan, P., and Nonnis-Marzano, F., An improved protocol for the production of AFLP markers in complex genomes by means of capillary electrophoresis, Journal of Animal Breeding and Genetics 122, 62–68, 2005. 115. Congiu, L., Fontana, F., Patarnello, T., Rossi, R., and Zane, L., The use of AFLP in sturgeon identification, Journal of Applied Ichtiiology 18, 286–289, 2002. 116. Han, K. P. and Ely, B., Use of AFLP analyses to assess genetic variation in Morone and Thunnus species, Marine Biotechnology 4 (2), 141–145, 2002. 117. Maldini, M., Marzano, F. N., Fortes, G. G., Papa, R., and Gandolfi, G., Fish and seafood traceability based on AFLP markers: Elaboration of a species database, Aquaculture 261 (2), 487–494, 2006. 118. Asensio, L., Gonzalez, I., Fernandez, A., Rodriguez, M. A., Lobo, E., Hernandez, P. E., Garcia, T., and Martin, R., Application of random amplified polymorphic DNA (RAPD) analysis for identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets, Journal of Food Protection 65 (2), 432–435, 2002. 119. Bektas, Y. and Belduz, A. O., Molecular characterization of the whiting (Merlangius merlangus euxinus nordmann, 1840) in Turkish Black Sea coast by RAPD analysis, Journal of Animal and Veterinary Advances 6, 739–744, 2007. 120. Bossier, P., Authentication of seafood products by DNA patterns, Journal of Food Science 64 (2), 189–193, 1999. 121. Wright, J. M., DNA fingerprintig in fishes, in Biochemistry and Molecular Biology of Fishes, Hochachka, P. W. and Mommsen, T. Elsevier, Amsterdam, the Netherlands, 1993, pp. 58–91. 122. Beacham, T. D., Lapointe, M., Candy, J. R., McIntosh, B., MacConnachie, C., Tabata, A., Kaukinen, K., Deng, L. T., Miller, K. M., and Withler, R. E., Stock identification of Fraser River sockeye salmon using microsatellites and major histocompatibility complex variation, Transactions of the American Fisheries Society 133 (5), 1117–1137, 2004. 123. Kasapidis, P. and Magoulas, A., Development and application of microsatellite markers to address the population structure of the horse mackerel Trachurus trachurus, Fisheries Research 89 (2), 132–135, 2008. 124. Zatcoff, M. S., Ball, A. O., and Sedberry, G. R., Population genetic analysis of red grouper, Epinephelus morio, and scamp, Mycteroperca phenax, from the southeastern U.S. Atlantic and Gulf of Mexico, Marine Biology 144 (4), 769–777, 2004.
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125. Nielsen, E. E., Hansen, M. M., and Meldrup, D., Evidence of microsatellite hitch-hiking selection in Atlantic cod (Gadus morhua L.): Implications for inferring population structure in nonmodel organisms, Molecular Ecology 15 (11), 3219–3229, 2006. 126. Yu, H. T., Lee, Y. J., Huang, S. W., and Chiu, T. S., Genetic analysis of the populations of Japanese anchovy (Engraulidae: Engraulis japonicus) using microsatellite DNA, Marine Biotechnology 4, 471–479, 2002. 127. Chistiakov, D. A., Hellemans, B., and Volckaert, F. A. M., Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics, Aquaculture 255 (1–4), 1–29, 2006. 128. Withler, R. E., Rundle, T., and Beacham, T. D., Genetic identification of wild and domesticated strains of chinook salmon (Oncorhynchus tshawytscha) in southern British Columbia, Canada, Aquaculture 272, S161–S171, 2007. 129. Rengmark, A. H., Slettan, A., Skaala, O., Lie, O., and Lingaas, F., Genetic variability in wild and farmed Atlantic salmon (Salmo salar) strains estimated by SNP and microsatellites, Aquaculture 253 (1–4), 229–237, 2006. 130. Koedprang, W., Na-Nakorn, U., Nakajima, M., and Taniguchi, N., Evaluation of genetic diversity of eight grouper species Epinephelus spp. based on microsatellite variations, Fisheries Science 73 (2), 227–236, 2007. 131. McCusker, M. R., Paterson, I. G., and Bentzen, P., Microsatellite markers discriminate three species of North Atlantic wolffishes (Anarhichas spp.), Journal of Fish Biology 72 (2), 375–385, 2008. 132. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K., Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain-reaction, Genomics 5 (4), 874–879, 1989. 133. Rehbein, H., Kress, G., and Schmidt, T., Application of PCR-SSCP to species identification of fishery products, Journal of the Science of Food and Agriculture 74 (1), 35–41, 1997. 134. Muyzer, G., Dewaal, E. C., and Uitterlinden, A. G., Profiling of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reaction-amplified genes-coding for 16s ribosomal-RNA, Applied and Environmental Microbiology 59 (3), 695–700, 1993. 135. Lockley, A. K. and Bardsley, R. G., Novel method for the discrimination of tuna (Thunnus thynnus) and bonito (Sarda sarda) DNA, Journal of Agricultural and Food Chemistry 48 (10), 4463–4468, 2000. 136. Kwok, S., Kellog, D. E., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J., Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies, Nucleic Acids Research 18, 999–1005, 1990. 137. Hird, H., Goodier, R., Schneede, K., Boltz, C., Chisholm, J., Lloyd, J., and Popping, B., Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication, Food Additives and Contaminants 21 (11), 1035–1040, 2004. 138. Infante, C., Crespo, A., Zuasti, E., Ponce, M., Perez, L., Funes, V., Catanese, G., and Manchado, M., PCR-based methodology for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products, Food Research International 39 (9), 1023–1028, 2006. 139. Infante, C. and Manchado, M., Multiplex-polymerase chain reaction assay for the authentication of the mackerel Scomber colias in commercial canned products, Journal of the AOAC International 89 (3), 708–711, 2006. 140. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H., Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase, Proceedings of the Natural Academy of Sciences 88, 7276–7280, 1991. 141. Itoi, S., Nakaya, M., Kaneko, G., Kondo, H., Sezaki, K., and Watabe, S., Rapid identification of eels Anguilla joponica and Anguilla anguilla by polymerase chain reaction with single nucleotide polymorphism-based specific probes, Fisheries Science 71 (6), 1356–1364, 2005. 142. Wittwer, C. T., Herrmann, M. G., Moss, A. A., and Rasmussen, R. P., Continuous fluorescence monitoring of rapid cycle DNA amplification, Biotechniques 22, 134–138, 1997.
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143. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R., Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions, Bio/Technology 11, 1026–1030, 1993. 144. Brodmann, P. D. and Moor, D., Sensitive and semi-quantitative TaqMan™ real-time polymerase chain reaction systems for the detection of beef (Bos taurus) and the detection of the family Mammalia in food and feed, Meat Science 65, 599–607, 2003. 145. Sawyer, J., Wood, C., Shanahan, D., Gout, S., and McDowell, D., Real-time PCR for quantitative meat species testing, Food Control 14, 579–583, 2003. 146. Rodriguez-Lazaro, D., Lombard, B., Smith, H., Rzezutka, A., D’Agostino, M., Helmuth, R., Schroeter, A. et al., Trends in analytical methodology in food safety and quality: Monitoring microorganisms and genetically modified organisms, Trends in Food Science & Technology 18 (6), 306–319, 2007. 147. Rosel, P. E. and Kocher, T. D., DNA-based identification of larval cod in stomach contents of predatory fishes, Journal of Experimental Marine Biology and Ecology 267 (1), 75–88, 2002. 148. Kiesling, T. L., Wilkinson, E., Rabalais, J., Ortner, P. B., McCabe, M. M., and Fell, J. W., Rapid identification of adult and naupliar stages of copepods using DNA hybridization methodology, Marine Biotechnology 4 (1), 30–39, 2002. 149. Kochzius, M., Nolte, M., Weber, H., Silkenbeumer, N., Hjorleifsdottir, S., Hreggvidsson, G. O., Marteinsson, V. et al., DNA microarrays for identifying fishes, Marine Biotechnology 10 (2), 207–217, 2008. 150. Shumaker, J. M., Metspalu, A., and Caskey, C. T., Mutation detection by solid phase primer extension, Human Mutation 7 (4), 346–354, 1996. 151. Kurg, A., Tonisson, N., Georgiou, I., Shumaker, J., Tollett, J., and Metspalu, A., Arrayed primer extension: Solid-phase four-color DNA resequencing and mutation detection technology, Genetic Testing 4 (1), 1–7, 2000. 152. Sanchez, J. J. and Endicott, P., Developing multiplexed SNP assays with special reference to degraded DNA templates, Nature Protocols 1 (3), 1370–1378, 2006.
Chapter 36
Veterinary Drugs Anton Kaufmann Contents 36.1 36.2 36.3 36.4
Introduction ..................................................................................................................713 Veterinary Drugs Used in Aquaculture .........................................................................714 Origin of Veterinary Drug Residues ..............................................................................716 Analytical Techniques Used for the Analysis of Veterinary Drugs .................................717 36.4.1 Biological Test Systems.....................................................................................717 36.4.2 Instrumental Analytical Approaches ................................................................717 36.5 Veterinary Drugs Used in Aquaculture .........................................................................721 36.5.1 Bactericidal Drugs (Antibiotics) .......................................................................721 36.5.2 Antimycotica ................................................................................................... 727 36.5.3 Tranquilizers ................................................................................................... 728 36.5.4 Antiparasitica .................................................................................................. 728 36.5.5 Anthelmintic Drugs .........................................................................................729 36.5.6 Hormones ....................................................................................................... 730 36.6 Future Developments ................................................................................................... 730 References ................................................................................................................................731
36.1
Introduction
The term antibiotics was originally reserved for substances, produced by fungi or bacteria, which are capable to kill microorganisms. Semisynthetic or synthetic substances lethal to microorganisms are often included under the term antibiotics. The following agents cannot be considered antibiotics: Anthelmintica (active against parasites like nematodes), antimycotica (against moulds 713
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and fungi), and hormones (used to control the sex ratio of males to females of fishes). However, all these substances can be conveniently grouped under the term “veterinary drugs.”
36.2 Veterinary Drugs Used in Aquaculture Aquaculture is a multibillion industry with a production value of more than 70 billion US$ in 2004 [1]. Sixty-nine percent of this production volume is located in China and 22% in the rest of Asia-Pacific [1]. These figures might be misleading, since a country like Ecuador exports almost its entire shrimp production, while only a fraction of the aquaculture output of China is being exported. Still, Asia is strongly dominating the aquaculture industry. This relates not only to the production volume but also to the technology, which is increasingly developed in Asia and then spreads to other production regions. Therefore, it is hardly possible to overemphasize the importance of Asia. From the Western perspective, aquaculture in Asia is still associated with rice fields stocked with fish, which fertilize the rice plants and provide valuable food proteins to the farmers at the same time. Although, such a symbiotic form of aquaculture still exists, it is definitely not responsible for the enormous growth of cultured fish in Asia. Aquaculture is a veritable industry, requiring large investments and enormous know-how to be successful. This is the major reason why aquaculture is hardly practiced in Africa [1]. Tilapia, which came originally from the great African lakes are produced in much larger quantities in the Philippines and Indonesia than in sub-Saharan Africa. This has mostly to do with the lack of infrastructure in sub-Saharan Africa (transportation, reliable electricity supply, skilled manpower, etc.). Many fish farms have more similarities with an industrial site than with a traditional fishpond. There are a number of reasons for this change. First, traditional fishponds permit only a low production volume. Second, lack of oxygen, remaining feeds, and produced excrements, are factors, which limits the number of fishes per volume of water. Third, traditional fishponds are difficult to sterilize as required after an outbreak of a disease. They might be infested by snails, which can be the host of many parasites and diseases that infect fishes and even humans. Using concrete basins is more expensive, but superior in many ways. It is easier to transfer fishes from one basin to another, and they can be more easily cleaned and sterilized if necessary. Maintenance of essential parameters like oxygen concentration, pH, ammonia levels, temperature, and the like can be controlled with less effort, permitting higher fish densities and faster growth rates. Still aquaculture can be a high-risk business. The outbreak of disease has led to huge production losses and even to the collapse of the industry in certain regions [1,2]. Fishes and shrimps are susceptible to a number of diseases caused by bacteria, virus, or fungus. Furthermore, they are prey to many parasites like protozoan, trematodes, and helminths. The infection pressure caused by such bacteria, virus, or parasites might be present for a long time without causing any harm to the fish or shrimps. However, stress factors, such as low oxygen concentration, changes of pH or temperature, might weaken cultured fishes and shrimps and lead to widespread infection with possible lethal consequences. The likelihood of infections increases when production shifts from extensive to semi-intensive and intensive farming. High fish density deprives the fish of space that can lead to lesion. Even minor injuries of the skin or scales can weaken its physical barriers, permitting an infection or penetration of parasites. Fish infected by viral diseases like viral hemorrhagic septicemia (VHS) develop hemorrhages and bleeding gills, making them very susceptible to secondary bacterial infections. New disease or new strains of known disease have regularly appeared in the past and are likely to be a threat in the future [3]. Diseases have spread in the past quickly from one country to another, and have even crossed continents. One reason for such a spread is the increasing specialization within the aquaculture.
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Broodstock and postlarvae are most often purchased outside the farm. In some poorer countries like Bangladesh [1] it might be obtained wild-caught. This practice carries the inherent risk of introducing infected juveniles into the farm. A number of species cannot yet be reproduced without using wild-caught broodstock. An example is the black Tiger Prawn (Penaeus monodon), cultures of which rely on wild-caught breeders [1]. The risk associated with the introduction of such wild-caught breeders has caused a shift in many countries to the Pacific White Shrimp (Penaeus vennamei), which is commercially available as “specific pathogen-free” broodstock. It is generally accepted that aquaculture diseases are preferably prevented by prophylaxis like proper sanitary conditions, sterilization of ponds, healthy juveniles, immunization, and good aquaculture practices. Still the sudden emergence of diseases can have enormous financial consequences for the involved aquaculturist [4]. As a result, bacterial and parasitic infections are often treated with veterinary drugs. While there is a significant number of veterinary drugs permitted for treating food-producing animals, such as cattle, swine, and fowl, there are only very few drugs allowed for the use in aquaculture. One major problem is the definition of withdrawal times (time between the end of a treatment and the clearance of the drug from the organism) for aquaculture species. The metabolism and consequently the elimination rate of a drug is dependent on the body temperature of an animal. While the body temperature of mammals and birds are a rather constant parameter, fishes and shrimps are cold blooded. Furthermore, aquaculture is not a traditional way of farming in Europe and the United States. This might be the reason why little pressure was exerted on regulatory agencies to register more veterinary drugs, permitted for the use in aquaculture. In the past, antibiotics were often used in a way that cannot be called responsible. The focus was on quick results and less on sustainable aquaculture practices. This has led to the emergence of resistance among bacteria [2–4] which forced farmers to increase the concentration of the antibiotic or shift to another drug. Although some drugs are explicitly prohibited in certain drugproducing countries, they have been used because of weak legal enforcement. Banned drugs like chloramphenicol and nitrofurans have found a widespread use in China and Southeast Asia. In the year 2001/2002, food safety laboratories in Europe discovered that a large percentage of fishes and shrimps from Asia contained significant residue concentrations of these drugs. Consequently, shipments from these countries to Europe were only released after the particular lot has been thoroughly analyzed by an accredited laboratory and declared free of nitrofurans and chloramphenicol. This had enormous consequences to many farmers in Asia, some of which utilized these drugs on a regular basis. The local authorities in the drug-producing countries could not adequately respond because of a lack of technology and trained manpower. Some drug-producing countries had virtually no laboratories to control veterinary drug residues. There were cases where analytical methods designed for the assay of medicated food were used for the determination of veterinary drug residues in fish or shrimps. Since the concentration of veterinary drugs in medicated feed is rather in the low percent range, all animals tested with these insensitive methods showed “no residues.” This situation has dramatically changed in many countries where highly sophisticated analytical instruments are now operated by skilled chemists. There was indeed progress and shipments contaminated with chloramphenicol or nitrofuran significantly dropped. However, some 2 years later a new problem arose. Enrofloxacin, a veterinary drug belonging to the group of chinolons was found in many shipments. After removing a number of shipments from the market, “residue-free” products were again rather the rule and not the exception. Soon another weave of contaminated Asian seafood was discovered. It was the malachite green, an antimycotica, which has also a long history of usage in European aquaculture. This development is shown in Table 36.1 that lists the percentage of samples with detectable residues of
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Table 36.1 Percentage of Samples Tested Positive for Veterinary Drug Residues by the Official Food Authority of the Canton of Zurich between the Year 2003 and 2006 Year Product
2002
2003
2004
2005
2006
62% (n = 8)
13% (n = 53)
0% (n = 2)
0% (n = 69)
Shrimps Vietnam
0% (n = 3)
3% (n = 38)
—
—
0% (n = 13)
Shrimps Thailand
8% (n = 12)
0% (n = 18)
—
—
0% (n = 2)
Nitrofurans Fish Vietnam
0% (n = 29)
Enrofloxacine Fish Vietnam
—
—
0% (n = 2)
38% (n = 69)
10% (n = 29)
Shrimps Vietnam
—
—
—
—
0% (n = 13)
Shrimps Thailand
—
—
—
—
0% (n = 2)
—
—
—
48% (n = 69)
0% (n = 29)
Malachite green Fish Vietnam
Note: The figures indicate the percentage of positive samples for a given drug within the specified product group and year. The number in brackets refers to the number of tested samples.
nitrofurans, enrofloxacin, and malachite green over a period of 5 years. All samples were obtained from the local market in Switzerland; measurements were made at the official food authority of Zurich. Only data from two export countries are listed. This reflects their importance as exporter of seafood to Switzerland as well as the history of previous positive findings. Besides these widespread antibiotics, one case with a very high concentration of sulfadimidine (1140 mg/kg) and one sample with penicillin residues (Dicloxacilline) was discovered.
36.3 Origin of Veterinary Drug Residues It seems to be logical that veterinary drug residues are the unequivocal proof that such substances have been deliberately used to prevent or treat a disease of the particular aquatic animals. However, there were cases where this “axiom” was violated. The increasing sensitivity of analytical methods is capable in detecting minute amounts of drug concentrations, which can be possibly caused by pre- or postharvest contamination. Some veterinary drugs have extremely long depletion times. Malachite green (respectively its metabolite leuco-malachite green) was reported to deplete in European eel within 100 days from initial 700 to 15 mg/kg [5]. Such long depletion periods bear the risk that juvenile fish treated with malachite green can still contain residues after being harvested. If such fishes are purchased from an external hatchery, the aquaculturist who raises the juvenile fish might not even be aware of the presence of residues.
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Low levels of antibiotics were observed in commercially available feed [6]. Feed mills produce a variety of unmedicated and medicated products. Some antibiotics are permitted for certain animals, which are not used for food production. In such mills, carryover can occur if the equipment is not carefully cleaned after the production of a medicated feed batch. There have been reports about the contamination of crabs during processing. Workers employed in processing facilities in Southeast Asia were reported to use commercially available hand creams containing chloramphenicol [6]. Analytical chemistry has made great progress concerning sensitivity and selectivity. This has strongly reduced the likelihood of false positive findings. Problems can arise when a veterinary drug undergoes fast and extensive metabolism in the animal. Some of such drugs, e.g., nitrofurans produce relatively small breakdown products which are used as indicator that the parent substance has been administered. Semicarbazide is the indicator (metabolite) of nitrofurazone. This small molecule was found to originate also from other treatments, ingredients, packing material, and natural sources. The details will be given in the section “Nitrofurans.” The analysis of metabolites instead of the parent drug is not necessarily an unreliable way of detection. Problems only arise if metabolites that contain less diagnostic information than the parent drug are produced, e.g., as caused by breaking the molecular structure and a corresponding loss of structural information.
36.4 Analytical Techniques Used for the Analysis of Veterinary Drugs 36.4.1 Biological Test Systems The presence of an antibacterial drug inhibits the growth of microorganisms and can be utilized for their detection [7]. Some tests based on this principle are commercially available, while others require the user to prepare growth media. Generally, a drop of meat extract is put on a growth media plate, which undergoes an incubation period later. Antibacterial drugs will cause an inhibition zone around the position of the drop. The diameter of which, correlates to the microbial inhibition activity of the sample. Such tests are not very selective; however, the use of growth media with different response toward various inhibitors can give information about the type of inhibitor (class of antibiotics) present. The mentioned tests can potentially detect any antibacterial compound, however, they can respond with widely varying sensitivity toward different antibiotic groups. Good sensitivity was reported for penicillins, yet sulfonamides or nitrofurans produce weak or even no inhibition. Inhibition tests are commonly used for large-scale screening. Higher sensitivity and selectivity are obtained by immunoassay test systems [8]. Immunoassay methods like enzyme-linked immunosorbent assays (ELISA) permit the fast and highly selective screening and semiquantification of various veterinary drugs. The technique is well suited for processing large number of samples. The high selectivity caused by the recognition of an epitope of the analyzed drug is both an advantage and a limitation. Although there are reports about the development of drug class specific techniques [9], ELISA is still recognized as a “one drug one test” approach.
36.4.2 Instrumental Analytical Approaches The early days of veterinary drug residue analysis has been dominated by microbiological tests. Chemical methods were slower, less sensitive, and more cumbersome. A low concentration of
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residues has to be detected in the presence of an overwhelming number and concentration of endogenous compounds, calling for some form of sample cleanup and chromatographic or electrophoresis separation prior to the detection. Unlike pesticides, most veterinary drugs are rather large and polar molecules, which in many cases prevent their separation by gas chromatography (GC). Liquid chromatography (LC) is definitely more suited for the analysis of veterinary drugs than GC. An exception remains, the area of hormone analysis, which is still dominated by GC, coupled to mass spectrometry (GC–MS). There has to be an extraction and cleanup step prior to any separation and quantification. Depending on the group of veterinary drug, different extraction procedures are employed. They might require a polar, nonpolar, highly buffered, or strongly acid environment. This variability creates problems for designing multiresidue methods. Matrix bound drugs such as aminoglycosides require low pH for quantitative extraction. Such an environment will induce degradation of many other drugs such as penicillins and tetracyclines. The proposed alternatives to liquid extraction are matrix solid phase dispersion extraction (MSPD) and accelerated solvent extraction (ASE). MSPD is a manual, rather labor-intensive approach, while ASE is limited to thermostabile analytes. The desorption of incurred drugs from the matrix is not always easy, because some drugs are bound to certain cell compartments. Quantitative or near-quantitative extraction can require one or two re-extraction steps. Drugs from fishes and shrimps are more easily extracted than from matrices such as mammalian kidney or liver. However, shrimp extracts were often found to produce excessive foam or viscous emulsion that can create difficulties in the following cleanup steps. The extract (after centrifugation) can seldom be directly injected into an analytical instrument. Depending on the selectivity of the used analytical technique and the sensitivity requirements, cleanup, and concentration steps are required. Modern methods of veterinary drug cleanup rely heavily on solid phase extraction (SPE). Often used are reversed phase techniques such as C-18 cartridges or more modern polymeric materials such as OASIS. Such novel materials do not suffer from irreproducible silanol activities, causing nonspecific losses of basic analytes. This approach of cleanup is not orthogonal if a reversed phase LC separation follows. However, a reversed phase SPE step removes peptides and proteins, which would otherwise precipitate on the analytical column, reducing separation performance, and cause signal suppression if LC–MS is used. Acid and basic drugs are often purified by the use of an anion or cation exchanger cartridges. Such a cleanup can be highly selective, producing clean extracts. However, it is very difficult to optimize an ion-exchanger cleanup to recover many different drugs with satisfactory recovery rates. Some compounds will only be insufficiently retained, while others might not be quantitatively eluded. The underlying problem can be traced to the different pK values of the analytes, each of which requires a different pH value for best retention and elution. Hence, multimethods covering more than one drug group utilize more often the less selective reversed phase SPE cartridges. Assay of pharmaceutical formulations are often performed with LC and ultraviolet detection (LC–UV). Such a configuration, however, provides only insufficient selectivity and sensitivity for most trace analysis purposes. More widely used are fluorescence detectors (LC–FL). They permit higher sensitivity and selectivity. Unfortunately only a number of veterinary drugs possess an inherent fluorescence, e.g., chinolons. Most drugs require therefore a pre- or postcolumn derivatization before detection becomes feasible. Still LC–FL is not very satisfactory for some drugs such as aminoglycosides, nitrofurans, nitroimidazoles, and the like. The advent of LC coupled to MS (LC–MS) opened new frontiers for the analysis of veterinary drug residues. Among the many developed interfaces, only electrospray (ESI), atmospheric pressure chemical ionization (APCI), and photoionization are in use today. It was soon discovered that LC–MS is not as selective and sensitive as desired. Adducts produced by the mobile phase and endogenous compounds of equal nominal mass were recognized as relevant interferences. Hence, most of the veterinary drug analysis is performed by
Veterinary Drugs
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LC coupled to tandem mass spectrometry (LC–MS–MS). The mass selection of a precursor ion, the induced fragmentation and the selective monitoring of one or more derived product ions delivered a previously unknown level of selectivity and consequently sensitivity. Within the EU, procedures have been defined (identification points) to prevent the reporting of false positive findings. Sufficient identification points for a positive confirmation can be earned if a drug can be detected by monitoring two independent MS–MS transitions. The area ratio of these transitions and the retention time of the peaks have to correspond to the standard. Confirmations are also possible by other techniques than MS or MS–MS, however, it might require the use of several different methods (ELISA, HPLC–FL, etc.) to earn the required confidence level (identification points). To compare selectivity and sensitivity of various LC technologies, a blank fish sample was processed by a published method [10] and spiked to obtain a concentration of oxytetracycline and tetracycline corresponding to 100 mg/kg, which corresponds to the maximum residue level (MRL) of this drug. This sample was analyzed with different detection techniques. Figure 36.1 gives an impression about the selectivity provided by LC–UV and LC–FL after postcolumn derivatization [10]. Figure 36.2
UV-detection 8 Tetracycline
mAU
6 Oxytetracycline 4 2 0 –2 0
5
10
15
20
min
FL-detection 8
LU
Tetracycline
Oxytetracycline
6 4 2 0 –2 0
5
10
15
20
min
Figure 36.1 Chromatograms of a fish sample containing 100 mg/kg oxy- and tetracycline. Higher selectivity and more stabile baseline are obtained by postcolumn derivatization and fluorescence (bottom) detection than by UV (top) detection.
720 ◾ Handbook of Seafood and Seafood Products Analysis 461 3.33e5
Oxytetracycline
%
90
SIM –10 1.00
2.00
3.00
4.00
5.00
2.00
3.00
4.00
5.00
90 %
Oxytetracycline
6.00 7.00 MRM of 7 channels ES+ 461 > 426 4.71e3 MS–MS
–10 1.00
6.00
7.00
Time
Figure 36.2 Chromatograms of a fish sample containing 100 mg/kg oxy- and tetracycline. Measurements were made by a quadrupole. Single stage quadrupole (top) give lower selectivity and sensitivity than triple quadrupole (bottom). The MS–MS transition depicted represents the MRM trace of oxytetracycline.
compares a single ion resolution MS trace obtained from a quadrupole instrument versus a MS–MS trace measured by the same instrument. Instead of using MS–MS as a tool for increasing selectivity, high resolution MS can produce similar results. Figure 36.3 depicts two reconstructed LC-TOF chromatograms. The chromatogram at the top uses a unit mass resolution of 1 Da. This degree of resolution is typically provided by LC–MS (quadrupole). The bottom chromatogram shows the same time of flight (TOF) chromatogram where a narrower mass window (0.02 Da) was extracted. Both chromatograms use the same scale. Note signal intensity is not affected, while a significant reduction of endogenous interferences and noise are observed. LC–MS–MS is a quantitative technique although in many cases it requires sophisticated calibration procedures to avoid or compensate ion source related matrix effects caused by endogenous compounds present in the sample. Ion trap instruments permitting MS–MS experiments have been used less frequently than tandem mass spectrometers, probably due to the larger standard deviation of measured mass peaks and the relatively long dwell times. The older instruments suffered from limited trap capacities (space charging). This might be less an issue with modern instruments. The recent introduction of linear traps has probably opened new opportunities. The more classical sector instruments are considered too bulky, slow, and expensive for most veterinary drug analysis. Multiresidue methods for veterinary drugs are not yet as widespread as in the field of the pesticide analysis. Th is is related to the widely varying chemical and physical properties of the different veterinary drugs, as indicated by the chemical structures given in Section 36.5.1. Veterinary drugs cover the whole polarity spectra, making it difficult to ensure a complete extraction from the tissue and separation in one chromatographic run. Some analytes lack functional groups required for a sensitive and selective detection. Others, such as nitrofurans, are covalently bound to tissues and require a liberation step. Still there were intensive efforts to
Veterinary Drugs
%
100
◾
721
461.156 300
TOF 1 Da Oxytetracycline
0 2.00
4.00
6.00
8.00
100 TOF 0.02 Da
10.00 461.156.0.02Da 300
%
Oxytetracycline
0
Time 2.00
4.00
6.00
8.00
10.00
Figure 36.3 Chromatograms of a fish sample containing 100 mg/kg oxy- and tetracycline. Measurements were made by an UPLC-TOF instrument. A 1 Da mass extraction window (corresponding to single stage quadrupole resolution) is shown at the top. The selectivity provided by TOF is shown by the chromatogram depicted at the bottom, where a narrow mass extraction window of 0.02 Da is shown.
include not only several members of one drug class, but also several different drug classes in one analytical method. Multimethods capable to analyze more than one class of veterinary drugs are almost exclusively based on LC–MS(/MS), specifically by triple quadrupole instruments. A promising approach is the use of emerging high-resolution techniques like TOF or possible Orbitrap. High resolution improves the selectivity as compared to a unit resolution single stage quadrupole. An important advantage of the mentioned high-resolution technique is the provided full scan signal. Th is permits the postanalysis extraction of any desired mass trace. Triple quadrupole rely on the multireaction mode (MRM) which requires the preanalysis definition of analyte specific transitions (precursor ion, product ion mass, and appropriate collision energy). In the case of multimethods, the setting up of dozens of transitions reduces sensitivity because of the reduced dwell time. Th is problem can be reduced by defining time programmed MRM acquisition windows. However, drifting analyte peak retention times can complicate such an approach. Readjustments of time windows are required, if one or more peaks move out of a predefined retention time window.
36.5 Veterinary Drugs Used in Aquaculture 36.5.1
Bactericidal Drugs (Antibiotics)
Sulfonamides represent a group of antibacterial drugs that are exclusively produced in a synthetical manner (Figure 36.4). They are antibacterial and antiprotozoal and show activity against a broad-spectrum of Grampositives and Gram-negatives. These substances have been in use for more than 50 years, which is probably the reason for widespread resistance of animal pathogens to sulfonamides. However, due
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Handbook of Seafood and Seafood Products Analysis O H2N
S
NH2
Sulfanilamide
O
CH3
N
O
Sulfamethazine H 2N
S O
Figure 36.4
N H
N
CH3
Sulfonamides.
to their low price, sulfonamides are still widely in use. They are reported to be often administered in combination with “potentizers” such as ormetoprim and trimethoprim that enlarges their therapeutic range. There are virtually hundreds of different sulfonamide drugs described in the literature, some of which are registered for veterinary use. Sulfonamides are rather stable molecules (pH and temperature) that facilitates their analysis. Significant concentrations of metabolites are not observed, unless in urine, where the N-acetyl metabolites dominate. There are a number of microbiological tests (ELISA, Charm, etc.) available, which cover one specific sulfonamide or are capable for the detection of generic sulfonamide structures [9]. Extraction with medium polar solvents is exhaustive and detection facilitated by the presence of functional groups, which even permits UV detection. The derivatization of the amino group (pre- or postcolumn) allows sensitive fluorescence detection. LC–MS–MS in the positive ESI mode permits quantification of sulfonamides [11] together with potentiators [12]. Tetracycline is an antibiotic produced by Streptomyces. Semisynthetic derivates such as chlortetracycline or doxycycline have enlarged this drug group. Tetracyclines are broad-spectrum drugs with activity against Gram-positives and Gram-negatives. Due to their activity and low price, tetracyclines have been widely used in aquaculture and animal husbandry. Consequently, resistance is a common problem (Figure 36.5). Tetracyclines are significantly less stable than sulfonamides, they form epimeres as well as complexes with metals, which complicate quantification. There is an extensive set of substance specific microbiological tests available to detect tetracyclines at residue levels. Extraction of tetracyclines is based on acid aqueous buffers containing complex agents such as oxalic acid and EDTA. UV detection is often unsatisfactory, fluorescence after postcolumn derivatization improves specificity and sensitivity [13]. Again, LC–MS–MS (positive ESI mode) is currently the most often employed instrumental analytical technique. Chinolones are a group of synthetically produced bactericidal drugs. They are active against a broad spectrum of animal pathogens, especially Gram-negatives. Chinolones are a relative new group of bactericidal drugs, many of which are reserved for the use of human treatment (Figure 36.6).
Veterinary Drugs
HO
723
CH3
H3C R
◾
N
CH3 H
H
R = H Tetracycline
OH
NH2
R = CI Chlortetracycline
OH OH
O
OH
O
O
Figure 36.5 Tetracyclines. CH3
N
O
O
Oxolinic acid
COOH O
H3C
N N
N
Enrofloxacin
COOH
F O
Figure 36.6 Chinolones.
Residues of enrofloxacin and its metabolite ciprofloxacin were found in many shrimps and fish samples from Southeast Asia in the years 2005 and 2006. Chinolones are relative stable molecules, most of which show intensive fluorescence. Hence, LC fluorescence detection is often employed while LC–MS–MS (positive ESI mode) is preferred for confirmation purposes [14].
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Penicillin is a substance produced by the mould Penicillium notatum and Penicillium chrysogenum. It was the first discovered antibiotic and its activity extends against Grampositives and Gram-negatives. Its long use has led to extensive resistance. A number of pathogens produce beta-lactamase that inhibits the action of penicillin. A number of semisynthetic penicillin derivates are available such as the aminopenicillins (amoxicillin, ampicillin) and the more modern cephalosporins. Cephalosporins are not affected by beta-lactamase. Several generations of these drugs are now available, most of which are reserved for human use only (Figure 36.7). Penicillins are very labile molecules, easily degraded by enzymatic or chemical hydrolysis. Qualitative and semiquantitative detection can be achieved by a variety of microbiological test systems. Extraction is preferably done with polar solvents. Deproteinization achieved by adding acetonitrile or tungstate, e.g., is an important step of every analytical method. The amphoteric character of penicillins can cause severe asymmetrical LC peaks in insufficiently buffered mobile phases. Detection requires derivatization or LC–MS–MS [7]. Nitrofurans are a purely synthetic class of antibiotics. They show a wide spectrum of activities against many microorganism. Their uses have been banned or strongly limited due to their possible carcinogenic and mutagenic potential. While most nitrofurans were originally developed for the human use, one such as Furanace (Nifurpirinol) was designed as a chemotherapeutic for fish. This drug is not anymore permitted for the use of fish intended for human consumption, yet it is widely used in the ornamental fish business.
O H
H S
N H
CH3 Penicillin G CH3 O COOH
N
O
N
H N
H
N
S N H N
S
S
CH3
O N
COOH Cefazolin
Figure 36.7 Penicillins and cephalosporines.
N
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Nitrofurans fed to animals are quickly metabolized and escape analytical detection [6] if the parent drug is searched. This is probably the reason why these banned drugs have not been detected for a long time, although they were heavily used. Only the discovery that metabolites are covalently bound to tissue proteins permitted the liberation, derivatization, and consequently detection of nitrofuran residues [15]. The detectable metabolites, which are considered as markers, are void of the nitrofuran structure typical to the parent drug as shown in Figure 36.8. These relatively small metabolites were initially not expected to be possible endogenous matrix components. This assumption turned out to be wrong in the case of nitrofurazone that produces semicarbazide as marker molecule. Semicarbazide was shown to be produced by the hypochlorite treatment of food [16]. It is a degradation product of azodicarbonamide used as flour improving agent and as blowing agent used for gaskets of certain food jars [16]. Furthermore, semicarbazide was reported to be an endogenous compound in Finnish crayfish [17]. All the published nitrofuran methods employ a very similar approach for the derivatization and quantification. Samples are hydrolyzed and derivatized by the use of hydrochloric acid and nitrobenzaldehyde. Processing includes liquid or SPE which is followed by LC–MS–MS separation and detection. The required performance limit of 1 mg/kg leaves almost only MS as detection technique. Quantification is preferably done by using isotopic labeled standards which are now readily commercially available for four different nitrofurans. Chloramphenicol is a very effective antibiotic, which has a long history of use in human and animal treatment. The drug was reported to have very serious, life-threatening side effects. Although, this occurs very seldom, there is apparently no safe concentration of the drug. This
O2N
H N
O
H N
NH2
NH2
H2N
N
O
O Nitrofurazone
Semicarbazide (metabolite)
O
O O
O O2N
O
N
N
N
N
AMOZ (metatbolite) OH N
O
N H2N
Furaltadone
O 2N
O
O
Nifurpirinol
Figure 36.8 Nitrofurans including their metabolites.
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Handbook of Seafood and Seafood Products Analysis CI
OH H N
CI
Chloramphenicol
O OH
O2N
CI
OH H N
CI Florfenicol
O
O F
S H3C
O
Figure 36.9 Phenicols.
led to the ban of this drug for human and veterinary use in most countries of the world. Florfenicol, a derivative of chloramphenicol was developed and registered for the use in aquaculture (Figure 36.9). Chloramphenicol is a small, rather polar molecule. The major analytical challenge is posed by the low minimum required performance limit (MRPL) of 0.3 mg/kg, which has to be met by the utilized analytical method. Commercial ELISA tests are widely used for screening while confirmation was done after derivatization by GC–MS [18]. LC–MS–MS (negative ESI) seems to replace GC–MS since it does not require derivatization and produces excellent sensitivities [19,20]. Macrolides and lincosamides are a group of semisynthetic antibiotics that are active against Gram-positives. They are often used against microorganisms having developed a resistance against penicillins. Macrolides are often derivatized and detected by LC–FL, however, LC–MS–MS seems to produce best results in terms of selectivity and sensitivity [21] (Figure 36.10). Aminoglycosides are polar compounds which are used against aerobic Gram-negative bacteria. Aminoglycosides were reported to show a narrow therapeutic range, due to their toxicity against the animal treated. Amikacin and gentamycin were used for ornamental fish and in aquaculture. Aminoglycosides require harsh (low pH) extraction conditions to be released from the sample. Most published methods use cation exchange SPE for sample processing. Aminoglycosides degrade on GC columns and elute unretained from most LC columns. Moreover, they have no chromophoric groups, which make derivatization compulsory. Only LC–MS–MS permitted the development of multimethods, covering more than one or two aminoglycosides [22]. Chromatographic retention is often achieved by using reversed phase columns conditioned with volatile ion pair agents (perfluorinated carbonic acids) or HILIC columns with relatively high volatile salt (ammonia formiate) concentrations. MS–MS is vastly superior to other detection techniques like derivatization and fluorescence detection (Figure 36.11). Nitroimidazoles, such as nitrofurans are banned drugs, which are suspected to be human carcinogens and mutagens. Still these drugs (metronidazole and ipronidazole) were used in aqua-
Veterinary Drugs
◾
727
CH3 N
H3C
CH3 CH
HO H N O
OH
O OH
Lincomycin SCH3 OH
Figure 36.10
Lincomycin. HOH2C HO
O
OH
H2N
HO
HO O
OH
CH2NH2
O H2N
OH
O NH2 Kanamycin A
Figure 36.11
Aminoglycosides.
culture for prophylactic and therapeutic treatments of diseases. Nitroimidazoles undergo extensive metabolism and are preferably detected as hydroxylated metabolites. LC–MS–MS permits the quantification and confirmation of nitrimidazole residues without requiring derivatization [23] (Figure 36.12).
36.5.2
Antimycotica
Fungal and protozoal infections of fish can be treated by the use of triphenylmethane dyes. Known members of these families are malachite green and crystal violet. Although effective and often used in aquaculture, these compounds are not registered in most countries. Residues of malachite green are very persistent and can be detected months after the application of the drug is stopped [5]. Malachite green is generally detected in the leuco form. Older analytical methods using LC–UV detection, utilize the chromophoric changes when oxidizing leuco-malachite green to malachite green. The required low detection limits are best achieved by LC–MS–MS (Figure 36.13).
728 ◾
Handbook of Seafood and Seafood Products Analysis CH3 O2N
CH3
N
Dimetronidazole
N
O2N
CH3
NH2
CH3
O2N
N O
OH N
N Ronidazole
Figure 36.12
N
O
DMZOH (metabolite)
Nitroimidazoles including metabolites.
CH3
H 3C N+
N H3C
Figure 36.13
Malachite green
CH3
Malachite green.
36.5.3 Tranquilizers Tranquilizers are used in aquaculture to sedate fishes and reduce mortality during transport and handling procedures. Reported was the use of benzocaine and tricaine [24] in aquaculture. The residue target organ for tranquilizers applied to mammals is the kidney. Since this organ from fish or shrimps is not eaten and mostly not available to the residue laboratory, analysis has to focus on muscle tissues where degradation occurs within hours. Detection is only possible if the drug has been applied directly prior catching, slaughtering, and freezing (Figure 36.14).
36.5.4
Antiparasitica
Avermectins act against parasites like sea lice. Members of this group (emamectin and ivermectin) were used to treat salmons and trout against such parasites. The marker residue for emamectin benzoate is emamectin B1a that has been detected by LC–FL [25] (Figure 36.15).
Veterinary Drugs
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729
O
CH3
O
Benzocaine 1116
H2N O
O
Tricaine
O S NH2
Figure 36.14
Tranquilizers.
Figure 36.15
Avermectins (emamectin B1).
HO O
36.5.5 Anthelmintic Drugs Benzimidazoles, as for example albendazole, are active against a broad-spectrum of intestinal helminth infection. These drugs were developed to treat mammalians, however activity against fish parasites is observed as well.
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Handbook of Seafood and Seafood Products Analysis
Figure 36.16 Benzimidazoles (albendazole).
Albendazole is extensively metabolized in fish, forming albendazole sulfoxide, albendazole sulfone, and albendazole-2-aminosulfone. Quantification is hampered by a lack of commercial availability of reference substances. LC–FL was reported to be used for the determination of albendazole [26] (Figure 36.16).
36.5.6 Hormones Hormones are used in aquaculture for sex reversal of newly hatched fry. Tilapia males are known to grow faster and larger than females [27], hence sex reversal is of economical interest. Such treatment is applied on juvenile fishes, hence residues of the applied hormones are not anymore likely to detect when the fishes reach the market. Besides, there remains the possibility that hormones are used as growth promoters. The analysis of hormones was a domain of GC–MS, however, newer papers increasingly often report LC–MS–MS with APCI interfaces [27] (Figure 36.17).
36.6 Future Developments Aquaculture is probably still in its infancy. There is an enormous potential for growth in such markets where consumers are not yet accustomed to fish or shrimp or do not have yet the purchase power to add such food onto their plates. On the other hand, the current fish harvest from the world oceans cannot probably be further increased. Overfishing will likely even lead to shrinking OH H
CH3
H
H
H
O Methyltestosterone
Figure 36.17
Hormones (methyltestosterone).
CH3
Veterinary Drugs
◾
731
harvests in the near future. All these are factors that point to the increasing importance of aquaculture. Aquaculture will likely serve different markets. There is the fish supply for poor countries, where fish is primarily a source of essential proteins. On the other hand, there are developed countries, which will likely develop a fancy for high end products, including exclusive aquatic species or fish produced under a certified organic environment. It is obvious that producers supplying these two market segments will have different opinions concerning the use of veterinary drugs. Therefore, it is to be expected that products produced for these two segments, will not always be consequently separated. A producer who caters for the European and U.S. market might be unable to supply the volume of fish as specified by contracts with his customers. Consequently, he or she might be tempted to fill the gap with fish coming from his neighbor’s ponds, which were intended for the local market. Considering the fact that processing plants cater to the local and the export markets, there is the likelihood that some fishes or shrimps intended for the low-end market will end up as a high-end shipments. Therefore, there is a continuing need to control antibiotic residues in the production regions as well as in the importing countries. With the more widespread use of aquaculture, the treat for lethal infections will stay or even increase. It is very difficult to envision the possibilities of future prevention or therapy methods. Certainly, increasing research will increase our knowledge of how aquatic species should be kept and fed. Improved breeding will lead to species that are less likely to develop disease. This might even include genetically modified fishes and shrimps. Likewise, there will be also further developments in the field of immunization. A marked progress for the immunization of fish has been achieved, however, immunization of shrimps was not very successful. There is the speculation that shrimps have a rather “primitive” immune system, which prevents the successful development of immunization strategies [4]. Progress is also to be expected in the field of residue analysis. Multimethods covering several groups of veterinary drugs are not yet commonly used. The widely different chemical and physical properties of the various drugs make it difficult to analyze many analytes by a single analytical method. Progress has to be made on two different fronts. First a generic extraction procedure has to be established, which quantitatively liberates incurred veterinary drugs from the tissue, followed by a cleanup intended to remove matrix compounds like peptides, proteins, and fats, without otherwise strongly affecting analyte recoveries. Detection will most likely rely on LC– MS–MS or LC-TOF. Modern LC–MS–MS is suitable for multiresidue methods. However, the more analytes are monitored, the shorter the MS dwell times have to be chosen. This does not only affect sensitivity, but also decreases reproducibility due to higher variation of the peak areas. TOF does not show such limitations. However, the current available resolution provides mass selectivity, which is above LC–MS, but still below LC–MS–MS. Technical developments and engineering improvements might ease the limitations for both MS–MS and TOF, or might even permit the use of ultra high-resolution MS in the routine residue analysis environment.
References 1. Nomura I., State of the world aquaculture 2006, FAO Fisheries technical paper 500, FOA, Rome, Italy, 2006. 2. Serrano P., Responsible use of antibiotics in aquaculture, FOA Fisheries technical paper 469, FOA, Rome, Italy, 2005. 3. Pillay TV.R. and Kutty M.N., Aquaculture Principles and Practices, 2nd edn., Blackwell Publishing, Oxford, U.K., 2005.
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4. Stickney R.R., Aquaculture: An Introductory Text, CABI Publishing, Oxfordshire, U.K., 2005. 5. Kuiper R.V., Scherpenisse P., and Bergwerff A.A., Persistence of residues of malachite green in European eel after water-born exposure of juvenile eels, Poster Euroresidue IV, Veldhoven, the Netherlands, 2000. 6. Kennedy G., Antibiotic residues in aquaculture systems, Lecture at Annual Conference of the Shellfish Association of Great Britain, London, 2004. 7. Oka H., Nakszawa H., Harada K., and MacNeil J., Chemical Analysis for Antibiotics Used in Agriculture, AOAC International, Airlington, TX, 1995. 8. Kurtz D., Skerritt J., and Stanker L., New Frontiers in Agrochemical Immunoassay, AOAC International, Airlington, TX, 1995. 9. Franek M., Diblikove I., Broad-specificity immunoassays for sulfonamide detection: Immunochemical strategy for generic antibodies and competitors, Anal. Chem. 78, 1559, 2006. 10. Kaufmann A. and Pacciarelli B., Bestimmung von Rückständen von Tetracyclinen in Lebensmittlen, Mitt. Lebenms. Hyg., 90, 167, 1999. 11. Bogialli S., Curini R., Di Corcia A., Nazzari M., and Saperi R., A liquid chromatography-mass spectrometry assay for analyzing sulfonamide antibioticals in callte and fish muscle tissues, Anal. Chem., 75, 1798, 2003. 12. Potter R.A., Burns B.G., Van De Riet J.M., North D.H., and Darvesh R., Simultaneous determination of 17 sulfonamides and the potentiators ormetoprim and trimethoprim in salmon muscle with liquid chromatography with tandem mass spectrometry detection, JAOAC, 90, 343, 2007. 13. Brillantes S., Tanasomwang V., Thongrod S., and Dachanantawitaya N., Oxytetracycline residues in Gian Freshwater Prawn, J. Agric. Food Chem., 49, 4995, 2001. 14. Schneider M., Vazquez-Moreno L., and Barraza R., Multiresidue determination of fluoroquinolones in shrimp by liquid chromatography-fluorescence-mass spectrometry, JAOAC, 88, 1160, 2005. 15. Leitner A., Zöllner P., and Lindner W., Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A, 939, 49, 2001. 16. Hoenicke K., Gatermann R., Hartig L., and Mandix M., Formation of semicarbazide (SEM) in food by hypochlorite treatment: Is SEM a specific marker for nitrofurazone abuse?, Food Addit. Contam., 21 526, 2004. 17. Saari L. and Peltonen K., Novel source of semicarbazide: Levels of semicarbazide in cooked crayfish samples determined by LC/MS/MS, Food Addit. Contam., 21, 825, 2004. 18. Shen H. and Jiang H., Screening determination and confirmation of chloramphenicol in seafood, meat and honey using ELISA, HPLC-UVD, GC-ECD, GC-MS-EI-SIM and GCMS-NCI-SIM methods, Anal. Chim. Acta, 535, 33, 2005. 19. Kaufmann A. and Butcher P., Quantitative liquid chromatography/tandem mass spectrometry determination of chloramphenicol in food using sub-2 mm particulate high-performance liquid chromatography columns for sensitivity and speed, Rapid Commun. Mass Spectrom., 19, 3694, 2005. 20. Van de Riet J., Potter R., and Christine-Fougere M., Simultaneous determination of residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in farmed aquatic species, JAOAC, 86, 510, 2003. 21. Horie M., Harumi T., and Kazuo T., Determination of macrolide antibiotics in meat and fish by liquid chromatography-electrospray mass spectrometry. Anal. Chim. Acta, 492, 187, 2003. 22. Kaufmann A. and Maden K., Determination of 11 aminoglycosides in meat and liver by liquid chromatography with tandem mass spectrometry, JAOAC, 88, 1118, 2005. 23. Mottier P., Huré I., Gremaud E., and Guy P., Analysis of four 5-nitroimidazoles and their corresponding hydroxylated metabolites in egg, processed egg, and chicken meat by isotope dilution liquid chromatography tandem mass spectrometry, J. Agric. Food Chem., 54, 2018, 2006. 24. Scherpenisse P. and Bergwerff A., Determination of residues of tricaine in fish using liquid chromatography tandem mass spectrometry, Anal. Chim. Acta, 586, 407, 2007.
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25. Kim-Kang H., Bova A., Crouch L., and Wislocki P., Tissue distribution, metabolism, and residue depletion study in atlantic salmon following oral administration of [3H] emamectin benzoate, J. Agric. Food Chem., 52, 2108, 2004. 26. Shaikh B., Rummel N., and Reimschuessel R., Determination of albendazole and its major metabolites in the muscle of atlantic salmon, tilapia, and rainbow trout by high performance liquid chromatography with fluorescence detection, J. Agric. Food Chem., 51, 3254, 2003. 27. Chu P., Lopez M., Serfling S., and Gieseker C., Determination of 17a-methyltestosterone in muscle tissues of tilapia, rainbow trout, and salmon using liquid chromatography-tandem mass spectrometry, J. Agric. Food Chem., 54, 3193, 2006.
Chapter 37
Differentiation of Fresh and Frozen–Thawed Fish Musleh Uddin Contents 37.1 Introduction ..................................................................................................................735 37.2 Examination of the Opacity of the Eye Lens ................................................................ 736 37.3 Measurement of the Electrical Resistance ......................................................................737 37.4 Determination of the Hematocrit Value and Examination of Erythrocytes.................. 738 37.5 Enzymatic Methods ......................................................................................................739 37.6 Spectroscopic Analysis...................................................................................................742 37.7 Conclusions ...................................................................................................................747 References ................................................................................................................................748
37.1 Introduction Product authenticity is an emerging area of concern within the food industry. Given the perishable nature of fish, extension of its shelf life is a requirement of normal trading. However, frozen fish usually have a much lower market price than fresh fish; therefore the substitution of frozen– thawed for fresh fish is a significant authenticity issue. According to the Food and Agricultural Organization (FAO) and the Japan Agriculture Standard (JAS) regulations, labeling should state that the fish has been frozen and must not be refrozen as usual, and frozen–thawed fish must be labeled as previously frozen [1,2]. These have become regulatory requirements in most countries nowadays. It is generally accepted that fresh fish (or fillets/portions) and frozen–thawed fish are types of products which should be differentiated [8]. Fresh fish is, indeed, understood as being fish freshly caught or which has been chilled and stored for a short period at normal refrigeration 735
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Handbook of Seafood and Seafood Products Analysis
temperature prior to purchase or use. For storage over longer periods freezing is normally utilized. However, while frozen storage is effective in protecting against microbiological deterioration of fish meat, its physicochemical and organoleptic properties suffer [16]. The consumer perception of such fish is inferior to that of the fresh material and this is reflected in the price which it realizes. In practice, a considerable number of frozen fish are thawed in fish shops, stored on ice, and sold as unfrozen fish without being labeled as such. For the benefit of the consumer and prevention of unfair competition in the trade of fishery products, correct labeling of frozen–thawed fish or fillets is desirable. Freezing is one of the main processes currently used for extending the shelf life of fish because it inhibits microbial growth and reduces biochemical and physical spoilage during storage. But freezing can alter the quality of frozen fish in comparison to the unfrozen, fresh product. Physical and chemical changes in proteins that occur during frozen storage may result in texture deterioration, which negatively affects the functional, nutritional, and sensory properties of the fish. In addition, lipid oxidation during long-term storage can lead to rancid flavor and is thought to enhance protein denaturation–aggregation reactions. However, when freezing, storage, and thawing are done properly, the sensory properties of the product are very similar to those of fresh fish. Consumers are unlikely to notice the changes that occur in frozen fish, but many prefer fresh fish despite its higher cost. For this reason, the authentication of fresh fish is important to the consumer because the substitution of frozen–thawed products for fresh is a frequent, fraudulent practice. Unfortunately, it is not easy to distinguish between a thawed fish and a fresh fish, because their physical and chemical characteristics are very similar, and much effort has been expended in devising methods to establish whether or not a fish has been previously frozen [5]. Control of labeling is only possible if rapid and reliable methods exist, which allow food control authorities to distinguish between fresh and frozen–thawed fish. A summary of the methods developed for differentiating between fresh and thawed fish are reviewed in this chapter.
37.2
Examination of the Opacity of the Eye Lens
The lens of the fish eye consists of the central part (medulla) and an outer layer, the cortex. The medulla of fresh fish is transparent, but freezing of the fish leads to turbidity; this was first used for differentiation of frozen–thawed fish from fresh fish by Love [3]. Later on the technique of examination was improved by other research groups [4–6]. Figure 37.1 shows the transparent and turbid medulla of fresh and frozen–thawed red sea bream. Yoshioka and Kitamikado [4] also
Fresh
Frozen–thawed
Figure 37.1 Transparent and turbid medulla of fresh and frozen–thawed red sea bream.
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737
found that the small medullae of eel and flatfish did not become opaque as a result of freezing and thawing. On the other hand medullae of some horse mackerel (9 of 112) become opaque after 7 days ice-stored in a chill room [6].
37.3
Measurement of the Electrical Resistance
The measurement of the electrical resistance of fish is widely used to evaluate its freshness [5,7]. During spoilage of fish the electrical resistance of the tissues (skin, muscle) steadily decreases due to the destruction of the membrane system. As membranes are also destroyed by freezing and thawing, the electrical resistance of thawed fish is as low as that of spoiled fish. This can be used to identify thawed fish unless the sample is spoiled [8]. Two instruments are available on the market: the Torrymeter and the Fish Tester. They have different scales (Torrymeter: 0–16; Fish-Tester: 0–100), but for both instruments the readings are positively correlated with freshness, i.e., high values indicate high quality and thawed fish is characterized by values near zero. The Torrymeter (Figure 37.2) was successfully used for differentiating fresh and frozen–thawed fish [5,9]. Duflos et al. [5] evaluated Torrymeter to
Figure 37.2
Torrymeter reading taking from red sea bream.
738 ◾ Handbook of Seafood and Seafood Products Analysis Table 37.1 Torrymeter Measurements for Fresh and Thawed Samples of Whole Fish and Fillets (Skin and Gut Cavity Sides) Fillets (Skin Side)
Whole Fish
Fillets (Viscera Side)
n
Mean
SD
Mean
SD
Mean
SD
Fresh whiting
60
10.97
2.88
7.63
2.98
8.13
2.98
Fast-frozen whiting
60
0.25
0.29
2.54
1.65
2.33
1.64
Slow-frozen whiting
60
0.11
0.30
2.75
1.48
4.92
2.30
Fresh plaice
60
13.22
0.86
10.08
0.67
9.46
2.25
Fast-frozen plaice
60
2.74
2.16
6.11
1.84
5.56
1.58
Slow-frozen plaice
60
3.77
2.64
3.94
3.47
6.17
4.00
Fresh mackerel
60
8.85
3.30
5.00
2.02
5.00
1.22
Fast-frozen mackerel
60
0.02
0.04
1.75
2.13
1.33
2.15
Slow-frozen mackerel
60
0.35
0.71
1.89
1.77
2.44
2.22
Source: Duflos, G. et al., J. Sci. Food Agric., 82, 1341, 2002. SD, Standard deviation.
classify fresh and frozen–thawed using three species: plaice (Pleuronectes platessa), whiting (Merlangus merlangus), and mackerel (Scomber scombrus). They also compared the results obtained following slow and rapid freezing and investigated how spoilage affects the Torrymeter measurements. The results obtained in this study are summarized in Table 37.1. The mean values of 60 measurements for each storage method for whole fish frozen rapidly or slowly were close to zero and were significantly below (p < 0.01) those of fresh fish. A smaller but still significant difference was also noted between fresh and thawed fillets (p < 0.01). There were no differences between the values recorded on the viscera and skin sides of a fillet. The values for fresh fillets were below those for whole fresh fish, whereas the values for frozen fillets were higher than for whole frozen fish; the difference between fresh and frozen was greater for whole fish than for fillets. The values for whole fish and fillets of all three species (whiting, plaice, cod) were not significantly influenced (p < 0.05) by the method of freezing (slow by storage or fast in a cell). Th is study revealed that the Torrymeter is a reliable indicator for whole fish rather than fillets. In a later study Okazaki [9] also recommended Torrymeter could be a reliable indicator for whole fish rather than fillets. However, the drawback of using Torrymeter is, if the fish are stored in refrigerated seawater for longer time or treated with salt, these instruments cannot be applied for all fish species like herring [8,9].
37.4
Determination of the Hematocrit Value and Examination of Erythrocytes
Both of these methods rely on the destruction of the red blood cells due to freezing and thawing. Obviously it is quite difficult to apply on those fish fillets possessing only minimal amounts of blood or red muscle, e.g., fillets from cod, redfish, and many other white-fleshed species.
Differentiation of Fresh and Frozen–Thawed Fish
739
Hematocrit value
Needle
Blood collector
◾
Needle before Centrifugation After centrifuged centrifuged (12,000 rpm, 5 min)
Hematocrit value
Fresh
Frozen–thawed
Figure 37.3 Hematocrit value determination procedure for Pacific mackerel.
The hematocrit value is defined as the proportion of the erythrocytes on blood volume (Figure 37.3). It is expressed as percent of volume and determined by centrifugation of blood. The hematocrit value of frozen–thawed fish (carp, red sea bream, Pacific mackerel, saury, yellowfin tuna, and skipjack) was zero, whereas fresh fish gave values between 21% and 48%, depending on the freshness and the species of fish [4,10]. Yoshioka and Kitamikado [11] reported about the differentiation of frozen–thawed fillets from fresh fillets by microscopic examination of stained erythrocytes. A small volume of blood (20 μL of less) was withdrawn with a capillary pipette from the central or dorsal aorta of fillets from red sea bream or Pacific mackerel, smeared on a slide glass, dried, stained with Giemsa solution, and inspected microscopically. Besides, a small amount of dark surface muscle was taken from the samples and processed as described above. All erythrocytes were destroyed in frozen–thawed samples, but intact erythrocytes were found in the blood and dark muscle of fresh and refrigerated samples (Figure 37.4). The method was also successfully applied to commercial fillets of several other fish species including small pelagic fish sardine and large tuna [12].
37.5
Enzymatic Methods
Among the techniques developed for differentiation between fresh and frozen–thawed fish or fillets, enzymatic assays are widely studied compared to other physical or physiological techniques. The cells of fish muscle and their organelles are destroyed by freezing and thawing of muscle [13–16]. Enzymes located inside the particles or bound to the membranes are released into thaw drip and press juice [13,17]. When muscle of fresh fish was carefully extracted with isotonic solution (0.25–0.30 M sucrose), only low activities of lysosomal and mitochondrial enzymes were detectable in the soluble fraction (supernatant); the same treatment applied to frozen–thawed fish muscle gave considerably
740
◾ Handbook of Seafood and Seafood Products Analysis
(a)
Blood collection
Blood-smeared slide 5°C for 4 days
Stained slide observation 5°C for 14 days
Fresh
(b)
Frozen–thawed (frozen at –20°C for 4 or 14 days)
Figure 37.4 (a) Microscopic examination procedure of stained erythrocytes. (b) Intact and destroyed erythrocytes observed for fresh and frozen–thawed Pacific mackerel.
enhanced activities of these enzymes in this fraction [14]. The appearance of originally particlebound enzymes in drip, press juice of isotonic extract has been used by a number of research groups to differentiate between fresh and frozen–thawed fish fillets [8,18,19]. The enzymatic methods involve assays of mitochondrial enzymes (β-hydroxyacyl-CoAdehydrogenase or HADH [20–22], l-malate-NADP-oxidoreductase or malic enzyme [23], aspartate-aminotransferase [24], glutamate oxaloacetate transaminase [25], lipoamide-reductase and 5′ AMP deaminase [26], lactate dehydrogenase [13,26], cytochrome C oxidase [14,27], succinate dehydrogenase [28], fumarase and glutamate dehydrogenase [13], l-malate dehydrogenase [29], enzymes extracted from blood cells β-N-acetylglucosaminidase [17,30]) and lysosomal enzymes (α-glucosidase and β-N-acetylglucosaminidase [8,18,31], acid phosphatase [13,18], β-galactosidase and β-glucuronidase [13]). The freezing of fish results in the leakage of these enzymes into the exudates and hence an increase in tissue enzymatic activity which can be measured. An extensive review showed that the best results are obtained by spectrophotometric assays for α-glucosidase, β-N-acetylglucosaminidase, and HADH, by fluorimetric assays for β-N-acetylglucosaminidase of blood, and by electrophoretic measurements for glutamate oxaloacetate transaminase and malic enzyme. The value of the enzymatic methods resides in the fact that they are applicable to fi llets as well as to whole fish. From these numerous enzymatic techniques, Duflos et al. [5] re-evaluated three selected enzymatic assays (α-glucosidase, β-N-acetylglucosaminidase, and HADH) to classify fresh and
Differentiation of Fresh and Frozen–Thawed Fish
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741
frozen–thawed fish and fillets. Three species of fish from commercial fishing boats were used in their study: plaice (P. platessa), whiting (M. merlangus), and mackerel (S. scombrus). A homogeneous batch of each species was stored at 0°C in ice or at −20°C after slow freezing by direct storage in a cold room or fast freezing in a cell. All samples were analyzed after 24 h at freezing temperature. The results obtained in this study are summarized in Table 37.2. α-Glucosidase was assayed in different fish of equivalent and satisfactory freshness (Table 37.2). The species effect is quite notable, as it was difficult to define a cutoff value for mackerel: The standard deviations were too great compared with the differences between the values for thawed mackerel and fresh mackerel. For whiting and plaice the means for fresh fish differed significantly from those for frozen–thawed fish (Student’s t test, p < 0.05). The values noted for fresh and thawed whiting and plaice indicated cutoff values of 0.15 for whiting and 0.5 for plaice, above which it can be asserted that the sample had been frozen. β-N-Acetylglucosaminidase assay was not useful for characterizing the freezing process, since the difference between the means was insufficient to distinguish reliably between fresh and frozen–thawed samples (Table 37.2). In all three species this enzyme seems ill suited to establishing whether or not the fish has been frozen. HADH activities in all three species differed substantially between fresh fish and frozen fish (Student’s t test, p < 0.05). The standard deviations, though, were large, especially for frozen fish. The analysis of results for plaice was facilitated by very low values for fresh samples. It seems difficult to define cutoffs above which the fish had undergone slow or rapid freezing, as the standard deviations are quite high. There was no significant difference between the two methods of freezing. Of these three enzyme activities, those of HADH and principally α-glucosidase were indicative of whether or not the fish had been frozen (Table 37.2).
Table 37.2 Mean Activities (mg−1 Protein) of a-Glucosidase, b-N-Acetylglucosaminidase, and b-Hydroxyacyl-CoA-Dehydrogenase Extracted from Fillets Whiting
α-Glucosidase
β-N-Acetyl glucosaminidase
β-Hydroxyacyl-CoAdehydrogenase
Plaice
Mackerel
n
Mean
SD
Mean
SD
Mean
SD
F
30
0.083
0.029
0.265
0.117
0.42
0.357
SF
30
0.389
0.108
0.68
0.125
0.547
0.444
FF
30
0.254
0.105
0.783
0.219
0.711
0.349
F
30
0.52
0.378
0.81
0.459
0.469
0.253
SF
30
0.745
0.639
0.92
0.867
0.395
0.271
FF
30
0.708
0.691
0.85
0.528
0.687
0.502
F
30
17.73
5.86
5.44
4.6
24.4
18.42
SF
30
56.86
34.91
45.56
31.39
34.2
25.59
FF
30
46.5
9.14
39.9
23.36
50.84
25.49
Source: Duflos, G. et al., J. Sci. Food Agric., 82, 1341, 2002. F, fresh; FF, fast frozen; SF, slow frozen; SD, standard deviation.
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Handbook of Seafood and Seafood Products Analysis
37.6 Spectroscopic Analysis Recent developments of nondestructive/noninvasive spectroscopic techniques are also employed to differentiate fresh and frozen–thawed fish and shell fish [32–34]. Front-face fluorescence spectroscopy combined with chemometric tools is investigated [33] as a rapid technique to differentiate frozen–thawed from fresh fish. For the frozen–thawed samples, they evaluated two speeds of freezing and thawing (fast and slow). The fluorescence emission spectra of tryptophan (excitation: 290 nm, emission: 305–400 nm) and nicotinamide adenine dinucleotide (NADH) (excitation: 340 nm, emission: 360–570 nm) were recorded directly on samples. Based on principal component analysis (PCA), NADH spectra gave good discrimination results between fresh and frozen–thawed fish compared to those of tryptophan fluorescence spectra. They concluded that NADH fluorescence spectra may be considered as a promising probe for the reliable differentiation between frozen–thawed and fresh fish. Nuclear magnetic resonance (NMR) imaging combined with histology study also performed to separate fresh and frozen–thawed fish [32]. The results reported in this study demonstrate that the magnetic resonance imaging (MRI) protocol developed provides measurements of NMR parameters that are consistently reliable and reproducible. Moreover, it allows differentiation of frozen–thawed trout from fresh trout on whole, intact fish. The variations in NMR parameters (especially the relaxation time, T2, and the diff usion coefficient, D, with the frozen storage duration) are in agreement with histological observations, and permit the characterization of structural changes in tissue induced by the freezing process. The technique also demonstrates that most damage occurs upon freezing. To differentiate fresh and frozen–thawed fish, various techniques proposed by several researchers [5,8,35] have already been discussed above; however, most of those methods are either time consuming, destructive, or have limitations for practical use. Therefore, the need exists for a method which is capable of differentiating between fresh and frozen–thawed fish or fillets. Ideally, any such method should be nondestructive and rapid as well as reliable. In the seafood industry, near-infrared (NIR) spectroscopy is widely used as a nondestructive rapid technique for quantitative and qualitative analysis of fish and related products [36–39]. NIR spectroscopy has become established during the last decade as one of the most important tools of modern industrial analysis, especially for online and inline analysis. The reasons for this are that it yields essentially real-time results, is reagentless and nondestructive, and can yield information about model compliance [40]. Dry extract spectroscopy by infrared reflection (DESIR) of fresh and frozen–thawed fish was performed on extracted meat juices and fresh and frozen–thawed fish were differentiated [16]. Uddin et al. [41] proposed nondestructive visible/NIR spectroscopy to investigate whether fish have been frozen–thawed. Compared to DESIR, no extraction is needed and no wastes are produced in visible/NIR spectroscopy using the fiber optic probe, which would be an eco-friendly instrumental technique. In this study, 108 fresh red sea bream (Pagrus major) were used. For fresh and unfrozen fish, 54 samples were used soon after arrival while the second lot of 54 fish was kept at −40°C, respectively. After 30 days of frozen storage, the fish were removed and thawed overnight at 5°C and then evaluated as frozen–thawed samples. The fish samples were scanned using a NIRSystems 6500 spectrophotometer equipped with a surface interactance fiber optic accessory. The red sea bream were measured at a location just behind the dorsal fin, midway on the epaxial part. Spectra were recorded in the wavelength range 400–1100 nm at 2 nm intervals. The spectra were stored in optical density units log (1/T ), where T represents the percent of energy transmitted (Figure 37.5). Among the total of 108 samples, 54 of them fresh and 54 of them frozen-then-thawed, were then divided into a modeling set and a prediction set. The modeling set contained 35 samples for the fresh and 35 for the frozen fish. Twenty-seven of these
Differentiation of Fresh and Frozen–Thawed Fish
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743
1.8 1.6 Fresh/unfrozen log 1/T
1.4 1.2 1.0 0.8 Frozen–thawed 0.6 700
800
900
1000
1100
Wavelength (nm)
Figure 37.5
Fresh and frozen–thawed red sea bream spectra.
samples were picked as each odd numbered sample in the order of recording, and the remaining eight samples were selected randomly. Thus, a total of 38 samples for both fresh and frozen fish (19 samples each) were allocated to the prediction set. Sample spectra for both sets were treated in exactly the same way with second derivative or multiplicative scattering correction (MSC), or no treatment was applied at all. There are many ways to explore data structures, recognize patterns, and classify samples according to some distance measures. In the study by Uddin et al. [41], the classification methods called soft independent modeling of class analogy (SIMCA) as defined by Wold [42] and linear discriminant analysis (LDA) using PCA scores [43] were used. The former method is based on disjoint PCA models where for each group an independent PCA model is constructed which is then used to classify new, unknown samples. The latter one, in our case, uses the so-called score values of PCA results as input variables to the LDA. By performing PCA first, the number of variables is reduced and the variables become independent. By doing so, only a small fraction of the information is lost. This is important, since LDA requires the number of samples to be considerably higher than the number of variables to have a statistically meaningful classification. For a classification to be successful two things are needed. First, the number of samples belonging to the same group should be as similar as possible and second, the groups should be as far away from each other as possible. The major effect of freeze-thawing treatment involves a gross change in the total absorbance after freezing and thawing; this arises from changes in light scatter presumably arising from alterations in the physical structure of at least the surface layer of fish [41]. In Figure 37.6 the PCA score plot clearly shows that the fresh (right side) and the frozen– thawed (left side) samples are well separated. For this model, only one factor was enough to separate the two groups. As it can also be seen that the frozen–thawed samples have a more compact structure, i.e., data points are closer to each other while in the fresh samples, the group is not so well defined (larger spread of data points). A similar separation was also observed in DESIR analysis of fresh and frozen–thawed fish that was performed on the sample juices [16,44]. Using the results of this exploratory stage for all the spectral treatments applied, two independent PCA models (900–1098 nm wavelength range with original absorbance spectra) were generated with the modeling sets and then were used to build SIMCA models. There are several powerful advantages
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PC2
0.1
–4
–3
–2
0.0 –1
1
2
3
4
–0.1 –0.2 PC1
Figure 37.6 Two-dimensional PCA score plot of all 108 red sea bream samples. Samples of the left side of the ordinate axis are frozen samples (triangles), while those on the right are fresh samples (circles).
of the SIMCA approach compared to methods like cluster analysis. First, SIMCA is not restricted to situations in which the number of objects is significantly larger than the number or variables as is invariably the case with classical statistical techniques. This is not so with the present bilinear methods, which are stable with respect to any significant imbalance in the ratio of objects/variables, be it either many objects with respect to variables or vice versa. Because of the score-loading outer product nature of bilinear models, the entire data structure in a particular data matrix will be well modeled even in the case where one dimension of the data matrix is much smaller than the other. Another advantage is that all the pertinent results can be displayed graphically allowing exceptional insight regarding the specific data structure behind the modeled patterns. SIMCA models were applied to the prediction set, and results of the prediction can be best visualized by plotting the sample-to-model distances for all samples as shown in Figures 37.7 and 37.8. These plots are called Coomans plots [41], which show orthogonal (transverse) distances from all new objects (samples) to two selected models (classes) at the same time. In Figure 37.7, the two groups are well defined and separated. All prediction samples are close to the group that they should belong to. However, not every sample is within the membership limits for both the modeling and the prediction samples. As can be seen, some samples are located in the upper right quadrant, indicating that they are not included in the defined models. No sample is in the lower left quadrant, meaning that no sample was classified to both groups simultaneously. The upper left and lower right quadrants define samples which belong to a specific group. In Figure 37.8 however, where the sample spectra were subjected to MSC transformation, modeling and classification seem much less clear. The two groups are very close; in fact, they almost overlap even at the modeling stage. Th is means that the MSC transformation removed information, i.e., scattering, on which the previous model is based, therefore models are not that far apart. However, the units in Figure 37.8 are by one to two orders of magnitude smaller compared to those of Figure 37.7, which explains why samples of the two groups are closer and more scattered. The sample distance, which is by an order of magnitude smaller compared to the previous model, between groups is important in terms of reliability or robustness of the model. Th is does not necessarily mean that the classification accuracy is worse, as indicated in Table 37.3, where results are summarized for models with original absorbance and MSC-transformed spectra. The
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Sample distance to model frozen
0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 0.000 0
0.02
0.04 0.06 Sample distance to model fresh
0.08
0.1
Figure 37.7 Coomans plot for discrimination between fresh and frozen–thawed red sea bream. Spectra were submitted to SIMCA without any treatment. (•) Fresh modeling samples; (■) frozen–thawed modeling samples; (+) fresh prediction samples; (▲) frozen–thawed prediction samples.
Sample distance to model frozen
0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 0.0000 0
0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 Sample distance to model fresh
Figure 37.8 Coomans plot for discrimination between fresh and frozen–thawed red sea bream. Spectra were MSC treated. (•) Fresh modeling samples; (■) frozen–thawed modeling samples; (+) fresh prediction samples; (▲) frozen–thawed prediction samples. The horizontal and vertical gray lines are class membership limits calculated at a 5% confidence limit.
same proportions of samples to none or to both groups are classified correctly meaning that the classification accuracy is the same for both models, however, the model using original absorbance spectra has a higher reliability. Th is is also an important model feature, since the model is more stable against random errors or interferences from any source.
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Table 37.3 Discrimination Results between Fresh and Frozen–Thawed Prediction for Red Sea Bream Samples Using the SIMCA Method Spectural Transformation
Kind of Sample
Classified Correctlya
Classified to Noneb
Classified to Bothc
No. of PCsd
None
Fresh
63
37
0
1
Frozen
84
16
0
1
Totale
73
27
0
—
Fresh
63
37
0
1
Frozen
84
16
0
3
Totale
73
27
0
—
MSC
a
b
c
d e
Proportion (%) of samples which were classified to the correct model at the 5% significance level. Proportion of samples which were classified to none of the models at the 5% significance level. Proportion of samples which were classified to both models at the 5% significance level. Number of PCs which were used for making class model. Proportion of fresh and frozen samples combined.
Table 37.4 Discrimination Results between Fresh and Frozen–Thawed Prediction for Red Sea Bream Using LDA with PCA Scores as Input Variables Group Proportion Correct
Type of Fish
Type of Fish
Fresh
Frozen
Fresh (%)
Frozen (%)
Overall Proportion Correct (%)
None
19
19
100
100
100
MSC
15
16
79
84
81
Spectural Transformation
a
N Correct in Groupsa
The number of correctly classified samples out of the 19 prediction samples for the fresh and frozen–thawed red sea bream groups, respectively.
As regards to LDA, the results are much more clear-cut as seen in Table 37.4. To perform modeling and classification in the same wavelength range (900–1098 nm), spectral transformation and prediction samples were used for LDA analysis as well. It is clear from Table 37.4 that the model using the original absorbance spectra achieved much better classification accuracy (100%) for the prediction samples; however, the results obtained using the MSC-treated spectra are considerably worse, indicating again that scattering is the information that makes classification work. For fresh fish, as the cellular structure is intact, when NIR light enters the fresh fish, cells not only absorb the light but change its direction until the light reaches the next cell. This process may continue until all light is absorbed or until the light emerges at the other side of the sample. This
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multiple change in the direction of light is called scattering, which increases the distance that the light travels from the entry point to the exit point of the sample. On the other hand, when freezing and thawing is done, the cell membrane is damaged, causing the leakage of the intracellular contents into the extracellular space. Thus, there is a much smaller number of cells that can scatter light as light travels through the sample, reducing the distance that the light has to cover. It is interesting to note that frozen–thawed samples were slightly better classified than fresh ones, but as the number of samples is limited drawing conclusions is as yet premature. This method maximizes the ratio of between-class variance to the within-class variance in any particular data set thereby guaranteeing maximal separatability. It is possible to achieve better accuracy and reliability by using a custom-made fiber optic probe or improved sample presentation. Uddin et al. [34] also attempted to authenticate between fresh and frozen–thawed shellfish by NIR spectroscopy. They tried with scallop (Patinopectin yessoensis) and Kuruma prawn (Penaeus japonicus) using the two-dimensional PCA as well as SIMCA and LDA. However, it was not successful to classify fresh and frozen–thawed clearly for those species. To certify the labeling which states that the fish has been frozen–thawed or unfrozen/fresh, Uddin et al. [45] conducted a separate study using visible/NIR spectroscopy where 100 samples of fresh Pacific saury (Cololabis saira) were used as fresh or frozen–thawed (different freezing storage temperatures: −5°C, −20°C, −30°C, −40°C, −80°C and different freshness level before freezing: 0, 2, 4, 8, 10, 15 days ice storage). They observed different spectral pattern for fresh and frozen– thawed samples especially in the lower wavelength region between 700 and 850 nm. MSC-treated spectra achieved relatively better classification accuracy for the prediction samples. Different storage temperatures of fishes had no marked effect on the separation of fresh and frozen–thawed samples while the classification accuracy became worse with lower prefreezing freshness level. They concluded that visible/NIR spectroscopy can certify the labeling system of unfrozen or frozen–thawed fish rapidly and also suitable for online or at-line processing control. Nevertheless, the results are promising that a rapid measurement method can be developed to detect practices such as when frozen–thawed fish are sold as fresh fish.
37.7 Conclusions To differentiate fresh and frozen–thawed fish, measurement of the electric properties of fish tissues, visual inspection of the eye lens, judgments of the integrity of red blood cells by microscopy, or estimation of the hematocrit value were proposed. In practice, proposed methods cannot be applied to those fish or fillets possessing no blood, eye lens, or skin. In the case of whole fish, those techniques are reliable and some of them are rapid, however, if fish are stored refrigerated for a longer time in terms of freshness, these techniques might not be reliable for all fish species. For differentiation of fillets, one of the above-mentioned enzymatic methods may be used. Which type of enzyme (located in the mitochondria, lysosomes, or red blood cells) gives the best results depends to some extent on the fish species. On the other hand, by considering the universal, noninvasive and nondestructive nature of the NIR spectroscopy, its speed, and the robustness of the instruments commercially available today, the disadvantages indicated above may be overcome in the near future. The number of scientific papers and the successes of international congresses on this theme is evidence of this fact especially in seafood analysis. As mentioned earlier, the authenticity, safety, and quality of seafood has been of particular concern in recent years, NIR spectroscopy could be a nondestructive testing method allowing reproducible and rapid assessment, something that has not been available in the past.
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References 1. FAO. 1982. Reference manual to codes of practice for fish and fishery products. FAO Fish, Circ. 750, 217. 2. JAS (Japan Agricultural Standard). 2000. Quality labeling standard for perishable foods. Notification No. 514. 3. Love RM. 1956. Post-mortem changes in the lenses of fish eyes. II. Effect of freezing, and their usefulness in determining the past history of the fish. J Sci Food Agric. 7: 220–226. 4. Yoshioka K, Kitamikado M. 1983. Differentiation of freeze-thawed fish from fresh fish by the examination of medulla of crystalline lens. Bull Jpn Soc Sci Fish. 49: 151–155. 5. Duflos G, Le Fur B, Mulak V, Becel P, Malle P. 2002. Comparison of methods of differentiating between fresh and frozen-thawed fish or fillets. J Sci Food Agric. 82: 1341–1345. 6. Okazaki E, Yamashita Y, Uddin M. 2006. Classification of fresh and frozen-thawed fish–a review. Refrigeration. 81: 175–181. 7. Sakaguchi M, Murata M, Kim JB. 1989. The effects of repeated freeze-thaw cycle on torrymeter readings of carp fillets. Nippon Suisan Gakkaishi. 55: 1665–1669. 8. Rehbein H. 1992. Physical and biochemical methods for the differentiation between fresh and frozenthawed fish or fillets. Ital J Food Sci. 2: 75–86. 9. Okazaki E. 2003. Unpublished data. National Research Institute of Fisheries Science (NRIFS). Yokohama, Japan. 10. Uddin M. 2003. Unpublished data. National Research Institute of Fisheries Science (NRIFS). Yokohama, Japan. 11. Yoshioka K, Kitamikado M. 1988. Differentiation of freeze–thawed fish fillet from fresh fish fillet by examination of erythrocyte. Nippon Suisan Gakkaishi. 54: 1221–1225. 12. Yamashita Y. 2003. Personal communication. National Research Institute of Fisheries Science (NRIFS). Yokohama, Japan. 13. Rehbein H. 1979. Development of an enzymatic method to differentiate fresh and sea-frozen and thawed fish fillets. Z Lebensm Unters-Forsch. 169: 263–265. 14. Karvinen VP, Bamford DH, Granroth B. 1982. Changes in muscle subcellular fraction of Baltic herring (Clupea harengus membras). J Sci Food Agric. 33: 763–772. 15. Yoshioka K. 1983. Differentiation of freeze–thawed fish from fresh fish by the determination of hematocrit value. Bull Jpn Soc Sci Fish. 49: 149–151. 16. Uddin M, Okazaki E. 2004. Classification of fresh and frozen-thawed fish by near-infrared spectroscopy. J Food Sci. 69: C665–668. 17. Yuan CS, Yoshioka K, Ueno R. 1988. Differentiation of frozen-thawed fish from unfrozen fish by determination of neutral β-N-acetylglucosaminidase activity in the blood. Bull Jpn Soc Sci Fish. 54: 2143–2148. 18. Rehbein H, Kress G, Schreiber W. 1978. An enzymic method for differentiating thawed and fresh fish fillets. J Sci Food Agric. 29: 1076–1082. 19. Vincenzo S, Francesco F, Giovanna P. 1985. A micromethod for the differentiation of fresh from frozen fish muscle. J Sci Food Agric. 39: 811–814. 20. Gottesmann P, Hamm R. 1983. New biochemical methods of differentiating between fresh meat and thawed, frozen meat. Fleischwirtschaft. 63: 219–220. 21. Hoz L, Yustes C, Camara JM, Ramos MA, Garcia de Fernando GD. 1992. β-Hydroxyacyl-CoAdehydrogenase (HADH) differentiates unfrozen from frozen-thawed crawfish (Procambarus clarkii) and trout (Salmo gairdneri) meat. Int J Food Sci Technol. 27: 133–136. 22. Fernandez M, Mano S, Garcia de Fernando GD, Ordonez JA, Hoz L. 1999. Use of β-hydroxyacylCoA-dehydrogenase (HADH) activity to differentiate frozen from unfrozen fish and shellfish. Eur Food Res Technol. 209: 205–208. 23. Gould E, Medler MJ. 1970. Fish and other marine products-test to determine whether shucked oyster have been frozen and thawed. J Assoc Anal Chem. 53: 1237–1241.
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24. Chhatbar SK, Velankar NK. 1977. A biochemical test for the distinction of fresh fish from frozen and thawed fish. Fish Technnol. 14: 131–133. 25. Salfi V, Fucetola F, Pannunzio G. 1985. A micromethod for the differentiation of fresh from frozen fish muscle. J Sci Food Agric. 36: 811. 26. Damodaran ND, Gopakumar K. 1990. Effect of freezing and thawing on press juices enzyme activity in the muscle of farmed fish and shellfish. IIF-IIR, Commission C2, Aberdeen, 3: 229–233. 27. Barbagli C, Crescenzy GS. 1982. Influence of freezing and thawing on the release of cytochrome oxidase from chicken’s liver and from beef and trout muscle. J Food Sci. 46: 491–496. 28. Frigerio R, Ardemagni A, Cantoni C. 1980. Variazoni quantitative della succino-deidrogenasi durante la lavorazione di molluschi. Arch Vet Ital. 31: 162–166. 29. Salfi V, Fucetola F, Verticelli V, Arata P. 1986. Optimized procedures of biochemical analysis for the differentiation between fresh and frozen thawed fish products. Test of mitochondrial malate dehydrogenase. Ind Alim. 25: 634. 30. Kitamikado M, Yuan CS, Ueno R. 1990. An enzymatic method designed to differentiate between fresh and frozen-thawed fish. J Food Sci. 55: 74–76. 31. Nilsson K, Ekstrand B. 1993. The effect of storage on ice and various freezing treatments on enzyme leakage in muscle tissue of rainbow trout. Z Lebensm Untersuch Forsch. 197: 3–7. 32. Foucat L, Taylor RG, Labas R, Renou JP. 2001. Characterization of frozen fish by NMR imaging and histology. Am Lab. 33: 38–43. 33. Karoui R, Thomas E, Dufour E. 2006. Utilisation of a rapid technique based on front-face fluorescence spectroscopy for differentiating between fresh and frozen–thawed fish fillets. Food Res Inter. 39: 349–355. 34. Uddin M, Kimiya T, Okazaki E. 2008. Near infrared spectroscopy-a rapid and non-destructive technology in seafood analysis. 5th World Fisheries Congress, Key Note Paper No. 4D1 (7). Yokohama, Japan. 35. Rehbein H, Cakli S. 2000. The lysosomal enzyme activities of fresh, cooled, frozen and smoked salmon fish (Onchorhyncus keta and Salmo salar). Turkish J Vet Animal Sci. 24: 103–108. 36. Lin M, Mousavi M, Al-Holy M, Cavinato AG, Rasco BA. 2006. Rapid near infrared spectroscopic method for the detection of spoilage in rainbow trout (Oncorhynchus mykiss) fillet. J Food Sci. 71: S18–S23. 37. Uddin M, Ishizaki S, Okazaki E, Tanaka M. 2002. Near-infrared reflectance spectroscopy for determining end-point temperature of heated fish and shellfish meats. J Sci Food Agric. 82: 286–292. 38. Uddin M, Okazaki E, Ahmed MU, Fukuda Y, Tanaka M. 2006. NIR spectroscopy: A nondestructive fast technique to verify heat treatment of fish-meat gel. Food Control. 17: 660–664. 39. Uddin M, Okazaki E, Fukushima H, Turza S, Yumiko Y, Fukuda Y. 2006. Nondestructive determination of water and protein in surimi by near-infrared spectroscopy. Food Chem. 69: 491–495. 40. Uddin M, Turza S, Okazaki E. 2007. Rapid determination of intact sardine fat by NIRS using surface interactance fibre probe. Int J Food Engine. 3 (6): Art. 12. 41. Uddin M, Okazaki E, Turza S, Yumiko Y, Fukuda Y, Tanaka M. 2005. Non-destructive visible/NIR spectroscopy for differentiation of fresh and frozen–thawed fish. J Food Sci. 70: C506–C510. 42. Wold S. 1976. Pattern recognition by means of disjoint principal components models. Pattern Recognit. 8: 127–39. 43. McLachlan GJ. 1992. Discrimimant Analysis and Statistical Pattern Recognition. Chichester, U.K.: John Wiley & Sons. 44. Uddin M, Okazaki E, Fukuda Y. 2005. Classification of fresh and frozen–thawed fish by dry extract spectroscopy by infrared reflection. NIR News. 16: 4–7. 45. Uddin M, Okazaki E, Kobayashi Y, Yamashita Y, Kimiya Y, Omura Y. 2006. Identification of Fresh and Frozen-Thawed Pacific Saury by Visible/NIR Spectroscopy. The Japan-Korea Joint NIR Symposium. Paper No. PJ13. Seoul, Korea.
Chapter 38
Spectrochemical Methods for the Determination of Metals in Seafood Joseph Sneddon and Chad A. Thibodeaux Contents 38.1 Introduction ..................................................................................................................752 38.2 Spectrochemical Methods .............................................................................................752 38.2.1 Atomic Absorption Spectrometry .....................................................................752 38.2.1.1 Theory..............................................................................................752 38.2.1.2 Instrumentation ...............................................................................753 38.2.1.3 Cold Vapor Atomic Absorption Spectrometry..................................756 38.2.2 Atomic Emission Spectrometry ........................................................................756 38.2.2.1 Theory..............................................................................................756 38.2.2.2 Instrumentation ...............................................................................757 38.2.3 Inductively Coupled Plasma-Mass Spectrometry..............................................759 38.2.4 Practice of Analytical Atomic Spectroscopy .....................................................760 38.2.5 Sample Preparation for Metal Determination in Seafood by Spectrochemical Methods ................................................................................761 38.3 Selected Application of Spectrochemical Methods in Seafood.......................................762 Acknowledgments ....................................................................................................................769 References ................................................................................................................................769
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Introduction
The surface of the earth is covered by approximately 70% of water (seas, lakes, rivers, etc.) and seafood is a major source of food for the majority of the inhabitants of the earth. It also has a large economic factor for many communities around the world. As well as anthropogenic sources, the seas have been found to be a dumping ground or last refuge for many potential pollutants including metals. The metals in the seas will be taken up by the seafood and can enter the human food cycle potentially causing serious health hazards. Many countries have enacted laws and warnings regarding the minimum concentration of many metals in seafood. This has attracted considerable interest and desire in determining metals in various seafood. While there are numerous analytical techniques for metal determination such as various electrochemical methods of voltammetry, coulometry, neutron activation analysis, and the like, this chapter will be confined to spectrochemical or atomic spectroscopic techniques. These are the most widely used and accepted techniques for the determination of metals in seafood.
38.2 Spectrochemical Methods Interaction of energy (light) with matter (gaseous atoms) produce three closely related, yet separate atomic phenomena, namely atomic absorption (AA), atomic emission (AE), and atomic fluorescence (AF). In these techniques, the atoms are detected by optical means. A closely related technique is that of plasma source-mass spectrometry, in particular inductively coupled plasmamass spectrometry (ICP-MS). In this case, the atoms are detected by mass spectrometry. These techniques are collectively known as atomic spectroscopy or spectrochemical techniques and have detection limits for metals and metalloids ranging from mg/mL to ng/mL and even as low as pg/ mL. They have been used to detect metals and metalloids in solids, liquids, and gasses in just about every conceivable matrix including biological, clinical, environmental, food and drugs, petroleum products as well as seafood. The object of this chapter is to give the reader an overview of spectrochemical techniques, including instrumentation and general analytical performance. It is not intended to be comprehensive or discuss the areas on the fringe of atomic spectroscopy. It is beyond the scope of this chapter to describe in detail these techniques and the reader is referred to a number of texts that provide detailed discussion of these four analytical phenomena [1–4]. This chapter provides an overview of atomic absorption spectrometry (AAS), atomic emission spectrometry (AES) with inductively coupled plasma (ICP) as the excitation source, and ICP-MS. Despite some early promise, atomic fluorescence spectrometry (AFS) has failed to live up to its potential and will not be discussed in this chapter. An additional technique will be described namely cold vapor (mostly coupled with AAS) as this has extensive use in Hg determination in seafood.
38.2.1
Atomic Absorption Spectrometry
38.2.1.1
Theory
Atomic absorption involves the impingement of light of a specific wavelength onto gaseous atoms. This causes a valence electron in the atom to be raised from a lower energy level to a higher energy level (called an electronic transition). When the energy of the photon is identical to the energy difference between the lower and higher energy level of the atom, then absorption will occur. The
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intensity of this transition is related to the original concentration of the ground state atoms. This can be represented as follows: T = I /I o
(38.1)
where T is the transmittance I is the intensity of the light source passing through the sample zone Io is the intensity of the light source before it passes through the sample zone The sample zone or path length, b, is relatively long to maximize the amount of light absorbed by the atoms. The amount of light absorbed will depend on the AA coefficient, k. This value is related to the number of atoms per cm3 in the atom cell, n the Einstein probability for the absorption process; and the energy difference between the two levels of the transition. In practice these are all constants, which are combined to give one constant, called the absorptivity, a. k is related exponentially to the transmittance as follows: T = I /I o = e − kb
(38.2)
In practice, the absorbance, A, is used in AAS and is related log arithmetically to the transmittance as follows: A = − logT = − log I /I o = log I o /I = log 1/T = kb log e = 0.43 kb
(38.3)
The Beer–Lambert law relates A to the concentration of the metal in the atom cell, c, as follows: A = abc
or
A = eobc
(38.4)
where a is the absorptivity in L/g-cm eo is the molar absorptivity in L/mol-cm b is the atom cell width in cm AAS involves the measurement of the drop in light intensity of Io to I (depending on the concentration of the metal). Current and modern instrumentation automatically converts the logarithmic value into A. Absorbance is a unit less number, typically, 0.01 to 2.0. In practice, it is better to work in the middle of this range (recommended 0.1–0.3 A) as the precision is poorer at the extremes due to instrumental noise. The most intense transition from the ground state to the first excited state (resonance transition) is the most widely used transition because it is the most sensitive. The origins of atomic spectra and detailed discussion are available elsewhere [5] AAS was discovered independently by Walsh, Alkemade, and Melatz in the early to mid 1950s.
38.2.1.2
Instrumentation
A typical AA system consists of six basic parts: a light source, atomizer, sample introduction system, wavelength selection device, a detection system, and a readout system. All the components
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are conveniently packaged in a complete benchtop unit and are connected to a computer for control, sample preparation, data reduction, and printout. There are numerous commercial instrumentation available with cost ranging from a small compact flame AAS of around $10,000.00 to a top-of-the-line multimetal flame/furnace AAS system with automatic sample introduction and data station of around $100,000.00. A detailed description of light sources (hollow cathode lamp and electrodeless discharge lamp), wavelength selection devices (monochromator), sample introduction systems (pneumatic nebulizers), detection systems (photomultiplier tubes, PMT) and readout (connected to external computers) parts are described elsewhere [1–4]. Sample introduction is very important in AAS (and AES and ICP-MS) and is discussed in detail elsewhere [6]. A short discussion on the atomizer is included in this chapter.
38.2.1.2.1 Atomizer The only widely used and accepted atomizers in AAS are the flame and graphite furnace. In flame AAS, the sample is (usually) introduced into the flame as a fine mist or aerosol. Flames consist of an oxidant and a fuel. The most widely used flames in AAS are air-acetylene (air is the oxidant and acetylene the fuel) and nitrous oxide-acetylene (nitrous oxide is the oxidant and acetylene the fuel). These flames are called combustion flames. Other flames, called diff usion flames, have been proposed but are not widely used. The primary object of the flame is to dissociate molecules into atoms. Air-acetylene (2500 K) does this readily and efficiently for about 40–50 metals in the periodic table. The other 10–20 metals in the periodic table require the hotter nitrous oxide-acetylene flame (3200 K). A long thin flame is desirable in AAS for maximum sensitivity. The graphite furnace atom cell or electrothermal atomizer (ETA) for AAS was commercially developed in the late 1960s. Their principal advantage over flame atomizers are the improvement in sensitivity, typically 10–100×, the ability to use microvolumes (2–200 mL) and micromass solid (few mg) sampling, and in situ pretreatment of the sample. However, ETAs are prone to interferences, particularly from alkali and alkaline earth halides and requires a more complex (and subsequently more expensive) system. The use of an electrically heated tubular furnace was first reported by King in 1905 but for analytical chemistry, the work and system developed by L’Vov around 1960 is regarded as the forerunner of present day ETAs. It consisted of a carbon electrode in which the sample was applied and a carbon tube that could be heated by electrical resistance. The initial design used a supplementary electrode for preheating the furnace, lined the carbon tube with tungsten or tantalum foil to minimize vapor diff usion, and purged the system with argon to prevent oxidation of the carbon. Later work involved direct heating of the sampling electrode by resistance heating and the tube was made of pyrolytic carbon. After heating the tube to an elevated temperature, the sample electrode was inserted into the underside of the tube, and vaporization of the sample was confined to the tube where AAS measurements were made. The system was difficult to operate and the reproducibility could be poor. In 1967, Massmann described a heated graphite atomizer (HGA) which was commercially developed by the Perkin–Elmer Corporation and proved the forerunner for all current commercial ETAs. An isothermal type furnace system proposed by Woodriff at around the same time was considered more difficult to commercialize although recent work has shown the advantage of atomization under isothermal conditions. The Massmann system was typically 50 mm long and 10 mm diameter graphite tube, which was heated by electrical resistance, typically 7–10 V at 400 amps. An inert gas, usually argon or nitrogen, flowed at a constant rate of around 1.5 L/min and the entire system was enclosed in a water jacket. A microliter sample was deposited through an
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entry or injection port in the center of the tube and could be heated in three stages by applying variable current to the system; drying to remove the solvent, ashing or pyrolysis to remove the matrix, and finally atomization of the element. Careful control of the temperature was required in order to obtain good reproducibility. In 1969, West and coworkers developed a rod or filament atomizer. It consisted of a graphite filament of 40 mm in length and 2 mm diameter, supported by water-cooled electrodes, and heated very quickly by the use of current of 70 amps at 10–12 V. Shielding from the air was achieved by a flow of inert gas around the filament. While primarily developed for AAS, West and coworkers showed the potential of the system for AFS. The West filament was the forerunner for the mini-Massmann atomizer developed commercially by Varian Associates. A commercial system was called the carbon rod atomizer (CRA 63). Its main advantage was in the somewhat simpler and less complex design compared to the HGA, low power requirements (2–3 kW), and fast (∼2 s) heating rate. There were differences between this system and the West filament, principally by drilling a hole in a solid cylindrical graphite tube and later using a small cup or crucible between two spring loaded graphite rods. The system was proposed for low microliter volumes, typically 1–20 mL. In general, detection limits and increased interferences were found using the CRA type system compared to the HGA and this type of system has been discontinued from around the mid-1980s and not currently commercially available. A typical schematic furnace AAS system is shown in Figure 38.1. Most current commercial furnaces are similar to that shown in Figure 38.1.
Dosing hole
+
Graphite furnace power supply
Optical temp. sensor
Water-cooled electrode
–
Quartz window
Source beam
To detection system
Graphite tube Internal gas flow
Argon supply
Figure 38.1
Typical GFAAS system.
Cooling water inlet
External gas flow
Cooling water exit
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Graphite furnace AAS has essentially the same instrumentation as flame AAS except for the (a) atom cell, and (b) sample introduction system. An additional need in furnace AAS is faster electronics to process the transient and faster generated signal compared to flame AAS. In practice, AAS usually has the fast electronics capability and most commercial systems have the flame and furnace as interchangeable. Typical volumes used with a graphite furnace are from 1 to 200 mL. This volume can be conveniently introduced to the furnace by manual introduction using a micropipette. There are various dedicated micropipettes available for this range as well as adjustable micropipettes. In the late 1970s, it was suggested that the precision of furnace AAS could be improved by automatic introduction systems. This led to systems which could be added to furnace AAS for automatic sample introduction. It was shown that the precision was not significantly improved by automatic sample introduction but is now an accepted parts of furnace AAS particularly for unattended operation and when numerous samples are required to be analyzed. These systems can be incorporated into sample preparation stations. A monograph by Butcher and Sneddon [7] provides an in-depth coverage of areas in graphite furnace atomic absorption spectrometry (GFAAS) such as matrix or chemical modification to allow a higher ashing temperature without loss of the more volatile metals such as Cd; background correction techniques, in particularly various geometrical configurations of Zeeman effect (inverse ac longitudinal and inverse transverse ac and dc) for a more accurate measurement; graphite tube design and material that allow a more rapid heating of the furnace which improves the signal from more volatile metals; and platform atomization. Atomic absorption spectrometry has reached a maturity in the mid-1990s since its initial development in the mid-1950s. Current developments are in refinements and modest improvements, e.g., software.
38.2.1.3
Cold Vapor Atomic Absorption Spectrometry
The unique properties, high toxicity, and large use in many industrial processes of Hg has led to the development of the cold vapor accessory, most widely used in conjunction with AAS. Mercury has an appreciable vapor pressure at room temperature (0.16 Pa at 25°C). Mercury (in the ionic form) and in an acidic medium can be reduced by stannous chloride to produce ground state atomic mercury. After equilibration, the mercury vapor is swept from the reaction vessel with a carrier gas (argon, air, or nitrogen) into the optical path of an AA instrument for determination as a transient signal. Alternatively, a closed system will produce a steady state signal. The primary advantage of CVAAS for Hg determination is a low detection limit of sub-mg/mL. This can be lowered further using a dedicated commercial cold vapor atomic fluorescence spectrometry system (CV-AFS) where a detection limit in tens of ng/mL is possible. It should be noted that type and concentration of acid, chemical form (inorganic versus organic mercury), matrix components and other components such as the reducing agents can degrade these low detection limits. The use of stannous chloride as a reducing agent will not reduce organomercury compounds. Various pretreatment processes have been developed to overcome this potential problem.
38.2.2 38.2.2.1
Atomic Emission Spectrometry Theory
Atomic emission spectrometry involves the impingement of an external source of energy on ground state atoms. The radiation from these atoms is observed in AES.
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The probability of transitions from the given energy level of a fi xed atomic population was expressed by Einstein, in the form of three coefficients, termed transition probabilities, Aji (spontaneous emission), Bij (spontaneous absorption), and Bji (stimulated emission). These can be considered as representing the ratio of the number of atoms undergoing a transition to an upper level to the number of atoms in the initial or lower level and can be represented as follows: N j = No
gj exp [ −ΔE /KT ] go
(38.5)
where No is the number of atoms in the lower state (or ground state usually for analytical work) Nj is the number of atoms in the excited or upper level g j and go are the statistical weights of the jth (upper state) and o (ground state) DE is the difference in energy in Joules between these two states K is the Boltzmann constant (1.38066 × 10−23 J/K) T is the absolute temperature. If self-absorption is neglected, then the intensity of emission, Iem is I em = A ji hνji N j
(38.6)
where h is Planck’s constant (6.624 × 10−24 Js) nji is the frequency of the transition (DE = hn) Therefore, N is directly related to the concentration of the solutions as follows: Nj = N
gj exp [ −ΔE /KT ] go
(38.7)
The intensity emission of a spontaneous emission line, Iem is related to this equation (sometimes called the Maxwell–Boltzmann equation) as follows: I em = Aij h ν ji N
gj exp [ −ΔE /KT ] go
(38.8)
It can be seen that the atomic emission intensity is dependent on temperature and wavelength. Thus, a higher temperature at longer wavelength would give the most intense atomic emission signal. A plot of emission intensity against sample concentration will be linear. AES (and AFS) has linearity extending up to five to seven orders of magnitude compared to two to three orders of magnitude of AAS.
38.2.2.2
Instrumentation
The primary components of an atomic emission spectrometer (AES) are similar to that of AAS, although for optimum performance the components are different. The excitation source and atomization source are the same, most frequently a plasma.
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Argon
Spectrometer ICP torch Nebulizer
Pump
PMT Spray chamber
Microprocessor and electronics
To waste
Sample Computer
Figure 38.2 system.
Schematic of a typical inductively coupled plasma-optical emission spectrometry
A typical setup for ICP-OES is shown in Figure 38.2. The higher temperature of the plasma will lead to a richer spectrum with many more lines. In order to separate these lines and prevent or minimize spectral interferences, a high-resolution monochromator is required. The 0.20 nm grating typically used in AAS does not provide the required resolution. The most widely adopted system is the Echelle Monochromator, which uses high-diffraction orders and large angles of diffraction. Resolution is around 0.015 nm compared to around 0.2 nm for a typical AAS monochromator. Simultaneous multimetal determination requires a polychromator. During the development of the plasma as an excitation source in analytical AES, PMTs were first used. The PMT continues to be used in ICP-OES, particularly for cost reduction and sequential determinations, as shown in Figure 38.2. However, they have very limited use in multichannel systems and AES quickly adopted systems capable of simultaneous multimetal analysis. In the early 1980s, the photodiode arrays (PDAs) and image sensing vacuum tubes, the so-called vidicons were used. A PDA consists of arrays (256, 512, 1024, 2048 elements arranged in linear manner) of photodiodes operated on a charge transfer storage mode. Each diode is sequentially integrated (several ms) after all the diodes have been integrated with the incident radiation. The current generated by each photodiode is proportional to the intensity of the radiation it receives. The sequential measurement of the current can occur many times a second under the control of a microprocessor. This digitized information can be stored in a computer for electronic processing and visual display. Diode array systems are excellent for studying transient signals such as those on the laser-induced plasma with gate delay generator systems. However, it does have somewhat of a limited resolution, usually 1–2 nm. Diode arrays are used in vidicons in the form of a spectrum. These are similar to a small television tube. The late 1980s through to the present has shown an interest in the use of the charge transfer device (CTD), specifically the charge coupled device (CCD) and to a lesser extent the charge injection device (CID). These are solid-state sensors that have integrated circuits. The charge generated by a photon is collected and stored in a capacitor. A typical pixel arrangement can be
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512 × 320 CCD (much larger arrangements such as 2000 × 2000 pixels have been constructed). The capacitor can be reversed biased by a positive voltage applied to the electrode, creating a potential well. The photons striking the array give electron-hole pairs and the electrons can be stored for a short time in the well. The amount of charge accumulated is a direct function of the incident radiation (and time) and is linear. The charge is shifted horizontally and down to a readout preamplifier which results in a scan of each row in series. CCDs are very useful where low levels of radiation are to be detected. At high levels, “blooming” occur that results in curvature of the response. A CID is a two-dimensional array of pixels. The photons generate positive charges below the negative well capacitors. Again, the amount of charge is proportional to the incident radiation. There is a rapid development of the CTD in spectrochemical analysis. The development of the ICP as an excitation source for analytical AES has been a major advancement in atomic spectroscopy. Its higher temperature has made this source the choice for many atomic spectroscopists work. The most common plasma source is the ICP, which was first developed in the mid-1960s by Fassel and coworkers at Iowa State University and Greenfield and coworkers at Albright and Wilson Ltd. in England. It became commercially available around the mid-1970s. A typical ICP consists of three concentric quartz tubes. These are frequently referred to as the “outer,” “intermediate,” and “inner or carrier gas” tubes. The outer tube can be of various sizes in the range 9–27 mm. A two or three turn induction coil surrounds the top of the quartz tube or torch and is connected to a radiofrequency generator. The coil is water-cooled. The argon, typically at a flow rate of about 1–2 L/min is introduced into the torch and the radiofrequency field operated at 4–50 MHz, most typically 27.12 MHz, and a forward power of 1–5 kW, typically 1.3 kW, is applied. An intense magnetic field around the coil is developed and a spark from a Tesla coil is used to produce “seed” electrons and ions in this region. This induced current flowing in a closed circular path, results in great heating of the argon gas and an avalanche of ions is produced. Temperatures in an ICP have been estimated to be around 8000–10,000 K. The high temperature necessitates cooling which is applied using argon to the outer tubes at flow rates as high as 17 L/min. The sample is introduced, usually as an aerosol, through the inner tube and is viewed at a distance of 5–20 mm above the coil. The advantages of the ICP include high temperature, long residence times, presence of no or few molecular species, optically thin, and few ionization interferences. The last decade has seen a tremendous amount of effort evaluating and understanding the ICP with numerous studies on mechanisms and characterizing variations of the system. The reader is referred to a recent book edited by Montaser and Golightly [8] that describes the current status of the ICP. Considerable improvement and refinement in plasma source-AES has occurred over the last decade. Improved detection limits have been achieved by rotating the plasma through 90° and the development of the miniature ICP. Considerable effort has been expended in the area of sample introduction (see earlier). Improved software has pushed ICP-AES into a well-established and frequently used technique; particularly for multimetal AES.
38.2.3 Inductively Coupled Plasma-Mass Spectrometry Since the early to mid-1980s, the ICP has been used as the ion source for mass spectrometry to determine metals. Its advantages include from two to three orders of magnitude improvement in sensitivity compared to traditional ICP-OES, the mass spectra of the metal are very simple and unique giving high specificity, inherent multimetal coverage, and the technique will measure
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Ion detector
Quadrupole mass filter
Plasma sampling interface
Ion lenses Skimmer Sampling cone cone including proton step
Inductively coupled plasma
Flasma tansh
Sample injection system
Nebulizer ETV
AF injection coil RF power Signal handling
Turbomolecular Turbomolecular Rotary pump pump pump
Optional
Sample FIA-HG Argon Laser probe
Optional
Data Processing
Figure 38.3 Typical Quadrupole ICP-MS system with various sample introduction options. ETV, electrothermal vaporization; LA, laser probe or ablation; FIA-HIG, flow injection analysishydride generation.
metal isotopic ratios. Disadvantages include potential spectral interferences from molecular species and the increased cost and complexity of instrumentation. An inductively coupled plasma-mass spectroscopy (ICP-MS) system consists of the ion source that is the ICP, an interface system, which consists of a sampling cone, a differentially pumped zone, and a skimming zone, ion lenses, a quadrupole mass spectrometer, and a detector. A schematic diagram of a typical ICP-MS is shown in Figure 38.3. Since its commercial availability sample introduction has been a fertile area of research in ICP-MS (as has ICP-OES) and the system in Figure 38.3 has several variations of commercially available sample introduction systems of electrothermal vaporization (ETV), laser ablation (LA), and flow injection analysis-hydride generation (FIA-HG). A less commonly used system involves the use of a high-resolution mass spectrometer as opposed to the quadrupole system. This system shows less resistance to molecular interferences. A detailed description of the instrumentation and the performance of ICP-MS is described elsewhere [4,8]. Essentially the ICP is in a horizontal position and works under atmospheric or normal pressure. Ions produced by this ICP are introduced to the MS through a small orifice, typically 1 mm diameter. The MS is a low pressure, typically at 10−5 to 10−6 Torr. ICP-MS has increasingly become the choice of many spectrochemical analysts.
38.2.4 Practice of Analytical Atomic Spectroscopy The choice of which analytical atomic spectroscopic technique to use will depend on the needs and expectations of the analyst, and the sample. There are many and varied commercial systems available or the analyst may decide that their needs are best suited to a laboratory-constructed system. The factors to be taken into consideration are the size of sample, whether it is a solid, liquid, or gas; the level to be detected; the accuracy and precision, which is acceptable; availability of a particular system; cost per sample, or the speed of analyses.
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Spectrochemical methods are techniques, which depend on the comparison of signals obtained from samples with those obtained from sample standards of known composition. In most cases, these standards are aqueous solutions of the metals of interest. However, the analysis of real samples is complicated by the fact that the metal of interest is present as part of a sample matrix. The matrix can cause interference in the analysis. Therefore, in analytical atomic spectroscopy much attention is paid to the possibility of interferences. This can lead to reduced or poor accuracy. Accuracy can be defined as how close the atomic spectroscopic analysis is to the “correct” answer. In a typical method development, accuracy will be established via several or many ways including standard additions, comparison of the results of the atomic spectroscopic analyses with the results from a different method, recoveries or spikes, or applying the atomic spectroscopic method to standard samples such as those supplied by the National Institutes of Science & Technology (NIST) (Gaithersburg, Maryland). A concern of analytical atomic spectroscopy is precision which can be defined as the repetitive analyses of a particular sample expressed as a percent. Precision will vary with many factors including sample, level to be determined, and choice of instrumentation. Finally, the detection limit is an important factor in analytical atomic spectroscopy. Current atomic spectroscopic techniques have detection limits in the mg/L to mg/mL range. However, lower detection limits are possible using newer and improved techniques in analytical atomic spectroscopy. The reader is referred to a recent book by Butcher and Sneddon [7] that describes the practice of graphite furnace AAS. Much of the advice and suggestions in the book could be equally applied to many areas of analytical atomic spectroscopy.
38.2.5 Sample Preparation for Metal Determination in Seafood by Spectrochemical Methods Most analyses are preferentially performed on solution samples. This can be attributed to the desire for a more homogeneous analysis sample, concern over whether a few micro- or milligrams will be representative of the bulk properties of a large solid sample, improved precision and accuracy is frequently obtained with solution samples (as opposed to solid samples), and the fact that most commercial instrumentation performs at an optimum with solution samples. Therefore, sample preparation remains an integral part of spectrochemical analysis. It is widely used in the preparation of seafood for metal determination by spectrochemical techniques. However, while the metal determination is most frequently performed on solutions, seafood results are most commonly reported as mg/g or in some cases as ng/g. Sample preparation can be conveniently divided into areas such as classical and microwave. Classical methods involve wet or acid decomposition and involves the use of various mineral acids (HNO3, H 2SO4, HClO4, and/or HF), and oxidizing agents (typically H 2O2) to effect dissolution of the sample. It can be performed on an open or closed system. Microwave digestion has rapidly become the choice for many digestion/dissolutions, particularly in seafood preparation [9]. It involves the use of 2450 MHz electromagnetic radiation to digest samples in a teflon or quartz container. Commercial systems are readily available and are conveniently automated and can digest up to 48 samples simultaneously under controlled temperatures conditions. A recent review describes sample preparation on spectrochemical samples for solid materials [10].
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Handbook of Seafood and Seafood Products Analysis
Selected Application of Spectrochemical Methods in Seafood
In the following section selected applications of spectrochemical methods applied to metal determination in various seafood is presented. This area has been discussed previously [11,12]. It is not meant to be comprehensive but show numerous results of several studies involving various seafood, spectrochemical techniques, and variety of metals determined. The results are summarized in Table 38.1.
Table 38.1 Selected Results of Metals in Seafood Metals
Samples Analyzed
Method
Comments
Reference
Cu, Fe, and Zn
Crawfish
ICP-AES
Microwave digestion procedure
[14]
As, Cd, and Pb
Tuna, salmon, shrimp, walleye, clams, oysters, and lobster
ICP-AES
Used single microwave digestion procedure
[16]
Cd, Zn, Cu, Al, and Fe
Scallop tissue
ICP-MS and ICP-AES
Used microwave assisted acid digestion
[17]
Na, Al, K, V, Co, Zn, Se, Sr, Ag, Cd, Ni, Pb, Mg, Mn, Fe, Cu, As, Ba, Hg, Ca, and Cr
Flounder, scup, and blue crab
Hg cold vapor and ICP-MS
Used microwave digestion with HNO3, H2O2, and HF
[18]
Cd, Hg, Pb, As, Se, Mn, Cu, and Zn
Brown rice and fish
ICP-MS and AAS
Used open digestion and did a comparison of the techniques
[19]
Al, Bi, Cd, Co, Cu, Ga, Mn, Ni, Pb, V, and Zn
Fish otoliths from the American eel
ICP-MS
[20]
Ag, Co, Cr, and Ni
Algae, crustaceans, and fish
ICP-MS
[21]
Ni, Cu, Zn, Cr, Cd, Pb, and V
Trivela mactroidea clams
ICP-OES and GFAAS
Pb and V done by GFAAS and others done by ICP-OES
[22]
As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pd, Se, and Zn
Mollusks
ICP-MS
Used nine kinds of Mollusks through a 1 year period
[23]
Spectrochemical Methods for the Determination of Metals in Seafood Table 38.1 (continued)
◾ 763
Selected Results of Metals in Seafood Samples Analyzed
Metals
Method
Comments
Reference
AFS and ICP-MS used for Pb and Hg, while AAS used for the other metals
[24]
Cd, Cr, Cu, Mn, Zn, Hg, Pb, and Ni
Mediterranean mussel
AFS, ICP-MS, and AAS
Pb, Cd, Fe, Cu, Mn, and Zn
Fish
GFAAS
Zn, Cu, Cd, and Pb
Benthic fish
Flame AAS
Looked at 20 different species of fish of the coast of Taiwan
[26]
Fe, Cu, Mn, Zn, and Pb
Fish
Flame and Graphite furnace AAS
Caught fish from the Black Sea and river Yesilimirmak
[27]
Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb, and Hg
Bathymodiolus mussels and grazer shrimp
ICP-MS
Looked at organisms from hydrothermal vents along the mid Altlantic Ridge
[28]
Hg, Cd, Mn, Pb, and Sn
Trout and Tuna
Zeeman GFAAS
Co and Cr
Tuna, scallop, mussels, fish, clam, sardine
Flame AAS
Used ultrasoundassisted acid extraction
[30]
Cu, Cd, Pb, Zn, Mn, Fe, Cr, and Ni
Nine fish species
AAS
Microwave digestion used
[31]
Cd, Cu, Fe, Zn, and Pb
Two fish species
AAS
[32]
Cu and Cd
Five fish species
AAS
[33]
Na, Mg, Ca, Fe, Cu, Mn, Ni, Cd, Cr, Pb
Oysters
Flame AAS
Used microwave digestion
[34]
As, Cd, Hg, and Pb
Carrot puree, fish muscle, mushroom, graham flour, scampi, and mussel powder
ICP-MS
Used pressure digestion. Looked at foodstuffs
[35]
[25]
[29]
Abbreviations: ICP-AES, inductively coupled plasma-atomic emission spectroscopy; ICP-OES, inductively coupled plasma-optical emission spectroscopy; ICP-MS, inductively coupled plasma-mass spectrometry; GFAAS, graphite furnace atomic absorption spectrometry; AAS, atomic absorption spectrometry; AFS, atomic fluorescence spectrometry.
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Crawfish or crayfish are widely consumed throughout the world, in particular in Louisiana, People’s Republic of China, and Far East, and their metal (and organic) concentrations has generated a great deal of interest [13]. Hagen and Sneddon [14] and Richert and Sneddon [15] have investigated the concentrations of several heavy metals in crawfish following microwave digestion. Hagen and Sneddon determined Cu, Fe, and Zn and found no differences between male and female species. Richert and Sneddon’s study was to determine the variation over a season (February through May) and involved monitoring on four separate occasions for two distinct sampling waters: one from natural run off from the nearby fields and other water from an underground source. Concentrations between the two sampling areas were not considered statistically significant. A single microwave digestion procedure was developed for use with a variety of seafood products by Sheppard et al. [16]. Inductively coupled plasma atomic emission and mass spectrometry were used to determine the levels of As, Cd, and Pb in samples of tuna, salmon, shrimp, walleye, clams, oysters, and lobster. The precision for 10 replicate analyses of clams was 2.1% for As at the 10.0 mg/g level, 5.6% for Pb at the 0.067 mg/g level, and 2.5% for Cd at the 0.079 mg/g level. Acceptable spike recoveries in each of the sample types were achieved using both detection methods. Results from two standard reference materials were in good agreement with certified values. A multimetal determination method for trace metals in scallop tissue samples was developed by ICP-MS and ICP-AES after microwave assisted acid digestion by Tamaru et al. [17]. A standard reference material of oyster tissue (SRM 1566b Oyster tissue) was analyzed to verify the method. Thirty metals (K, Na, In, Mg, Ca, Fe, AI, Cu, Mn, Ba, Sr, Cd, Ni, Co, Pb, Y, and rare earth elements) in the reference material could be determined. The concentrations of metals obtained were in good agreement with their certified and reference values and had good precision within relative standard deviations (RSD) of 4% except for Ni and Pb. This method was applied to determine concentrations of metals in a scallop tissue sample. Cadmium, In, Cu, Al, and Fe in the scallop tissue sample were obtained at 69.0,148, 19.0, 415, and 221 mg/g level, respectively. In particular, the bio-accumulation factors of Cd, which were estimated between the concentrations in scallop tissue sample and those in shale and seawater, were highest among the 30 metals determined. A microwave digestion method suitable for the determination of multiple metals in marine species was developed, using cold vapor atomic spectrometry for the determination of Hg, and ICP-MS for all of the other metals by Yang and Swami [18]. An optimized reagent mixture composed of 2 mL of HNO3, 2 mL of H202, and 0.3 mL of HF was used in the microwave digestion of about 0.15 g (dry weight) of sample and was found to give the best overall recoveries of metals in two standard reference materials. In the oyster tissue standard reference material (SRM 1566b), recoveries of Na, AI, K, V, Co, In, Se, Sr, Ag, Cd, Ni, and Pb were between 90% and 110%; Mg, Mn, Fe, Cu, As, and Ba recoveries were between 85% and 90%; Hg recovery was 81%; and Ca recovery was 64%. In a dogfish certified reference material (DORM-2), the recoveries of Al, Cr, Mn, Se, and Hg were between 90% and 110%; Ni, Cu, In, and As recoveries were about 85%; and Fe recovery was 112%. Method detection limits of the metals were established. Metal concentrations in flounder, scup, and blue crab samples from coastal locations around Long Island and the Hudson River estuary were determined. A study was conducted by Oshima et al. [19] to evaluate the applicability of ICP-MS for the determination of metals in brown rice and fish. Cadmium, Pb, Hg, As, Se, Mn, Cu, and Zn were determined by this method. An open digestion with HNO3 (Method A) and a rapid open digestion with HNO3 and HF (Method B) were used to solubilize analytes in samples, and these procedures were followed by determination by ICP-MS. Recovery of certified metals from standard reference materials by Method A and Method B ranged from 92% to 110% except for Hg (70%–100%). Analytical results of brown rice and fish samples obtained agreed with those obtained by AAS.
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The results of this study demonstrated that quadrupole ICP-MS provides precise and accurate measurements of the metals tested in brown rice and fish samples. Transition and heavy metals within the calcined otoliths of estuarine fish may represent valuable tracers of environmental exposures, allowing inferences on fatality, habitat use, and exposure to pollution. Accurate measurement of very low concentrations of these metals in otoliths by ICP-MS is often precluded by the interferences of predominant calcium matrix. Arslan and Secor [20] coupled a solid-phase extraction procedure to an ICP-MS instrument to overcome the matrix problems and improve the limits of detection. To test this novel application and the utility of otolith transition and heavy metals as tracers of habitat use, otoliths of American eel (Anguilla rostrata) captured from six locations (George Washington Bridge, Haverstraw, Newburgh, Kingston, Athens, and Albany, all in New York State) throughout the Hudson River estuary were analyzed for site specific differences expected due to varying environmental exposure. Several trace metals, including Al, Bi, Cd, Co, Cu, Ga, Mn, Ni, Pb, V, and In, were selectively extracted from otolith solutions and preconcentrated on a microcolumn of chelating resin. The concentrations of all metals in A. rostrata otoliths were above the limits of detection that ranged from 0.2 ng/g for Co to 7 ng/g for In. Differences in the metal composition of the otoliths among the groups were significant indicating different levels of exposure to environmental conditions. Discriminant analysis yielded an overall location classification rate of 78%. Aluminum, Bi, Cd, Mn, Ni, and V contributed most to the discriminant function. Samples collected at George Washington Bridge showed 100% discrimination from other locations, and higher levels of many transition and heavy metals, consistent with higher exposure to those metals in the most polluted region of the Hudson River estuary. Original results concerning Ag, Co, Cr, and Ni determination in marine biotope (sediment and water) and biocenosis (algae, crustaceans, and fish) collected in 2003, 2004, 2005, and 2006 from the Romanian Black seacoast ecosystem are presented by Chirila et al. [21]. The solid samples were carefully prepared (washed and dried) and subjected to dissolution with HNO3 and H202 in a Digesdahl device. Metal concentrations were determined by ICP-AES and applied to solid samples (sediment and biota). The levels of Ag varied from ND (not detectable) to 0.20, Co from 0.03–0.65, Cr from 0.49–22.44, and Ni from 0.32–28.13 mg/g. In water, the mean metal concentrations were Ag of 1.07, Co of 0.75 and Ni of 8.68 mg/L. LaBrecque et al. [22] performed a study using Trivela mactroidea clams which were handpicked directly from the marine sediment at 14 sampling sites along the Venezuelan coast of the state Miranda. Clam soft tissues were washed, dried, and ground into fine powder. For heavy metal analysis, the powder samples were digested with HNO3 and further with H202. Determination for Ni, Cu, In, Cr, and Cd were performed by ICP-OES while determination for Pb and V were made by GFAAS. The suitability of the ICP-OES method was assessed by analyzing mussel tissue standard reference material NIST -2976. Trace metal concentrations of 11–49 mg/g for Cu, 55–166 mg/g for In, <1–6.2 mg/g for Cr, 6–15 mg/g for Ni, 2–13.2 mg/g for V, <1–1.9 mg/g for Cd, and <1.5–4.9 mg/g for Pb were determined. These values were significantly lower than those obtained in a study 12 years ago on soft clam tissue from the same area. Mollusks living in seas can accumulate heavy metals, and may serve as excellent passive biomonitors. During a period of one year, bioaccumulation of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn was examined in nine kinds of mollusks (Rapana venosa, Neverita didyma, Scapharca subcrenata, Mytilus edulis, Amusium, Crassostrea talienwhanensis, Meretix meretrix, Ruditapes philippinarum, and Mactra veneriformis). These were collected at eight coastal sites along the Chinese Bohai Se by Wang et al. [23]. Metal concentrations were directly determined by ICP-MS. Two certified reference materials, dogfish muscle (DORM-2) and mussel (GBW 08571), were used to validate the methods, and the recoveries were within 83.72%–112.30% of the certified values.
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Bioaccumulation of metals varied strongly among sampling sites and species. Statistical analysis (one-way ANOVA) indicated that different species examined showed different bioaccumulation of metals, and perhaps they could be used as potential biomonitors to investigate the contamination levels of heavy metals. Principal component analysis (PCA) and correlation analysis were used to study the relationships between these heavy metals. The results showed that, in nine mollusks’ tissues, there are significant correlations between these metals in the adjacent group or subgroup in the periodic table of elements (metals). Heavy metal concentrations of Hg, Cd, Pb, Zn, Cu, Ni, Mn, and Cr in Mytilus galloprovincialis were investigated by Maanan [24] to provide information on pollution of the Safi coastal area in Morocco, since these metals have the highest toxic potential. The concentration of Hg and Pb was determined by AFS and ICP-MS methods, respectively, while the remaining metals (Cd, Cr, Cu, Mn, In, and Ni) were quantified by AAS. High Pb, Cd, Cr, and Hg levels were registered in tissue samples collected from two stations near the Jon Lihoudi and Safi city, while elevated concentrations of Mn and Zn (14.70–25.30 mg/kg and 570–650 mg/kg dry weight, respectively) were found in mussel specimens from Cap Cantin. The high levels of Ni found near the industrial area are of concern in terms of environmental health need frequent monitoring. The metal concentrations recorded at the clean stations may be considered as useful background levels to which to refer for comparison within the Atlantic coast. M. galloprovincialis are suitable biomonitors to investigate the contamination levels of heavy metals pollution face a different human activity in this coastal area of the Atlantic coast. The concentrations of heavy metals (Pb, Cd, Fe, Cu, Mn, and Zn) in fish samples were determined using GFAAS after dry and wet ashing methods by Tuzen [25]. Different matrix modifiers were used for the stabilization of the analyte. Good accuracy was assured by the analysis of biological reference materials. Recoveries were quantified for all metals studied (~95%). The RSD were less than 7% for all metals. Taiwanese consume a large amount of marine fish, most of which are collected from the coastal waters around Taiwan. Heavy metals are recognized as one of the most important pollutants, and their accumulations in the organisms were studied and monitored for the safety of seafood consumption in the coastal waters of Taiwan; however, its regulation was overlooked in the eastern region. Huang [26] evaluated the seafood consumption safety of the coastal fisheries in eastern Taiwan and established a baseline reference of the heavy metal levels in the fish of this region for the future monitoring of heavy metal pollution. Indium, Cu, Cd, and Pb concentrations were determined in muscles, gills, intestines, and livers of 20 benthic species of the most common fish caught from the coastal waters of eastern Taiwan using flame atomic absorption spectrometry (FAAS). Indium concentrations were the highest in the tissues, followed by Cu and Cd, and Pb being the lowest except in the gills. Among the tissues, liver showed the highest metal concentrations followed by intestine and gill, and was the lowest in the muscle. The concentrations of In, Cu, Cd, and lead in muscle ranged 2.0–6.2, 0.15–0.81, 0.02–0.12, and <0.02–0.15 mg/g wet weight, respectively The concentrations of the four metals in liver were at 16.9–59.1, 1.4–12.4, 0.11–1.16, and <0.02–1.09 mg/g wet weight, respectively. The concentrations of the heavy metals in the tissues varied significantly among species. Spottyback searobin Pterygotrigla hemistica and soldierfish Myripristis berndti contained in general higher concentrations of the metals in muscle and liver than other species of fish, respectively The metal concentrations of fish found in this study are similar to the metal levels of the fish caught from slightly polluted waters in other parts of Taiwan, while the metal concentrations in the authors’ fish muscle are far below the consumption safety tolerance set by most countries in the world. Therefore, no public health problem would be raised from the consumption of fish from the coastal waters of eastern Taiwan.
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The concentrations of trace metals in the fish species from the Black Sea and Yesilirmak River in Turkey were determined by Tuzen et al. [27] using FAAS and GFAAS after microwave digestion. The proposed method showed satisfactory recovery rates, detection limits, and standard deviations. The average metal concentrations (mg/g) of the five species varied in the following ranges: Fe 90.16–102.51; Cu 1.34–1.72; Mn 1.29–9.21; Zn 25.76–112.71; Pb 0.53–1.73; Cd 0.98–2.2; Cr 1.43–1.92, and Ni 2.90–9.36 mg/g, respectively. Kadar et al. [28] describes several features of the aquatic environment with the emphasis on the total versus filter-passing fraction (FP) of heavy metals in microhabitats of two typical deepsea vent organisms: the filter-feeder, symbiont-bearing Bathymodiolus, and the grazer shrimps Rimicaris/Mirocaris from the Mid-Atlantic Ridge (MAR). The concentration of 10 trace metals: Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb, and Hg was explored highlighting common and distinctive features among the five hydrothermal vent sites of the MAR: Menez Gwen, Lucky Strike, Rainbow, Saldanha, and Menez Hom that are all geochemical different when looking at the undiluted hydrothermal fluid composition. The drop off in the percentage of FP from total metal concentration in mussel and/or shrimp inhabited water samples (in mussel beds at Rainbow, for instance, FP fraction of Fe was <23%, Zn 24%, Al 65%, Cu 70%, and Mn 89%) as compared to noninhabited areas (94% of the Fe, 90% of the Zn, 100% of the other metals was in the FP fraction) may indicate an influence of vent organisms on their habitat’s chemistry, which in turn may determine adaptational strategies to elevated levels of toxic heavy metals. Predominance of particulate fraction over the soluble metals, jointly with the morphological structure and elemental composition of typical particles in these vent habitats suggest a more limited metal bioavailability to vent organisms as previously thought. It is evoked that vent invertebrates may have developed highly efficient metal-handling strategies targeting particulate phase of various metals present in the mixing zones that enables their survival under these extreme conditions. Direct solid sampling Zeeman GFAAS methods were developed by Detcheva and Grobecker [29] for the determination of Hg, Cd, Mn, Pb, and Sn in seafood. All metals except Hg were measured by a third generation Zeeman AAS combined with an automatic solid sampler. In 3-fieldand dynamic mode the calibrations concentration range was substantially extended and high amounts of analyte were detectable without laborious dilution of solid samples. The measurements were based on calibrations using certified reference materials of organic matrixes. In this case, solid certified reference materials were not available and calibration by aqueous standard solutions was proved an alternative. No matrix effects were observed under the optimized conditions. Results obtained were in good agreement with the certified values. Solid sampling Zeeman AAS was shown to be a reliable, rapid, and low-cost method for the control of trace metals in seafood. A rapid and sensitive method was proposed by Yebra-Biurrun and Cancela-Perez [30] for the determination of Cr and Co in seafood samples by FAAS combined with a dynamic ultrasoundassisted acid extraction and an online mini-column preconcentration. The use of dilute HNO3 as an extractant in a continuous mode at a flow rate of 3.5 mL/min and room temperature was sufficient for quantitative extraction of these trace metals from seafood. A mini-column containing a chelating resin was an excellent device for the quantitative preconcentration of Cr and Co prior to their detection. A flow-injection manifold was used as interface for coupling all analytical steps, which allowed the automation of the whole analytical process. A Plackett–Burman experimental design was used as a multivariate strategy for the optimization of both sample preparation and preconcentration steps. The method was successfully applied to the determination of Cr and Co in seafood samples. Trace metal content of nine fish species harvested from the Black and Aegean Seas in Turkey were determined by microwave digestion and atomic absorption spectroscopy (MD-AAS)
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by Uluozlu et al. [31]. Verification of the MD-AAS method was demonstrated by analysis of standard reference material (NRCC-DORM-2 dogfish muscle). Trace metal content in fish samples were 0.73–1.83 mg/g for Cu, 0.45–0.90 mg/g for Cd, 0.33–0.93 mg/g for Pb, 35.4–1 06 mg/g for In, 1.28–7.40 mg/g for Mn 68.6–163 mg/g for Fe, 0.95–1.98 mg/g for Cr, and 1.92–5.68 mg/g for Ni. The levels of Pb and Cd in fish samples were higher than the recommended legal limits for human consumption. Samples of Mugil cephalus and Mullus barbatus were collected in the Northeast Mediterranean coast of Turkey to determine the concentrations of Cd, Cu, Fe, Zn, and Pb in the liver, the gill, and the muscle tissues were determined by FAAS [32]. Except for Pb, highest levels of each metal were found in the liver and this was followed by the gill and the muscle in both species. Among the metals analyzed, Cu, Zn, and Fe were the most abundant in the different tissues while Cd and Pb were the least abundant both in M. cephalus and M. barbatus. Seasonal changes in metal (Cd, Cu, Pb, Fe, and Zn) concentration were observed in the tissues of both species, but these seasonal variations may not influence consumption advisories. In general, the highest concentrations were detected for all metals in summer. The Cd and Cu levels were determined by Erdogrul et al. [33] in a total of one hundred and twenty six fish samples which belongs to five fish species collected from Sir and Menzelet Dam Lakes in Kahramanmaras Province, Turkey by AAS. The concentrations of heavy metals were expressed as parts per million (ppm) wet weight of tissue. The mean levels of cadmium and copper in the muscle, the liver, and the gill tissues of Cyprinus carpio from the Menzelet Dam were found to be 0.27, 0.91, 1.49 and 0.94, 1.2, 1.05, respectively. The mean levels of Cd in the muscle tissues of Leuciscus cephalus from the Menzelet Dam were found 0.32 ppm, Cd was not found in tissues of the liver and the gill. The mean levels of Cu in the muscle, the liver and the gill tissues were found as 3.17 ppm, 1.19 ppm, 0.96 ppm, respectively. The mean levels of the Cd and Cu in muscle and gill tissues of Acanthobrama marmid from the Sir Dam were found to be 1.28, 2.64 and 0.72, 0.08, respectively. The levels of the Cd and Cu in muscle tissues of Cyprinus carpio from the Sir Dam were found 0.87 and 0.02 ppm, respectively. The mean levels of the Cd and Cu in the muscle and gill tissues of Chondrostoma regium from the Sir Dam were found to be 0.80, 2.62 and 0.67, 1.34 ppm, respectively. The mean concentrations of Cd in the muscle tissues of Silurus glanis were found to be 0.60 ppm. In the muscle of the Silurus glanis from the Sir Dam, Cu was not found. The Sir Dam is more polluted than the Menzelet Dam from the point of Cd but less polluted than the Menzelet Dam. From the point of Cu a relationship was determined between species and their habitat region in terms of the levels reflected metal residues. In this study, it was emphasized that the amounts of Cd and Cu in the samples were low, however, seas, lakes, rivers, soil, air, and consumed foods has to be routinely controlled. A comparison was made between microwave digestion and wet digestion methods for the determination of Na, Mg, Ca, Fe, Cu, Mn, Ni, Cd, Cr, and Pb in oyster with FAAS by Ren et al. [34]. Using microwave digestion method with a closed-vessel, the digestion could be done more rapidly and more effectively. The method could save reagents and display a lower background. It was available for biomonitoring of seawater and analysis of seafood. Thirteen laboratories participated in an inter-laboratory method performance (collaborative) study on a method for the determination of As, Cd, Hg, and Pb by ICP-MS after pressure digestion including a microwave heating technique [35]. Prior to the study, the laboratories were able to practice on samples with defined metal levels (pretrial test). The method was tested on a total of seven foodstuffs: carrot puree, fish muscle, mushroom, graham flour, simulated diet, scampi, and mussel powder. The metal concentrations in mg/kg dry matter (dm) ranged from 0.06–21.4 for As, 0.03–28.3 for Cd, 0.04–0.6 for Hg, and 0.01–2.4 for Pb. The materials used in the study were
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presented to the participants as blind duplicates, and the participants were asked to perform single determinations on each sample. The repeatability RSD for As ranged from 3.8% to 24%, for Cd from 2.6% to 6.9%, for Hg from 4.8% to 8.3%, and for Pb from 2.9% to 27%. The reproducibility relative standard deviation for As ranged from 9.0% to 28%, for Cd from 2.8% to 18%, for Hg from 9.9% to 24%, and for Pb from 8.0% to 50%. The HorRat values were less than 1.5 r for all test samples, except for Pb in wheat flour at a level close to the limit of quantitation (0.01 mg/kg). The study showed that the ICP-MS method was satisfactory as a standard method for metal determination in seafood.
Acknowledgments This work was supported, in part, by Merck undergraduate research program awarded to McNeese State University for 2005–2007. Partial support from Environmental Protection Agency, EPA-R82958401–1 is gratefully acknowledged.
References 1. J.D. Ingle and S.R. Crouch, Spectrochemical Analysis, Prentice Hall, Englewood, NJ, 1988. 2. L.H.J. Lajunen, Spectrochemical Analysis by Atomic Absorption and Emission, Royal Society of Chemistry, Cambridge, England, 1992. 3. S.J. Haswell, ed., Atomic Absorption Spectrometry: Theory, Design and Applications, Elsevier Science Publishers, Amsterdam, the Netherlands, 1991. 4. J. Nolte, ICP-Emission Spectrometry–A Practical Guide, Wiley-VCH, Hoboken, NJ, 2003. 5. I.I. Sobelman, Atomic Spectra and Radiative Transitions, second edition, Springer-Verlag, Berlin, Germany, 1992. 6. J. Sneddon, ed., Sample Introduction in Atomic Spectrometry, Elsevier Science Publishers, Amsterdam, the Netherlands, 1990. 7. D.J. Butcher and J. Sneddon, A Practical Guide to Graphite Furnace Atomic Absorption Spectrometry, John Wiley & Sons, New York, 1997. 8. A. Montaser and D.W. Golightly, eds., Inductively Coupled Plasmas in Analytical Atomic Spectrometry, second edition, VCH Publishers, Inc., New York, 1992. 9. H.M. Kingston and S.J. Haswell, eds., Microwave-Enhanced Chemistry, Fundamentals, Sample Preparation and Applications, American Chemical Society, Washingon DC, 1997. 10. J. Sneddon, C. Hardaway, K.K. Bobbadi, and A.K. Reddy, Sample preparation of solid samples for metal determination by atomic spectroscopy-an overview and selected recent applications, Applied Spectroscopy Reviews, (2006), 41(1), 23–42. 11. J. Sneddon, P.W. Rode, M.A. Hamilton, S. Pingeli, and J.P. Hagen, Determination of metals in seafood, Applied Spectroscopy Reviews, (2007), 42(1), 1–16. 12. J. Sneddon, Use of spectrochemical methods for the determination of metals in fish and other seafood in Louisiana. In The Determination of Chemical Elements in Food: Applications for Atomic and Mass Spectrometry, S. Caroli, ed., Chapter 14, John Wiley & Sons, Hoboken, NJ, 2007, pp. 437–454. 13. J.C. Richert and J. Sneddon, Determination of inorganics and organics in crawfish, Applied Spectroscopy Reviews, (2008), 43, 1–17. 14. J.P. Hagen and J. Sneddon, Determination of copper, iron and zinc in crawfish (Procambrus clarkii) by inductively coupled plasma-optical emission spectrometry, Spectroscopy Letters, (2009), 42(1), 58–61. 15. J.C. Richert and J. Sneddon, Determination of heavy metals in crawfish (Procambrus clarkii) by inductively coupled plasma optical emission spectrometry; a study over the season in Southwest Louisiana, Analytical Letters, (2008), 44(17), 3198–3209.
770 ◾ Handbook of Seafood and Seafood Products Analysis 16. B.S. Sheppard, D.T. Heitkemper, and C.M. Gaston, Microwave digestion for the determination of arsenic, cadmium and lead in seafood products by inductively coupled plasma atomic emission and mass spectrometry, Analyst, (1994), 119(8), 1683–1686. 17. M. Tamaru, T. Yabutani, and J. Motonaka, Multielement determination of trace metals in scallop tissue samples, Bunseki Kagaku (2004), 53(12), 1435–1440. 18. K.X. Yang, and K. Swami, Determination of metals in marine species by microwave digestion and inductively coupled plasma mass spectrometry analysis, Spectrochimica Acta, Part B: Atomic Spectroscopy (2007), 62(10), 1177–1181. 19. H. Oshima, E. Ueno, I. Saito, and H. Matsumoto, A comparative study of cadmium, lead, mercury, arsenic, selenium, manganese, copper and zinc in brown rice and fish by inductively coupled plasmamass spectrometry (ICP-MS) and atomic absorption spectrometry, Shokuhin Eiseigaku Zasshi (2004), 45(5), 270–276. 20. L. Arslan, and D.H. Secor, Analysis of trace transition elements and heavy metals in fish otoliths as tracers of habitat use by American eels in the Hudson River estuary, Estuaries (2005), 28(3), 382–393. 21. E. Chirila, T. Petisleam, I.C. Popovici, and Z. Caradima, ICP-MS utilization for some trace elements determination in marine samples, Chem. Oep, Constanta, Rom. Revista de Chimie (2006), 57(8), 803–807. 22. J.J. LaBrecque, L Benzo, J.A. Alfonso, P.R. Cordoves, M. Quintal, N. Manuelita, C.V. Gomez, and E. Marcano, The concentrations of selected trace elements in clams, Trivela mactroidea along the Venezuelan coast in the state of Miranda, Marine Pollution Bulletin (2004), 49(7–8), 664–667. 23. Y. Wang, L. Liang, J. Shi, and G. Jiang, Study on the contamination of heavy metals and their correlations in mollusks collected from coastal sites along the Chinese Bohai Sea, Environment International (2005), 31(8), 1103–1113. 24. M. Maanan, Biomonitoring of heavy metals using Mytilus galloprovincialis in Safi coastal waters, Morocco, Environmental Toxicology (2007), 22(5), 525–531. 25. M. Tuzen, Determination of heavy metals in fish samples of the middle Black Sea (Turkey) by graphite furnace atomic absorption spectrometry, Food Chemistry (2003), 80(1), 119–123. 26. W.-B. Huang, Heavy metal concentrations in the common benthic fishes caught from the coastal waters of eastern Taiwan, Fenxi (2003), 11(4), 324–330. 27. M. Tuzen, D. Mend, H.Sari, M. Suicmez, and E. Hasdemir, Investigation of trace metal levels in fish species from the Black Sea and the river Yesilirmak, Turkey by atomic absorption spectrometry, Fresenius Environmental Bulletin (2004), 13(5), 472–474. 28. E. Kadar, V. Costa, I. Martins, R.S. Santos, Ricardo and J.J. Powell, Enrichment in Trace Metals (Al, Mn, Co, Cu, Mo, Cd, Fe, Zn, Pb and Hg) of Macro-Invertebrate Habitats at Hydrothermal Vents Along the Mid-Atlantic Ridge, Hydrobiologia (2005), 548 191–205. 29. A. Detcheva, and K.H. Grobecker, Determination of Hg, Cd, Mn, Pb, and Sn in seafood by solid sampling Zeeman atomic absorption spectrometry, Spectrochimica Acta, Part B: Atomic Spectroscopy (2006), 61B(4), 454–459. 30. M.C. Yebra-Biurrun and S. Cancela-Perez, Continuous approach for ultrasound-assisted acid extraction-minicolumn preconcentration of chromium and cobalt from seafood samples prior to flame atomic absorption spectrometry, Analytical Sciences, (2007) 23(8), 993–996. 31. O.D. Uluozlu, M. Tuzen, D. Mendil, and M. Soylak, Trace metal content in nine species of fish from the Black and Aegean Seas, Turkey, Food Chemistry (2007), 104(2), 835–840. 32. H.Y. Cogun, A. Yuezereroglu, Oe. Firat, G. Goek, and F. Kargin, Metal concentrations in fish species from the Northeast Mediterranean Sea, Environmental Monitoring and Assessment (2006), 121(1–3), 431–438. 33. O. Erdogrul, D. Ates, and D. Ayfer, Determination of cadmium and copper in fi sh samples from Sir and Menzlet Dam Lake Kahramanmaras, Turkey, Environmental Monitoring and Assessment (2006), 117(1–3), 281–290.
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34. N. Ren, H.Li, Hong, Q. Zeng, and X. Xijiang, Determination of metal ions in oyster by microwave digestion of sample and flame atomic absorption spectrometry (FAAS), Huaxue Shijie (2005), 46(2), 83–85, 108, 117. 35. K. Julshamn, A. Maage, N. Amund, S. Hilde, K.H. Grobecker, L. Jorhem, and P. Fecher, Determination of arsenic, cadmium, mercury, and lead by inductively coupled plasma/mass spectrometry in foods after pressure digestion: NMKL inter-laboratory study, Journal of AOAC International (2007), 90(3), 844–856.
Chapter 39
Food Irradiation and Its Detection Yiu Chung Wong, Della Wai Mei Sin, and Wai Yin Yao Contents 39.1 Introduction ..................................................................................................................774 39.1.1 Foodborne Diseases..........................................................................................774 39.1.2 High Energy Irradiation for Food Preservation ................................................774 39.1.3 Development of Food Irradiation .................................................................... 775 39.1.4 Global Acceptance and Attitudes .................................................................... 779 39.1.5 Detection Methods for Irradiated Foods ......................................................... 780 39.2 Detection Methods .......................................................................................................781 39.2.1 Electron Spin Resonance Spectroscopy ............................................................781 39.2.2 Analysis of Radiolytic Chemicals .....................................................................782 39.2.2.1 2-Alkylcyclobutanones .....................................................................782 39.2.2.2 Volatile Hydrocarbons .................................................................... 784 39.2.2.3 o-, m-Tyrosine ..................................................................................785 39.2.2.4 Hydrogen and Carbon Monoxide Gases ..........................................785 39.2.3 DNA Methods .................................................................................................785 39.2.4 Luminescence.................................................................................................. 787 39.2.5 Microbiological Methods ................................................................................ 788 39.3 Conclusions ...................................................................................................................789 Acknowledgment .....................................................................................................................789 References ................................................................................................................................789
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39.1 Introduction 39.1.1
Foodborne Diseases
Diseases transmitted through contaminated food (or foodborne diseases) are always major social problems recognized by many national and international health authorities. Foodborne microbes such as Salmonella spp. and Escherichia coli O157:H7 are the primary cause of food poisoning in the United States and other industrialized nations, whereas Vibrio spp. especially V. cholerae, V. parahaemolyticus, and V. vulnificus caused a significant number of outbreaks and deaths in Asia and Latin America since the end of the last century [1,2]. In addition, foodborne diseases from parasites such as tapeworms, taxoplasmosis, and trichinosis are also of concern in developing countries. Foodborne diseases were responsible for a total of 76 million cases, 325,000 hospitalizations, and 5,000 deaths annually in the United States [3] and 2.4 million cases, 21,138 hospitalizations, and 718 deaths annually in England and Wales [4], respectively. However, there is a lack of adequate reporting mechanisms in both the developed and developing countries; only a very small proportion (less than 1%–10%) was said to be taken into account in the surveys [5]. The true incidence of such diseases is very difficult to determine and unavoidably results in an underestimated figure. Although, the real picture of the problem has remained a mystery, the situation has been well demonstrated by the World Health Organization (WHO) stating that approximately one in three people worldwide suffer annually from a foodborne disease and 1.8 million die from severe food and waterborne diarrhea [6].
39.1.2
High Energy Irradiation for Food Preservation
Two common ways have long been used to prevent the occurrence of foodborne diseases. Either physical methods such as heating or chemical methods such as adding salt as preservative are known to be extremely simple and effective to get rid of most pathogenic microbes and parasites. These well-accepted methods, however, are not deemed suitable to treat some solid foods, in particular raw meats, seafoods, and fresh fruits, because the texture, flavors, taste, and incurred ingredients would be irreversibly changed during the treatments. Nowadays, with the advancement of food-processing technology, there are more options and combinations of techniques available in food preservation. As described by a number of comprehensive reviews [7–12], the application of ionization radiation is regarded as one of the important new techniques in preserving hygienic quality of food in the food industry. Food irradiation is a process in which food matrices are exposed to high ionization energy gamma-ray produced from radionuclides (usually 60Co or 137Cs sources), fast moving electrons (maximum energy of 10 MeV), or x-rays (maximum of 5 MeV) from machines. These sources are permitted to be used in food treatment processes and have been adopted as a Codex Alimentarius General Standard [13]. However, the relatively low penetrating power of electron beams and the low efficiency of x-ray conversion limited their uses when compared to that of gamma irradiation. At present, cobalt-60, having higher penetrating power than that of cesium-137 is the preferred choice of irradiation source. It is estimated that 120 electron accelerator processing units [14] and more than 200 industrial 60 Co irradiation facilities [15] are being operated worldwide for sterilizing medical devices and for food irradiation. The penetrating high energy could damage the DNA of living cells through energy transfer and significantly reduce the number of bacteria, yeasts, and moulds in food. The mechanisms of these energy transfer actions are well understood and described in detail in the literature [16,17].
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The ionization radiation loses its energy to molecules of any matter (such as water, carbohydrates, fats, and proteins in food molecules) and leads to direct breakdown of molecules (primary effect) with the ejection of an electron and formation of a free radical: M → •M+ + eBoth the electron and the free radical are highly reactive and cause a cascade of further ionization reactions (secondary effect) along the track. A combination of the primary and secondary effect leads to the chemical decomposition of molecules that are exposed to radiation in the medium. The magnitude of the effects achieved is solely governed by the radiation dose being applied. The International System of Unit for radiation is the Gray (Gy) where 1 Gy is equal to 1 J of energy absorbed per kilogram of food mass. According to the Joint Expert Committee on Food Irradiation (JECFI) [18], there are three defined categories when food is irradiated at low, medium, and high doses: 1. Radurization at below 1 kGy: Prevents sprouting in vegetables, delays ripening in fruits, and inactivates parasites in insects and fish, kills or sterilizes insects in grains, or dried fish and fruits. 2. Radicidation at 1–10 kGy: Also termed as radiation-pasteurization, kills parasites and insects, reduces significantly the number of bacteria, yeasts, and moulds. 3. Radappertization at above 10 kGy: Eliminates all bacteria, achieves a complete sterilization of food. The potential applications of food irradiation at different doses over a variety of foods including seafood have been extensively studied. Many of the pathogenic microorganisms are evident to be sensitive to ionization radiation [19] and can be conveniently reflected by the D10 values. D10 values represent the dose necessary to reduce population of 90% of a particular species. D10 values are usually under 1 kGy for vegetative cells, yeasts, and moulds, and under 4 kGy for spore-forming species (Table 39.1). As shown in Table 39.2, while the shelf life of unirradiated fishery products is less than 10 days of acceptability at 0°C–2°C, irradiation at optimum dose could extend the shelf life to several weeks [20]. Apart from effective disinfestation, food irradiation also offers a distinct advantage of virtually not raising the temperature of the food being processed. It was estimated that the heat energy absorbed at 10 kGy is equivalent to 10 J/g, an amount of energy needed to increase 1 g of water by 2.4°C [21]. Hence, nutrient losses are often small and are substantially less than other methods of preservation such as canning, drying, and heat pasteurization and sterilization. The technique has been described as possibly the most significant contribution to public health to be made by food science and technology after the pasteurization of milk [24].
39.1.3
Development of Food Irradiation
Although the principle and concept of food irradiation have been known for a long time, the technology was not utilized in the food industry until the 1950s and is still regarded as a “new” technology. The first commercial use of food irradiation was reported in 1957 [10] where a spice manufacturer in Stuttgart, Germany employed electron beam to improve the hygienic quality
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Handbook of Seafood and Seafood Products Analysis Table 39.1
D10 Values of Some Common Food Pathogens
Organism
Matrix
D10 Value (kGy)
Vegetative Cells Campylobacter jejuni
Ground turkey
0.19
Escherichia coli (including O157:H7)
Ground beef
0.24–0.31
Listeria monocytogenes
Fish, shrimps
0.15–0.25
Salmonella paratyphi A
Oysters
0.85
Salmonella senftenberg
Liquid whole egg
0.47
Salmonella typhimurium
Roast beef, gravy
0.57
Streptococcus faecium
Shrimp
0.65–1.0
Staphylococcus aureus
Prawn, crabmeat
0.16–0.29
Vibrio cholerae
Clams, fish
0.14
Vibrio parahaemolyticus
Shrimp
0.11
Bacillus cereus
Mozzarella cheese
3.6
Clostridium botulinum type E
Beef stew
1.4
Clostridium perfringens
Water
2.1
Aspergillus flavus
Growth culture
1.0
Trichosporon cutaneum
Fresh sausage
1.0
Spores
Yeasts and Moulds
Source: Adapted from Miller, R.B., Electronic Irradiation of Foods, Springer Science, New York, 2005; Stewart, E.M., Biologist, 51, 91, 2004; Irradiation to control Vibrio infection from consumption of raw seafood and fresh produce, TECDOC Series No. 1213, IAEA, Vienna, Austria, 2001; Foley, D.M., Food Irradiation Research and Technology, Blackwell Publishing, Ames, IA, 2006.
of its products. Ironically, the irradiation machine had to be dismantled 2 years later because a new law prohibited such treatment for food. Like many other innovations, the adoption of irradiation for preventing foodborne diseases is slow and lengthy. In particular, the safety issues concerning human consumption of irradiated products have often been questioned. To promote successful implementation of the technique in the control of food pathogens, several national and international food control authorities have extensively studied this irradiation process under a variety of testing conditions over the past few decades. The research on the wholesomeness
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Table 39.2 Shelf Life of Some Seafood Items by Irradiation Items
Radiation Dose (kGy)
Storage Temperature (°C)
Shelf Life (Days)
Catfish
1–2
0
20
Salmon
1.5
2.2
20
Lake trout
3
0.6
26
Whitefish
1.5–3
0
15–29
Yellowperch
3
0.6
40–45
Clams
2
0.6
39
Crabs
2–2.5
0.6
28–42
Lobster
0.75
0
35
Mussels
1.5–2.5
3
42
Oysters
2
0
23
Scallops
0.75
0
28
Freshwater prawns
1.45
0
28
Tropical shrimps
1.5–2
3
42
Source: Adapted from Venugopal, V. et al., Crit. Rev. Food Sci. Nutr., 39, 391, 1999.
and safety of irradiated food is said to be the most extensive undertaking of food scientists in history. One of the most important international authorities is the coalition of international organizations like the Food and Agriculture Organization (FAO) of the United Nations, the International Atomic Energy Agency (IAEA), and the WHO [25]. They had sponsored the establishment of the International Consultative Group on Food Irradiation (ICGFI) which aimed to develop a practical framework for proper application, to evaluate regulations and to give expert advice related to food irradiation. With untiring efforts and unprecedented depth of investigations, the FAO/IAEA/WHO JECFI [18] in 1980 declared that “irradiation of any food commodity up to an overall average dose of 10 kGy causes no toxicological hazard; hence, toxicological testing of food so treated is no longer required.” Following the important declaration by JECFI was the issuance of “Codex General Standard for Irradiated Foods” and “Recommended Code for Practice for the Operation of Radiation Facilities Used for the Treatment of Foods” by Codex Alimentarius Commission [13] in 1983. Later in 1997, the joint FAO/IAEA/ WHO group conducted further studies on the dose higher than 10 kGy and confirmed it was safe. With the accumulation of reliable scientific information, many nations have shown intense interest in using the technology to treat foodstuff s as one of the effective tools to safeguard public health and safety and also an alternative to reduce the use of banned fumigants such as ethylene oxide. At present, there are more than 50 nations granted clearance for a range of food items (Table 39.3) and an estimated amount of 200,000–500,000 tons of foods is being treated with irradiation every year.
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Table 39.3 Countries with Commercial Radiation Processing Facilities Country
Irradiated Food Type
Algeria
Potato
Argentina
Cocoa, spices
Bangladesh
Dried fish, onion, potato
Belgium
Deep frozen food, dehydrated vegetable, spices
Brazil
Dehydrated vegetables, spices
Canada
Spices
Chile
Dehydrated vegetable, onion, poultry meat, spices
China
Apple, Chinese sausage, dehydrated vegetable, garlic, onion, potato, rice, tomato
Cote d’Ivoire
Cocoa bean, yarns
Croatia
Food ingredients, spices
Czech Republic
Dry food ingredients, spices
Cuba
Beans, onion, potato
Denmark
Spices
Finland
Spices
France
Dried fruits, frozen frog leg, poultry, shrimp, spices, vegetable seasonings
Hungary
Enzymes, onion, spices, wine cork
India
Spices
Indonesia
Rice, spices
Iran
Spices
Israel
Condiments, spices
Japan
Potato
Korea
Garlic powder, spices
Mexico
Dry food ingredients, spices
Netherlands
Egg, frozen and dehydrated vegetable, rice, spices
Norway
Spices
Poland
Garlic, onion
South Africa
Chicken, fish, fruits, meat, onion, potato, processed products, spices
Food Irradiation and Its Detection Table 39.3 (continued) Facilities
779
Countries with Commercial Radiation Processing
Country
Irradiated Food Type
Thailand
Enzymes, fermented pork sausages, onion, spices
Ukraine
Grain
United Kingdom
Spices
United States
Fruits, meat, poultry, spices, vegetable
Former Yugoslavia
Spices
39.1.4
◾
Global Acceptance and Attitudes
The number of nations on the approval list has kept increasing over the past decades, but the degree of acceptance of using radiation processing, to a certain extent, is still varying throughout the world. Application of irradiation as a method of controlling foodborne diseases mainly depends upon consumers’ attitude, regulatory actions, and the economic situation. The resistance and reluctance to accept the technique are thought to be comprised of a variety of factors. It has been reviewed [26] that food irradiation is perceived to be associated with nuclear radioactivity and therefore has often been opposed and challenged by numerous antinuclear groups, environmental protection activists, and some other food groups. Among the countless examples, the consumers in Europe Group stated that food irradiation should only be applied if other methods are not available or possible, and it should not be used as a substitute for poor hygiene and is not a low cost method [27]. Second, the general public has limited knowledge of the cause and prevention of foodborne diseases. These factors inevitably have negative impacts on consumer attitudes and eventually the legislative decision of policy makers toward food irradiation. The key to consumer acceptance for irradiated foods is education [28] and it has been shown in a study that there was a drastic change of consumer attitudes in irradiated foods in the United States between 1993 and 2003 [29]. Approximately 76% prefer to buy irradiated pork and 68% prefer to buy irradiated poultry; and more consumers were willing to buy irradiated products in 2003 than in 1993. A variety of recent market surveys also confirmed that a positive shift in attitudes toward irradiated foods could be achieved through the delivery of proper and accurate information to consumers [30,31]. Support from legislator and acceptance from the public and industry has helped the rapid growth of food irradiation in the United States [32] and in the Asia Pacific region [33]. Albeit the contributions of some members of the European Union (EU) like Belgium, France, and the Netherlands, the progress in Europe, meanwhile, is lagging behind [12]. For instance, food irradiation for specific categories of food (fruits, vegetables, cereals, tubers, spices fish, shellfish, and poultry) have been authorized since the early 1990s in the United Kingdom, however, the volume of irradiated foods in the retail markets is almost nonexistent [34]. Furthermore, in 1999, the European Parliament and the Council of EU issued Directives on irradiated food and the permitted commodities have been restricted to dried aromatic herbs, spices, and vegetable seasonings [35,36]. While the use of food irradiation as a distinct application for preventing outbreaks of foodborne diseases in red meats and seafood is gaining popularity across the world, it is rather remote for most of the countries in the continent and results from the European regulations.
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39.1.5
Detection Methods for Irradiated Foods
Although policies about food irradiation vary from one country to another, the availability of methods for identifying irradiated food is one of the crucial requisites for legitimate implementation of the irradiation regulations. Reliable methods provide scientific tools for upholding regulatory controls, checking compliance against labeling requirements, facilitating international trade, and reinforcing consumer confidence. However, detection is very difficult as the actual changes that present in irradiated foodstuffs are extremely small within the working irradiation doses of less than 10 kGy; and in many cases, the changes involved are far less than those of classic food treatment processes. It is not surprising that the overall progress on detecting food irradiation was not satisfactory in the early years. Since an agreement on promoting food irradiation adopted by delegates from 57 countries at the International Conference on the Acceptance, Control of and Trade in Irradiation Foods in 1988 [37], intensive research studies and cooperation on detection of irradiation have been supported nationally and internationally. With concerted actions from the Community Bureau of Reference, and the Joint Division of the FAO and the IAEA, a number of detection methods, on the basis of chemical, physical, biological, and microbiological changes, were successfully developed under the cooperation framework. Five methods were adopted as the European standards (EN1784–1788) in 1997; four of them were revised after some years of publication. Another five EN standards have also been published in 2002–2004 (Table 39.4). The methods using electron spin resonance spectroscopy (ESR) and thermoluminescence (TL) for detecting primary radiolytic radicals and the analysis of radiolytic 2-alkylcyclobutanones (2-ACBs) and hydrocarbons are more specific and conclusive, while others are convenient to be used as fast screening methods. This chapter discusses the technical Table 39.4 EN Protocols for the Detection of Irradiated Foods Protocols
Title
EN1784:2003
Detection of irradiated food containing fat. GC analysis of hydrocarbons.
EN1785:2003
Detection of irradiated food containing fat. GC/MS analysis of 2-ACBs.
EN1786:1997
Detection of irradiated food containing bone. Method by ESR spectroscopy.
EN1787:2000
Detection of irradiated food containing cellulose by ESR spectroscopy.
EN1788:2001
TL detection of irradiated food from which silicate minerals can be isolated.
EN13708:2002
Detection of irradiated food containing crystalline sugar by ESR spectroscopy.
EN13751:2002
Detection of irradiated food using photostimulated luminescence.
EN13783:2002
Detection of irradiated food using direct epifluorescent filter technique/ aerobic plate count.
EN13784:2002
DNA comet assay for the detection of irradiated foodstuffs. Screening method.
EN14569:2004
Microbiological screening for irradiated food using LAL/GNB procedures.
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information on some common and validated detection methods that are mostly reported in the literature. There are also a number of other methods available, which have been thoroughly reviewed elsewhere [38–40].
39.2
Detection Methods
39.2.1 Electron Spin Resonance Spectroscopy Electrons or radicals possess a magnetic moment that arises from their intrinsic spin and motion in the orbit. When placed in a magnetic field, the magnetic moment is proportional to the angular momentum of the electron. The torque exerted then produces a change in angular momentum which is perpendicular to that angular momentum, causing the magnetic moment to process around the direction of the magnetic field. The frequency at the precession is called Larmor frequency. When an external electromagnetic wave of the same frequency as the Larmor frequency is applied, a portion of the energy is absorbed and gives characteristic resonance signals. ESR, or electron paramagnetic resonance (EPR) spectroscopy is a nondestructive physical technique pertaining to the detection of such resonance signals. ESR had originally been used as a tool for postirradiation dosimetry, dating, and imaging [41–43], and was later proposed to be applicable to detect induced radicals in irradiated foods [44,45]. To allow accurate identification, the ESR signals must be stable or fairly stable during the usual storage time of foodstuff; and must be distinguishable from those of unirradiated substances. Such detection is not suited to foods containing high water content in which induced radicals will be rapidly and significantly removed. Foods having dry and hard compositions, on the other hand, are known to be a favorable environment for stabilizing free radicals, and subsequently prolong their life span. Therefore, confirmation of irradiation status in these materials has been widely determined by ESR including the recent studies on bones [46], crustaceans’ shell [47], beans [48], seeds [49], dried fruits [50], and spices [51]. At the early stage of the method development, the majority of the ESR work has been focused on the study of meat bones [52–54]. The ESR spectra were attributed to the CO33−, CO3−, CO2−, CO− radicals being trapped in the lattices of hydroxyapatite (Ca10(PO4)6(OH)2) [15], the major components of calcified materials. At approximately 0.5–1 kGy, the radiation-induced signals are clearly resolvable from those of nonirradiated bones with a characteristic asymmetric singlet (Figure 39.1). The shape of signals is basically similar for all bones and is observed in the region at g = 2.0010–2.0050 [53,56,57]. With the ESR method it was also possible to detect mechanically recovered meats, a product where flesh is separated from carcass through mechanical processes, by recovering the bone fragments using alcoholic alkaline hydrolysis [58]. The treatment allowed the ESR detection at an inclusion level of 10% (w/w) but recently the g = 2.0020 1 kGy 0 kGy mT 340
Figure 39.1
345
350
355
ESR spectra of nonirradiated and irradiated fish bone at 1 kGy.
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sensitivity has been improved to the level of 0.5% (w/w) [59]. For exoskeletons of crustaceans, such as mollusks [60] and cuttlefish [61], the ESR signals due to the Mn2+ and CO2− are very intense and allowing a detection limit of less than 0.5 kGy. On the other hand, complex ESR signals are derived from dried cellulose materials [48–51]. The multicomponent signals consist of the predominant peak of crystalline sugars centered at g = 2.003 as stipulated in the EN13708 method, and the weak cellulose peak, typically at g = 2.0045 [62]. The induced radicals in irradiated foods are stable in most cases and the signal intensities are found to be dose dependent. As a consequence, some workers proposed that ESR could be used as a quantitative procedure where dose–response curves were commonly applied to estimate the original dose in irradiated foods [63–65]. However, the chemical composition [66,67], storage conditions [50,67], and processing treatment [70] could influence the dose response, and one must take into account correction factors in order to obtain a reliable dose estimation.
39.2.2 39.2.2.1
Analysis of Radiolytic Chemicals 2-Alkylcyclobutanones
In the early 1970s, LeTellier and Nawar [71] had isolated and identified a group of cyclic ketones as radiolytic products from pure triglycerides irradiated at 60 kGy in vacuum. The compounds were known as 2-ACBs, having the same carbon number as their respective parent fatty acid molecules, that were formed via ionization and cyclic rearrangement processes (Figure 39.2). These compounds are found to be degraded by oxidation during storage, but their stability is long enough to be detected. The production of radiolytic 2-ACBs is one of the most debated issues for the safety of irradiated food as some experimental studies indicated that these compounds are cancer promoters [71–74], while others claimed no mutagenic and genotoxic effects [75–77]. Despite the arguments, 2-ACBs were not detected in the processes of microwave treatment, oven heating, ultraviolet irradiation, and high pressure treatments and conformed to be used as unique markers for irradiated fat-containing foods [78,79]. Since then, a large amount of research work was initiated to use 2-ACBs to detect irradiated foods. 2-Dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB), which are derived from the abundantly occurring palmitic and stearic acids in food items, are the most studied 2-ACB members. Positive identification of DCB and TCB were reported in irradiated chicken meat [80–82], lamb meat [84], ground beef [82,84], pork [82,85], quenelles [81], cheese [86], egg [80], fruits [81,86], melon seeds [87], fish
HO
CH3 n O
Ionization –e–
HO
+ O
H abstraction CH3 n
H
HO +e– CH3 n
CH3
HO HO
+ OH
Cyclization OH
HO
n
CH3 n
–H2O
CH3 O
n
Figure 39.2 Schematic transformation of free fatty acids to 2-ACBs during high energy irradiation. Palmitic acid (n = 11) forms 2-dodecylcyclobutanone (DCB) and stearic acid (n = 13) forms 2-tetradecylcyclobutanone (TCB).
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GC–MS response
[82,86,88], and dried shrimp [89]. The radiolytic 2-ACBs were extracted from foods by Soxhlet extraction, then purified, and isolated by column chromatography using Florisil [80,83,86,87,89], and detected using gas chromatography (GC)–mass spectrometry (MS), which was adopted as a standard protocol in EN1785 after being validated by a series of interlaboratory comparison studies [90]. Non-EN detection methods such as enzyme-linked immunosorbent assay (ELISA) [91] and TLC [93] have been used for fast screening, but these methods were subject to selectivity and sensitivity problems. In view of the tedious and time-consuming extraction procedures involved in EN1785, some workers proposed to replace the Soxhlet–Florisil chromatography by a supercritical fluid extraction (SFE). A SFE operated at its optimized conditions could extract low levels of DCB and TCB from samples within 30–60 min with good efficiency [81,84,88]. Others recommended the use of a fully automated accelerated solvent extraction method [82] for extracting 2-ACBs. A typical mass chromatogram of DCB and TCB, usually monitored at m/z 98 and 112 is shown in Figure 39.3. The amount of DCB and TCB produced is proportional to the irradiation dose and is also dependent on the food types and the presence of fatty acid precursors. The limits of detection for DCB and TCB were only at a dose of 0.5–1 kGy for red meats and seafoods [82,88,89] compared to that of 0.1 kGy for mango and papaya [86]. The low sensitivity in the former food types was attributed to the presence of interfering substances that had not been adequately removed. An inclusion of solid phase extraction using cation exchanger impregnated with silver ions after Florisil extraction was found to improve the sensitivity to about 0.1 kGy. Another study using pentafluorophenyl hydrazine as the coupling agent for DCB and TCB [93] was reported to enhance the sensitivity of detection by two to five times for chicken meat and pork samples.
TCB
DCB
GC–MS response
0 kGy
5 kGy
8.0
10
12
14
16
20 min
Figure 39.3 Total ion chromatogram showing detectable quantities of the DCB and TCB in black melon seed at 5 kGy. These radiation-induced compounds were not present in the nonirradiated sample.
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39.2.2.2
Volatile Hydrocarbons
Radiolytic cleavage of fatty acids (Cm:n), where m is the number of carbon atoms and n is the double bond, at a- and b-position of the carbonyl group leads to the formation of two characteristic volatile hydrocarbons; one has a carbon atom less than the parent fatty acid (Cm−1:n) and the other has two carbon atoms less and one extra double bond in position 1 (C m−2::n + 1). Although volatile hydrocarbons are also found in other nonirradiated foods, the detection of the hydrocarbon couple unambiguously indicated the presence of irradiation treatment [94,95]. Similar to that of 2-ACB, extracted hydrocarbons could be isolated by Florisil chromatography and detected by GC-FID or GC–MS (Figure 39.4). The method was validated through interlaboratory comparison [90,96] and adopted as a standard protocol in EN1784. Pentadecane (C15:0) and 1-tetradecene (C14:1), heptadecane (C17:0) and 1-hexadecene (C16:1), and 8-heptadecene (C17:1) and 1,7-hexadecadiene (C16:2) that respectively were generated from abundant palmitic, stearic, and oleic acids in foods were widely reported in beans [97], cereals [98], nuts [99], eggs [100], meats [101,102], shrimps, and seafood [89,103,104]. The radiolytic hydrocarbons found in those food matrices were relatively stable and varied from a week to several months postirradiation, and the respective concentrations were proportional to the dose applied. A comparative study [105] on investigating the profile of hydrocarbon markers in different foodstuffs showed that while C16:2 was commonly detected at the lowest practical dose in dairy products, fruits, beef, pork, chicken, and tuna, only 6,9-heptadecadiene (C17:2), owing to the different fatty acids composition, was the suitable marker for dried shrimp at the practical dose of 0.75–2 kGy. Another recent study [107] also showed that only C17:1, C16:2, C17:2, and 1,7,10-hexadecatriene (C16:3) were detected at 0.5 kGy in irradiated soybeans as the food consisted mostly of oleic (26%) and linoleic acids (49.6%). Therefore, in order to achieve the best performance of the detection method, it was recommended to check the fatty acid profile of the food type under study as the marker hydrocarbons would vary from one food to another [105]. The disadvantages of using Florisil chromatography were that it is tedious and the procedure was not very efficient in removing all lipid interference prior to detection, hence decreasing the sensitivity. Some workers proposed using argentation chromatography [107] for enrichment of
30 0 kGy
20 10
I.S.
C15: 0
30 20
C14: 1
C16: 2 C16: 1 C17: 0 C17: 1
10
10 kGy I.S.
10
20 min
Figure 39.4 Chromatograms for the hydrocarbons from nonirradiated and 10 kGy-irradiated dried shrimps. (From Kim, K.S. et al., J. Food Prot., 67, 142, 2004. With permission.)
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radiolytic unsaturated hydrocarbons from fruits and meats using a silver column; and the use of online coupled LC–GC [109] for fish and prawn samples to improve sample preparation and separation efficiency. Others developed a solid phase microextraction (SPME) and purge and trap method [109] to overcome the tedious extraction process. All those modifications might have good potential to enhance the detection capability of EN1784 where further investigation work and research are required.
39.2.2.3
o-, m-Tyrosine
Amino acids are vulnerable to the active radicals induced in irradiation and the end products might be used as markers for identifying irradiated protein-containing foods. An experiment showed that o-, m-, and p-tyrosine were produced upon irradiation of a phenylalanine solution and the yields were proportional to the applied dose [110]. As o- and m-tyrosine were nonnatural amino acids, some early studies have demonstrated the feasibility using these compounds as potential markers to detect food irradiation [111–114]. At almost about the same time, minute quantities of o-tyrosine were found to be present in nonirradiated food and thus diminished their potential usefulness for detecting irradiated foods. However, another research study showed that the background level of o-tyrosine determined by high performance liquid chromatography (HPLC) with florescence detection [115] in unirradiated shrimps was 19.3 mg/kg, which was 10 times less than those irradiated at 1 kGy and concluded that o-tyrosine was a reasonable marker to detect irradiated shrimps down to 1 kGy dose. The results were in good agreement with another similar study using HPLC with coulometric electrode array detection [116] to detect o- and m-tyrosine in irradiated shrimps. Unfortunately, the use of tyrosine isomers has not been thoroughly validated and the relevant literature information on other food matrices is very limited.
39.2.2.4 Hydrogen and Carbon Monoxide Gases Simple molecular inorganic gases produced upon the radiolysis of water and organic components (carbohydrates, lipids, proteins, etc.) in foods were proposed as versatile probes for irradiation detection. Radiolytic hydrogen was detected in pepper [117] by GC and in frozen chicken by a headspace analyzer based on a hydrogen-specific electronic sensor [118]. The latter work also claimed that hydrogen generated during the irradiation of frozen chicken at 5 kGy was measurable after storage for up to at least 6 months. Similar studies on radiolytic carbon dioxide in deboned frozen meats [119], spices, and dry grains [120] showed that this gas could also be well retained after irradiation and could serve as another reliable marker for detection. Another study [121] successfully used microwave heating and headspace GC to measure the level of hydrogen and carbon monoxide in frozen shrimps, cod slices, and deshelled oyster irradiated at 1–8.8 kGy. The radiolytic gases were detected up to 3 months after irradiation. The above studies demonstrated hydrogen and carbon monoxide are convenient for distinguishing some irradiated foods and could provide a rapid screening test. However, no definite conclusions can be drawn from negative results, which should be endorsed by other confirmatory tests.
39.2.3
DNA Methods
Since DNA is vulnerable to high energy irradiation, radiation-induced changes in DNA molecules could be used as a tool to detect the radiation treatment of foods. Upon irradiation, the
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double helical strands of DNA molecules usually break and form various fragments having lower molecular weight than their parent DNA. Östling and Johanson [122] first reported the experiment by mixing extracted DNA in buffer solution with low-melt agarose and subjecting to electrophoresis analysis. DNA fragments from irradiated cells showed longer migration distance from the nuclei and the unradiated cells presented no or little movement. The migration of the DNA fragments from cells giving the appearance of a comet, and the technique is commonly termed DNA comet assay. Using the approach, other workers [123,124] found that the extension of the comet tail correlated with the degree of cellular DNA damage, i.e., the radiation dose. As a consequence, DNA comet assay has proved its wide application in genotoxicity [125,126], environmental contamination monitoring [127–129], and fundamental research in DNA damage and repair [130,131]. The method was modified to detect irradiated foods [132] with the aid of sets of reference samples at 0–5 kGy for checking migration patterns. Because of the fast analysis time (<1 h), such assay was also applied as a screening to control imported foods in Sweden [133]. Another study [134] on DNA degradation of chilled fresh chicken explicated that the length and shape of the comets obtained could be used to estimate the dose. The observed comet tail was short for unirradiated cells, but it became longer and even separated from the comet head when the dose increased. At a very high dose, almost no DNA was left in the head, and the tail appeared as a cloud (Figure 39.5). Validation of comet assay through interlaboratory comparisons was satisfactory, with an average of over 95% test results correctly identified for seeds, dried fruits, spices [135], chicken, and pork [136] and other foodstuffs [137,138] and the procedure served as a screening in the EN method. However, DNA is known to be naturally degraded at room temperature through the activities of nucleases within the cells. EN 13784 stated that the application of DNA comet assay is limited to determining fresh or frozen foods, and not applicable to foods that have been subjected to various forms of physical and chemical treatments that resulted in DNA fragmentation. Any new type of foodstuffs shall also be tested by the method before unknown samples are analyzed. Since
Type
Comet
Corresponding dose (kGy)
1
0
2
1
3
5
4
10
5
>>10
Figure 39.5 Drawing of the comet types observed with increasing DNA degradation (types 1–5) in fresh chicken legs at 2°C–4°C. The darker regions represent higher amount of DNA. For comparison, the doses of ionizing radiation which produce similar comets are given. (From Cerda, H. and Koppen, G., Z. Lebensm. Unters. Forcsh. A, 207, 22, 1998. With permission.)
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then, a large number of food detections such as pork [139], poultry [140], beef [141], hamburger [142], papaya, and melons [143], using comet assay were reported. An unsuccessful study [144] on halibut, herring, saithe, plaice, and squid, however, might explain limited work on seafood using comet assay. The use of supercoiled mitochondrial DNA (mtDNA) was proposed [145] as a good alternative for comet assay because mtDNA is well protected by the mitochondrial wall from enzymatic reactions. mtDNA was shown to be very stable during prolonged storage at 4°C, but it was significantly reduced upon irradiation at 2–4 kGy [146]. Therefore, detection of mtDNA could be a good marker for irradiation. However, the method had not been extensively studied for irradiated food by other workers owing to the complication and time-consuming extraction process. Another approach relying on the immunological detection of DNA base changes was also proposed. Dihydrothymidine (DiHT) was known to be produced from thymidine base via radical reaction upon irradiation [147,148]. With the development of monoclonal antibody, novel ELISA assays for the detection of DiHT in prawns at 2 kGy and other irradiated food were reported [149,150]. The potential advantage of ELISA was its usefulness in crude food homogenates, which could reduce the extraction process and offer a rapid screening test. The method, however, would require more validated work in order to be applicable to other foodstuffs.
39.2.4
Luminescence
Electrons can be excited to higher energy states by absorption of ionization radiation, and trapped if the substance has a crystalline structure. When the trapped electrons are released and returned to the ground state, some of the energy appears in the form of light and causes the substance to luminesce. The release process could be stimulated by heat (TL) or light (photostimulated luminescence or pulsed infrared stimulation [PSL]). The trapped electrons can remain in the crystalline lattice for many years, the measurement of the energy emission reveals the ionizing radiation to which the substance had been exposed. The technique of TL has already been used for the radiation dosimetry in archaeological and geological dating [151]. In 1989, it was first reported to identify irradiated spices [152] by measuring the intensity of emitted photons over a range of temperature (glow curve). As an illustrative example shown in Figure 39.6 [153], typical glow curves of irradiated dried fish from 1 to 7 kGy, which were distinguishable from the nonirradiated sample, were observed to peak at about 150°C. The TL signals detected from earlier studies were thought to come from organic materials, but later studies [154,155] indicated the signals originated from the contaminated silicate minerals present in the samples. With the isolation and normalization of minerals, TL was extensively studied in detecting other food items that contain concomitant minerals such as fruits, vegetables, shellfish, shrimps, and prawns [156–160]. The TL method was the first confirmative method adopted in the United Kingdom for detecting irradiated foods and later as a European standard (EN 1788) in 1997, which was revised in 2001. TL is a very specific method for the confirmation of food irradiation, but it requires the tedious and skillful separation of silicates from the food matrices. These mandated procedures limited the usefulness of TL in routine surveillance examination. Furthermore, the mixture of endogenous inorganic and organic materials in food could inhibit high-temperature TL analysis. Therefore, a novel development was proposed to release the trapped charge carriers from the excited energy level using pulsed infrared stimulation (PSL), which allowed the detection in the presence of interfering organic materials, and eliminated the necessity of isolating inorganic materials [156,157]. With the employment of a simple instrument, the PSL measurement was claimed to produce a
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6000
TL signal intensity (a.u.)
3 kGy 5000
5 kGy 7 kGy
4000 3000
2000
1000
0 0
100
200
300
400
Temperature (°C)
Figure 39.6 Dose-dependent TL glow curves of minerals separated from irradiated sliced-dried Pollack at different doses. (From Kwon, J.H. et al., Radiat. Phys. Chem., 71, 81, 2004. With permission.)
fast qualitative analysis for irradiated food within 15–60 s [161]. PSL was shown to be a reliable screening method for herbs, species, and shellfish after the successful outcome of collaborative trials [162–165]; PSL was adopted as a European Standard (EN13751) in 2002.
39.2.5 Microbiological Methods Food irradiation is a process to eliminate/reduce the number of microbial flora that is present, and therefore the change of these microorganisms could be used to detect irradiated food. Through elaborate validations and verifications, two microbiological tests have been adopted by EU as the standard screening methods in 2002 and 2004. EN13783 relies on the difference between aerobic bacteria plate count (APC) and direct epifluorescent filter count (DEFT). APC is a convention method to show the number of viable bacteria capable of forming colonies on agar plate and the unit is expressed as cfu. DEFT is another standard count protocol used for enumerating the total number of live and dead microbes [166]. For unirradiated samples, the counts of APC are often comparable to those in DEFT. Conversely, APC is found to be significantly lower, usually in the range of 3–4 log units in irradiated food such as frozen meats [167], herbs and spices [168,169]. EN14569 is based on two different microbiological techniques, viz., the enumeration of viable Gram-negative bacteria (GNB) and immunological analysis of endotoxins contained in the GNB by Limulus Ameobocyte Lysate test (LAL) in the test samples. In general, GNB count is proportional to the LAL results. For a low GNB count with a high LAL result, it might indicate
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the presence of a large population of dead microbes caused by irradiation. Application of the technique in poultry meats [170,171] was found to be successful. Since the change of microbe loading in food could also be influenced by other physical and chemical treatments, both microbiological methods are not specific methods and any positive results obtained shall be confirmed by a specific test. Apart from the two EN standards, turbidimetric measurement for the number of bacteria present in the extracted medium was also proposed [172]. The method was claimed to provide a very good index for food irradiation and was simpler and quicker than that of the DEFT/APC method.
39.3 Conclusions Owing to the minute change of physiochemical properties in food matrices before and after irradiation, detection of food radiation was considered as an unreachable target two decades ago. With the concerted actions from various national and international organizations such as the Community Bureau of Reference and the Joint Division of the FAO, IAEA, and WHO, we observed an overflow of useful and validated methodologies that are applicable in food irradiation. One of the most noticeable events has been the approval of 10 validated standard methods by European Committee for Standardization from 1996 to 2004. ESR spectroscopy, TL glow curve studies, and the chromatographic analyses of hydrocarbons and 2-ACB methodologies provide reliable and confirmative information of the irradiation status in a wide variety of food items. Whereas, the DNA comet assay, PSL, and microbiological tests could offer a fast and inexpensive alternative for screening a large amount of samples. The research and development for food irradiation was at its climax in the 1990s but it showed signs of losing momentum in the 2000s. For example, the mandate of ICGFI ended in 2004 and no official arrangement was recommended to continue the mission of this active group. The downfall of ICGFI was an illustration of fading supports from federal governments of most leading countries. However, as stated by Ehlermann, the former Federal Research Centre for Nutrition, Germany, in his review of “Four decades in food irradiation” [173], “After the age of enlightenment it is time for a modern society to counteract myths, ideologies, superstition and to rely on facts and knowledge based on sound science. This must include the appreciation of processing food by ionization irradiation as a valuable and justified technology.”
Acknowledgment The authors would like to express their sincere thanks to Mr. Peter Brown of the Association of Public Analysts, the United Kingdom, for his invaluable technical comments to this manuscript.
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154. Autio, T. and Pinnioja, S. Identification of irradiated foods by the thermoluminescene of mineral contamination, Z. Lebensm. Unters. Forsh., 191, 177, 1990. 155. Autio, T. and Pinnioja, S. Eds., Leonardi, M., Raffi, J.J., and Belliardo, J. Identification of irradiated foods by the thermoluminescene of contaminating minerals, in Recent Advances on Detection of Irradiated Food, EUR-14315: Commission of the European Communities, Luxembourg, 1992, pp. 177–183. 156. Sanderson, D.C.W., Carmicheal, L.A., and Naylor, J.D. Recent advances in thermoluminescence and photostimulated luminescence detection methods for irradiated foods, in Detection Methods for Irradiated Foods – Current Status, McMurray, C.H., Stewart E.M., Gray, R., and Pearce, J. (Eds.), Royal Society of Chemistry, Cambridge, London, 1996, pp. 124–138. 157. Sanderson, D.C.W. et al. Luminescence detection of shellfish, in Detection Methods for Irradiated Foods – Current Status, McMurray, C.H., Stewart E.M., Gray, R., and Pearce, J. (Eds.), Royal Society of Chemistry, Cambridge, London, 1996, pp. 139–148. 158. Schreiber, G.A. et al. Methods for routine control of irradiated food: Determination of the irradiation status of shellfish by thermoluminescence analysis, Radiat. Phys. Chem., 43, 533, 1994. 159. Pinnioja, S. and Pajo, L. Thermoluminescence of minerals useful for identification of irradiated seafood, Radiat. Phys. Chem., 46, 753, 1995. 160. Carmichael, L.A. and Sanderson, D.C.W. The use of acid hydrolysis for extracting minerals from shellfish for thermoluminescence detection of irradiation, Food Chem., 68, 233, 2000. 161. http://www.gla.ac.uk/surrc/luminescene/psl.html. 162. Sanderson, D.C.W., Carmichael, L.A., and Naylor J.D. Photostimulated luminescence and thermoluminescene techniques for the detection of irradiated food, Food Sci. Technol. Today, 9, 150, 1995. 163. Sanderson, D.C.W., Carmichael, L.A., and Fisk, S. Establishing luminescence methods to detect irradiated food, Food Sci. Technol. Today, 12, 97, 1998. 164. Sanderson, D.C.W., Carmichael, L.A., and Fisk, S. Photostimulated luminescence detection of irradiated shellfish: International interlaboratory trial, J. AOAC Int., 86, 983, 2003. 165. Sanderson, D.C.W., Carmichael, L.A., and Fisk, S. Photostimulated luminescence detection of irradiated herbs, spices, and seasonings: International interlaboratory trial, J. AOAC Int., 86, 990, 2003. 166. Pettipher, G.I. et al. Rapid membrane filtration-epiflourescence microscopy technique for the direct enumeration of bacteria in raw milk, Appl. Environ. Microbiol., 39, 423, 1980. 167. Jones, K. et al. The DEFT/APC screening method for the detection of irradiated, frozen stored foods: A collaborative trial, Food Sci. Technol. Today, 10, 175, 1996. 168. Wirtanen, G. et al. Microbiological screening method for indication of irradiation of spices and herbs: A BCR collaborative study, J. AOAC Int., 76, 674, 1993. 169. Oh, K.N. et al. Screening of gamma irradiated spices in Korea by using a microbiological method (DEFT/APC), Food Chem., 14, 489, 2003. 170. Scotter, S.L., Beardwood, K., and Wood, R. Limulus ameobocyte lysate test/gram negative bacteria count method for the detection of irradiated poultry: Results of two interlaboratory studies, Food Sci. Technol. Today, 8, 106, 1994. 171. Scotter, S.L., Beardwood, K., and Wood, R. Detection of irradiation treatment of poultry meat using the Limulus amoebocyte lysate test in conjunction with a Gram negative bacteria count, J. Assoc. Publ. Anal., 31, 163, 1995. 172. Guatam, S., Sharma, A., and Thomas, P. Improved bacterial turbidimetric method for detection of irradiated spices, J. Agric. Food Chem., 46, 5110, 1998. 173. Ehlermann, D.A.E. Four decades in food irradiation, Radiat. Phys. Chem., 73, 346, 2005.
Chapter 40
Analysis of Dioxins in Seafood and Seafood Products Luisa Ramos Bordajandi, Belén Gómara, and María José González Contents 40.1 Introduction ................................................................................................................. 798 40.2 Sample Pretreatment and Recovery Studies .................................................................. 799 40.2.1 Sample Storage ................................................................................................ 799 40.2.2 Spiking and Recovery Studies ......................................................................... 799 40.3 Extraction Methods ..................................................................................................... 800 40.3.1 Soxhlet Extraction........................................................................................... 800 40.3.2 Solid Phase Extraction and Matrix Solid Phase Dispersion ............................. 800 40.3.3 Supercritical Fluid Extraction ..........................................................................801 40.3.4 Accelerated Solvent Extraction .........................................................................801 40.3.5 Microwave Oven ............................................................................................. 802 40.4 Cleanup Methods ......................................................................................................... 802 40.4.1 Lipid Removal ................................................................................................. 803 40.4.2 Isolation of Uncommon Chemical Interferences ............................................. 803 40.5 Fractionation/Group Separation ................................................................................... 803 40.6 Automation of Extraction and Cleanup........................................................................ 804 40.7 Instrumental Determination ........................................................................................ 805 40.7.1 GC Congener Separation ................................................................................ 805 40.7.2 GC Detectors .................................................................................................. 807 40.8 Bioanalytical Screening Methods ................................................................................. 808 References ............................................................................................................................... 809 797
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40.1 Introduction Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), two groups of Persistent Organic Pollutants (POPs), are structurally related chlorinated aromatic hydrocarbons which are generally referred to as “dioxins.” They are of great concern due to the extreme toxicity of the 2,3,7,8 chlorine substituted congeners and their presence in all compartments of the environment. PCDD/Fs are formed as by-products of a wide variety of chemical industry and combustion processes that contain chlorine and chlorinated aromatic hydrocarbon sources [1]. Due to their low water solubility, hydrophobicity, and resistance to degradation, these substances are found in a wide range of biological samples, and tend to accumulate in animal and human adipose tissues through the food web [2]. Among the 210 possible congeners, seven 2,3,7,8-substituted PCDDs and 10 PCDFs are generally considered the most persistent and toxic PCDD/F congeners, since they have toxic properties similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is the most toxic congener of these compounds [3–5]. For the general population, dietary intake is the main route of PCDD/F exposure, contributing to more than 90% of the daily exposure [6,7]. Public concern over the adverse health effects of these toxicants at this time has been intensified by a number of dioxin contamination incidents involving food and feedstuffs [8–10]. Recent reports concerning toxicological aspects have led to a revaluation of the tolerable daily intake (TDI) of dioxins [4] and have prompted wide-ranging efforts and the tightening of regulations to reduce dioxin release into the environment [11]. To prevent the health risk from dioxin exposure, the European Commission has recently established maximum permissible levels of dioxins and dioxin-like polychlorinated biphenyls (PCBs) in foods [12]; the minimal risk level (MRL) for fish and seafood is 8 pg of toxic equivalents (TEQs)/g fresh weight (including dioxin-like PCBs), except for eels (Anguilla anguilla) and their products, where it is 12 pg of TEQs/g fresh weight. As required by the current legislation regulating dioxins in foodstuffs, including seafood and seafood products, large numbers of samples have been analyzed, and a great deal of data concerning PCDD/F levels in foodstuffs is now available. All these studies show that human dietary dioxin intake has been decreasing in recent years, and that seafood and seafood products have received special attention due to their widespread consumption by the population and their high dioxin contents [13–25]. Although seafood and seafood products exhibit higher dioxin levels than any other food category [14,16,19,21,24,25], they are present in sub-ppb levels and their analysis is complex and challenging. At present, there is a need for cost-efficient, reliable, and rapid analytical alternatives to expensive methods involving the use of gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) so that food items can be routinely monitored to detect contamination at an early stage. Usually, the analysis of these compounds in fatty seafood tissues requires three main steps: extraction of the target analytes, cleanup of the extract obtained, and GC separation [26]. Several extraction and cleanup procedures are described in the literature, and which one is chosen depends on individual analytical laboratories. Soxhlet (SOX) [15,27,28], solid-phase extraction (SPE) [29], matrix solid phase dispersion (MSPD) [20,30], supercritical fluid extraction (SFE) [31,32], microwave-assisted solvent extraction (MASE) [33], and accelerated solvent extraction (ASE) (also named pressurized liquid (PLE) [34,35] have all been employed as extraction methods. Cleanup procedures, including open column chromatography on activated Florisil®, alumina, silica, carbon, and size exclusion chromatography (SEC) [26,36–39] are also currently used. Automatic online procedures combining extraction and cleanup methods to obtain extracts ready for
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GC analysis with the maximum extraction efficiency and overcoming matrix-related interferences have always been a target, but they are still being developed [40]. Over the last few years, the European Union (EU) has initiated a large-scale research project to develop new analytical methodologies for the determination of dioxins (most of them including dioxin-like PCBs) in food matrices to serve as alternatives to GC-HRMS. This last technique is taken as the benchmark for accurate and specific determination of these compounds in food samples as described in Environmental Protection Agency (EPA) and EU official methods [41–43]. GC-HRMS provides enough specificity and selectivity at concentration levels down to femtograms per gram for the analysis of these compounds, but it is a relatively expensive technique and requires qualified personnel. Because of that, alternative techniques such as GC coupled to ion trap mass spectrometry (GC-ITMS), working in tandem mode (MS/MS) [44–46], and also comprehensive two-dimensional GC (GC × GC) coupled to microelectron capture detection (mECD) [47] and time of flight (TOF) MS [48], have recently been validated for acceptance as an alternative to GC-HRMS. In addition, bioanalytical methods have improved considerably in sensitivity and selectivity to the extent that they can be used as screening methods to determine the total quantities of dioxin-like compounds [49]. Although most analytical methods for measuring dioxins in seafood and seafood products include dioxin-like PCBs, this chapter specifically focuses on those targeting the 17 toxic 2,3,7,8PCDD/Fs in seafood and seafood products. Attention has been paid to both, methods that are in current use and methods that have recently been developed for each step of the analysis from sample preparation to instrumental determination of these congeners.
40.2 Sample Pretreatment and Recovery Studies 40.2.1
Sample Storage
Seafood and seafood products collected in the field are usually preserved by freezing immediately, either in the field, on board ship or at the laboratory. Whenever possible the seafood should be dissected immediately and the individual tissues stored in individual packs of approximately the size required for analysis to minimize thawing of subsampling material. Seafood tissues are first macerated and then freeze-dried or ground with sodium sulfate and silica to reduce the water content and rupture cell walls and these are the most commonly used pretreatment procedures for seafood tissue matrices [20,29,50]. It should be noted that the concentration of the 2,3,7,8-PCDD/F congeners is generally at femtogram per gram. It is therefore necessary to analyze samples containing around 6 g of fat, which require large amounts of fresh sample (from 600 g for mussels [1% fat] to 30–40 g for salmon [20% fat]). In any case, that is much more than what is usually employed for the analysis of other POPs. For this reason, almost all sample pretreatment methods involve freeze-drying, which completely eliminates the water content and drastically reduces the sample size. On the other hand, the freeze-drying step takes 48 h, which considerably increases the total analysis time.
40.2.2 Spiking and Recovery Studies In PCDD/Fs analysis it is mandatory to use isotope dilution mass spectrometry (IDMS) for the final quantitative determination of the target 2,3,7,8-PCDD/Fs and the recoveries of the total analysis (extraction + cleanup + analytical determination) [41,43]. IDMS is the most elegant way to overcome
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the whole problem of sample recovery and quantification. The seventeen 13C12 2,3,7,8-PCDD/F labeled isotopes are added to the sample at a known concentration prior to extraction, as extraction standards for quantification. Two more 13C12-PCDD congeners (1,2,3,4-TCDD and 1,2,3,7,8,9HxCDD) are added to the extract at a known concentration prior to analytical determination, as instrumental standards. The ratio of the labeled and native compounds is measured by MS and automatically accounts for any losses in the procedure. Although it is not necessary to calculate the recoveries for quantification purposes, they are calculated as a quality parameter from the ratio of the labeled congeners in the extraction and recovery standards [13–22,24,25,43].
40.3 Extraction Methods The purpose of extraction step is to remove the bulk of the sample matrix and to transfer the fraction containing the analytes to a suitable solvent. Extraction techniques for fish and seafood are generally based on the assumption that lipophilic compounds such as PCDD/Fs predominantly occur in the fat fraction of the food matrix, and they are based on general methods for isolation of the lipid fraction from the sample matrix. Conventional extraction methods of extraction are SOX [15,27,28], SPE [29], MSPD [20,30], and SFE [31,32]. The need to change the nature of the solvent, the amount of solvent used, and the time required to undertake an extraction from a food matrix has driven the development of various techniques in recent years to challenge SOX extraction, which has been a long-standing and proven technique. Substantial progress has been made toward developing improved techniques such as MASE [33] and the most popular ASE or PLE [34,35]. Finally, other methods such as dialysis [51] have also been tested. Saponification under alkaline conditions (in the presence of ethanol and KOH) [52] followed by extraction with organic solvents is often employed for the analysis of large amounts (up to 100 g) of fat. However, this method is known to lead to degradation of dioxins in proportion to their chlorine content, and in the case of PCDFs to production of lower chlorinated PCDFs and ethoxy-PCDFs as artifacts [53]. Finally, it is worth noting that although the approach has not yet been studied extensively, some applications have already demonstrated the potential of sonication (USE) for food dioxin analysis [54].
40.3.1 Soxhlet Extraction One of the most frequently used liquid–solid extraction methods, developed in the late nineteenth century, is still routinely used for extraction of dioxins from seafood tissues [15,27,28]. However, the technique has a number of drawbacks, the most important of which are the large volume of solvent (200 mL for 100 g of tissues), the long extraction time (more than 18 h), the generation of dirty extracts that require extensive cleanup, and the impossibility of automation. In order to overcome these, alternative extraction strategies have been developed, offering analysts a choice of newer techniques such as SPE [29], MSPD [20,30], and more recently ASE [35].
40.3.2
Solid Phase Extraction and Matrix Solid Phase Dispersion
SPE is today a classic extraction system, thanks mainly to the popularization of SPE cartridges, which have been successfully applied to biological human fluids [29]. However, in the case of
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solid samples, SPE is less popular and has almost never been used to extract dioxins from seafood tissues because of the large amount of sample needed. On the contrary, MSPD, using open conventional glass chromatography columns, is very often used in routine analysis of seafood samples [20,22,24,30]. In MSPD, the sample is mixed or blended with an appropriate sorbent (e.g., C18, silica) until a homogeneous mixture is obtained; this mixture is packed into a column, from which the analytes of interest are eluted with a suitable organic solvent [20,24,30]. The extraction and first cleanup step are performed simultaneously, and most of the artifacts are eliminated. Because a large amount of sample is needed, the method compares unfavorably with SOX in terms of the amount of solvent required (around 400 mL).
40.3.3 Supercritical Fluid Extraction SFE is another classic method for seafood dioxin analysis, but not as popular as SOX and MSPD. SFE has attracted intense interest during the past 20 years, mainly for extraction of solid samples, because it offers short extraction times and minimum use of organic solvents [26,55]. Carbon dioxide (CO2) is mostly used as the extraction solvent because of its moderate critical temperature (31°C) and pressure (73 atm). In the 1990s, SFE instruments became available, enabling larger sample sizes and rendering it more suitable for wider applications. For seafood and seafood matrixes, fat retainers such as Florisil and silica are usually introduced in the extraction thimble to achieve a fat-free extract. Some applications for seafood dioxin determinations have been published [31,32,56]. Although SFE extraction is automated and offers a short extraction time and minimum use of organic solvents with no additional cleanup step before GC–MS, it is not widely used because of the large number of parameters that have to be optimized, especially in the analyte collection chamber, and the high cost of the equipment. Similarly to SPE, due to the large amounts of samples required (5–10 g of lipid equivalents) to be able to reach the low levels at which dioxins are present in food samples, the use of SFE for this purpose is scarce.
40.3.4
Accelerated Solvent Extraction
One of the most recent extraction methods used instead of SOX and MSPD is ASE or PLE, an alternative to the classic extraction methods [26,34,57,58]. It uses conventional liquid solvents at high pressures (150–200 psi) and temperatures (50°C–200°C) to extract solid samples quickly, and with much less solvent than conventional techniques. Seafood samples are placed in extraction cells, which are filled with an extraction solvent and heated. The sample is statically extracted for 5–10 min, with the expanding solvent vented to a collection vial. Following this period, compressed nitrogen is used to purge the remaining solvent into the same vial. The entire procedure is completed in 10–20 min per sample, and uses only 15–20 mL of solvent. Of special interest are applications dealing with selective extraction procedures, where integrated cleanup strategies are used to combine extraction and cleanup or fractionation to further simplify all the sample-preparation steps [34]. Fish and seafood have been the food matrices most commonly investigated using a fat retainer (alumina, silica, and Florisil) in the extraction thimble to achieve a fat-free extract; with satisfactory results for dioxins and dioxin-like PCBs [59–62]. As part of the DIFFERENCE project [63], Wiberg et al. [35] evaluated traditional extraction techniques vs. alternative techniques such as ASE for PCDD/F and dioxin-like PCB determinations in food and feed, including certified reference materials. They demonstrated that ASE is more of a quantitative
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extraction process than other conventional techniques. The ASE method in combination with HRMS detection meets the quality criteria for official control of dioxins in foodstuffs [43]. One of the recent developments is the combination of ASE with integrated carbon fractionation [64], in which dioxins can be fractionated and obtained in backward elution, and only a small, miniaturized multilayer silica column cleanup is required after ASE and before detection. Some attempts have also been made to combine ASE with automated cleanup systems, in particular the powerprep FMS system (which is discussed further in Section 40.4), and to construct a fully automated system (extraction plus cleanup); however, the results have not been satisfactory because ASE, as a dynamic system, requires the incorporation of a concentration phase prior to prep-FMS, rendering automation virtually impossible and considerably increasing the analysis times [65]. More extensive information about this technique can be found in the literature, where there are some reviews dealing exclusively with ASE for dioxins in foods [34] and biological matrices [66].
40.3.5 Microwave Oven MASE in the analysis of dioxins has been only recently introduced, and there are no published studies in which MASE was used for dioxin seafood extraction. In recent years, MASE has attracted growing interest, as it allows rapid extraction of solutes from solid samples by employing microwave energy as a source of heat, with an extraction efficiency comparable to that of classic techniques. The partitioning of the analytes from the sample matrix to the later extractant depends on the temperature and the nature of the extractant. Unlike conventional systems, microwaves heat the entire sample simultaneously without heating the vessel; thus the solution reaches its boiling point very rapidly and the extraction time is very short [33]. In view of the good results of MASE in the extraction of PCBs and DDTs [67] from biological tissues with only 8 mL of ethyl-acetate (1:1, v:v), this technique is very attractive for dioxin analysis in food samples. Its main drawbacks are the loss of more volatile solutes if the temperature of the vessel rises rapidly; and that the vessels need to be cooled to room temperature after extraction before they can be opened, which increases the overall extraction time. In addition, it is not possible to automate the procedure to incorporate cleanup steps.
40.4
Cleanup Methods
Analytical procedures for determination of PCDD/Fs in seafood samples involve sophisticated and tedious cleanup methods. Several steps are usually required to remove the bulk of coextractants (including lipids) in order to end up with an extract containing only PCDD/Fs, in which the analytes can be detected at the ultra trace levels at which they occur in seafood and seafood samples. The choice of a particular sequence of steps will depend very much on the analytical system that is finally used. Sample extraction, cleanup, and GC method together form a delicately balanced combination, each contributing to the ultimate specificity and selectivity. For the determination of dioxins, nearly all established schemes involve combinations of cleanup methods developed for the analysis of PCBs and organochlorinated pesticides (OCPs) (solid–liquid adsorption chromatography using Florisil, silica, and alumina, gel permeation chromatography [GPC], and high-performance liquid chromatography [HPLC]) in combination with an active carbon step to isolate the specific fraction containing the dioxins without chemical interferences. In many of the methods used today, the sample extraction and cleanup steps are combined “online” or “at-line,” and some are automated.
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Lipid Removal
Lipid removal is the first step in the cleanup process, and some other interferences are also usually eliminated. Several different methods have been used to remove lipids, including destructive methods such as sulfuric acid [28] or sodium hydroxide treatment [52], and nondestructive methods such as GPC [68] and dialysis [51,69]. GPC, which is sometimes referred to as SEC has been successfully applied to seafood tissues for POPs analysis using SX-3 Biobeds (200–400 mesh) in a range of column sizes and solvents. It can be fully automated and, unlike adsorption chromatography, it is also more suitable for the isolation of unknown contaminants on whose polarity or chemical functionality there is little information. The method can also handle a large mass of lipid in each sample (e.g., columns of ca. 500 × 25 mm ID can handle up to 500 mg of lipids) compared to adsorption columns that are limited to 50 mg of lipids per g of adsorbent [68,70]. Dialysis with semipermeable membranes (SPMs) in an organic solvent can separate other similar POPs from lipids. The method can eliminate more than 20 g of lipids in a single membrane with acceptable recoveries of internal standards, practically irrespective of the amount and type of lipid dialyzed. The method has been successfully used for dioxin analysis in a large variety of seafood species [69]. Although it is efficient, simple, and versatile and does not entail excessive solvent use, this procedure is not very often used for dioxin analysis because it is very time consuming (72 h). In addition, online coupling, either with extraction or with the following cleanup steps, is not possible.
40.4.2
Isolation of Uncommon Chemical Interferences
For dioxin analysis in a seafood matrix, an additional purification step is necessary to eliminate other interferences (including any other lipids). A combination of adsorbents (neutral, basic and acid alumina, silica, modified silica with acids and basics, and Florisil in multilayer or one-layer columns) and solvents with different polarities and dielectric constants are used to eliminate interferences [15,22,24,27]. It is well known to experts that the application of the extract to a strongly basic adsorbent (potassium or cesium hydroxides) silica gel with a low-polarity solvent hexane is very effective for removing trace residues of acidic compounds such as phenolic and carboxylic acids, and sulfonamide compounds [71]. On the other hand, the sulfuric acid-impregnated silica gel (20%–40%, w/w) is very effective in removing numerous types of compounds by dehydration, acid-catalyzed condensation, and oxidation reactions [72]. Alumina (basic, acid, and neutral) and Florisil are used, at different activation grades, mainly to eliminate all other lipids and other coextractants [37,39]. The literature gives no indication of preferences for any specific adsorbent or solvent, the choice of which depends more on the laboratory’s preferences than on performance. Cleaning up of seafood samples for dioxin analysis is a laborious and tedious task, which has to be validated. The combination of adsorbents and solvents chosen to obtain a clean extract without any dioxin loss before the GC–MS analysis is up to each laboratory.
40.5 Fractionation/Group Separation Normally, a group separation is necessary before final analysis of dioxins by GC-HRMS. At this stage, the cleanup extract may contain other similar organohalogen compounds such as PCBs. With the exception of nonortho PCBs, dioxins are present at substantially lower concentrations than the other POPs, and it is therefore necessary to separate dioxins from the bulk of POPs. The
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methods available for the isolation of POPs into separate fractions prior to GC analysis are based on the spatial planarity of dioxins to separate them as a distinct fraction. The available methods for fractionation have been extensively reviewed [36,73]. Open liquid chromatography columns of Florisil [37,74]; alumina [39] active carbon [30,75,76]; and graphitic carbon [38] are among the most widely used methods. In recent years, HPLC with either porous graphitic carbon (PGC) [77,78] or active carbon [5] and PYE (2-(1-pyrenyl) ethyldimethylsilylated silica gel) columns [79,80] has become more popular thanks to the inherent advantages of HPLC. Concejero et al. [81] studied the feasibility of employing four different carbons, Amoco PX-21, Carbosphere and Carbopack B and C, and one HPLC stationary phase, PYE (typically used for PCB and PCDD/F fractionation) for environmental studies. Recoveries for fractionation of the target compounds with all the sorbents studied were generally good and reproducibility satisfactory. All were able to isolate PCDD/Fs from PCBs, which could interfere in the final determination of the former by GC-HRMS. As a result, Carbopack B (as SPE cartridges) and PYE were considered the most valuable alternatives for simultaneous fractionation of PCDD/Fs and of the different classes of PCBs typically investigated in environmental studies. An additional merit of this HPLC stationary phase is the possible of automates. It is worth noting that Immunoaffinity Chromatography (IAC) using mono- and polyclonal antibodies specifically developed to recognize 2,3,7,8-CDD/Fs was considered a very attractive technique in the 1990s. Thanks to the good results achieved in the cleanup of aqueous samples (water and blood) for dioxin analysis [82], it was initially thought to be very promising. However, because of the need to inject fat-free extracts, the variability of the results and the presence of cross-reactions, only a few years later it had been forgotten as an alternative cleanup process for dioxin analysis.
40.6
Automation of Extraction and Cleanup
In view of the extreme difficulty and tediousness of the extraction + cleanup process in seafood dioxin analysis, there have been many attempts at automation, but so far no one has come up with an automated procedure for simultaneous extraction and cleaning up. The first attempt at a semiautomated at-line extraction/cleanup procedure was made by Smith et al. [71]. They developed a method for dioxin analysis of biological (including fish) tissues in two steps. In the first step, the extraction and a first cleanup step, using active carbon, were performed simultaneously. In the second step, the extract was applied to a second series of adsorbents contained in two tandem columns. Based on this general scheme, in 1997 was developed a semiautomated method for online extraction plus cleanup and fractionation of PCBs and PCDD/Fs [30]. Up to 6 g of fat can be extracted with this method, which is very useful in the case of fish, and in fact it has been successfully used to determine dioxins in seafood and seafood products [83,84]. However, despite its good performance, the method has not been widely used because there is no commercially available apparatus. The efficiency of the automated Power-Prep system (FMS, Waltham, MA) in purifying sample extracts for dioxin analyses has already been demonstrated in recent years for different type of matrices, including food [40,85]. The multistep procedure is based on the use of disposable multilayer silica columns, basic alumina and PX-21 carbon columns, which can be combined to suit the target analytes. This means that dioxins and nonortho PCBs can be isolated in a fraction with good recoveries, and several samples can be analyzed in parallel, even ones with
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high fat contents [86]. This method has become increasingly popular over the years; as the only commercially available apparatus, it is gradually making its way into all laboratories that perform large numbers of analyses. Some efforts have been made [65] to couple in an online ASE extraction step, but for the moment no satisfactory results have been achieved. While any laboratory can choose the method that best suits it for extraction and cleanup of seafood dioxins, there is no doubt that ASE as an extraction method and the Power-prep system for cleanup, both commercial products, are the only ones that, although not fully automated, permit large numbers of samples to be analyzed in the shortest possible time. With this combination, it is possible to handle 10 samples at once in both the extraction and the purification steps, and to deal with any food health emergency due to dioxin contamination of foodstuffs.
40.7
Instrumental Determination
As noted earlier, the choice of analytical procedure for extraction plus cleanup is up to each laboratory, if the analyte recoveries that they achieve are within the range laid down by the EU directive [43]. However, in the case of instrumental determination of the seventeen 2,3,7,8-PCDD/F congeners, the EU directive requires the use of GC-HRMS, which was the only method able to reliably determine dioxins at levels appropriate for food analysis. Some other methods, such as DR CALUX® bioassay and GC–MS/MS (ion trap detector [ITD]), are only officially accepted for screening purposes. The basic requirements for acceptance of analytical requirements [43] are high sensitivity (10−12 g), selectivity, accuracy, and low limits of detection. The most important specific requirements are recovery control by the addition of 13C12-PCDD/Fs as standards. The recoveries of the individual internal standards should be between 50% and 130%; the GC separation of the isomers should be <25% peak to peak; the identification should be performed according to EPA Method 1613 revision B and EU official method [43] using isotope dilution RGC/HRMS; and the difference between upper bound (not detected at limit of detection) and lower bound (not detected equal to 0) determination levels, should not exceed 20% for foodstuffs with about 1 pg WHOTEQ/g fat (only PCDD/F), and 25%–40% for foodstuffs with about 0.5 pg WHO-TEQ/g fat. Following a number of dioxin contamination incidents involving foodstuffs [8–10], there has been a tremendous increase in the demand for PCDD/Fs measurements in foodstuffs, including seafood and seafood products. Because of this, alternative and relatively inexpensive techniques such as GC-ITMS, working in tandem mode (MS/MS) [44,45], or GC × GC mECD [47] and GC × GC-ToFs [48], have recently been developed and validated in order to be accepted as an alternative to GC-HRMS for instrumental determination of PCDD/Fs.
40.7.1
GC Congener Separation
High-resolution gas chromatographic methods for analysis of PCDDs and PCDFs have been developed extensively in the last two decades and continue to progress today. GC isomer-specific separation of all 136 tetra- to octa-PCDD/Fs on a series of nine fused-silica capillary GC columns containing silicone stationary phases of diverse polarity (100% methyl, 5% phenyl methyl, 50% phenyl methyl, 50% methyl trifluoropropyl, 50%, 75%, 90%, and 100% cyanopropyl, and liquid crystalline smectic) was studied by Ryan et al. [87]. They showed that all 136 PCDD/F compounds, including the biologically important 2,3,7,8-substituted congeners, could be
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separated from each other mostly with two stationary phases. More recent studies have focused on separation of the seventeen 2,3,7,8-PCDD/F congeners from closely co-eluting isomers. Almost all methods found in the literature for seafood analysis use DB-5 stationary phase (5% diphenyl 95% dimethyl polysiloxane) (J&W Scientific, Folsom, CA) or equivalent [15,17,20,27]. Since the DB-5MS (J&W Scientific Folsom, CA) stationary phase (5% Silphenylene Silicone copolymer or Si-Arylene) has become commercially available, most laboratories use these products for dioxin analysis because their thermal stability is much improved as compared to DB-5 [88]. However, either of the two GC stationary phases can completely separate all seventeen 2,3,7,8-PCDD/F congeners, particularly the 2,3,7,8-TCDF. The EPA and European Standard methods [41–43] recommend the use of a second polar GC stationary phase such as DB-255 (50% cyanopropylmethyl 50% phenylmethylsiloxane), DB-Dioxin (44% methyl, 28% phenyl, 20% cyanopropyl polixiloxane) (J&W Scientific, Folsom, CA), Supelco SP-2330 (100% cyanopropyl polysiloxane), or equivalent as a complementary tool. Most recently, Fishman et al. [89], evaluated 13 different GC columns: HP-5MS (Agilent technology), Rtx-5MS and Rtx-Dioxin2 (Restek, Bellefonte, PA), Supelco Equity 5 and SP-2331 (Supelco, Bellefonte, PA), Factor Four VF-5MS and CP-Sil 8 CB LowBleed/MS (Varian, Walnut Creek, CA), DB-5, DB-5MS, DB-225, DB-XLB (J&W Scientific, Folsom, CA), ZB-5MS, and ZB-5UMS (Phenomenex, Torrance, CA) for separation of the 17 toxic dioxin congeners. Their conclusion was similar to that of Ryan et al. (15 years ago): all dioxins can be separated from closely eluting isomers using either of two sets of nonpolar and polar stationary phase combinations. On the other hand, all efforts made to improve the separation among target isomers in some specific stationary phases (i.e., 1,2,3,7,8- and 1,2,3,6,7-penta-chlorinated dioxins) using the Rtx-Dioxin-2 (Restek Bellefonte, PA) have failed [90]. In this context, multidimensional GC techniques such as heart-cut multidimensional GC (heart-cut MDGC) and lately comprehensive two-dimensional GC (GC × GC) are regarded as powerful alternatives to the one-dimensional GC to solve coelutions between dioxins and other similar compounds present in the extract. Heart-cut MDGC allows coeluting congeners on a precolumn to be transferred to a second capillary column with a different selectivity, improving the separation of the selected regions. A number of applications for PCDD/Fs analysis can be found in the literature [91]. However, when dealing with such complex mixtures, the number of heart cuts that can be made in one analytical run is limited to avoid coelutions in the second column. The need of reinjecting several times the same extract makes this technique time consuming, and is therefore a major drawback. Recently, GC × GC has been recognized as a powerful chromatographic technique for the resolution of complex mixtures. In this case, a modulation process transfers the entire effluent from the first column into the second one as consecutive narrow bands. Compared to heart-cut MDGC, a much higher peak capacity is obtained since the whole extract is subjected to two independent chromatographic separations by two sequential GC columns, without the need of reinjecting several times the same extract. In addition, the focusing effect that takes place during the modulation yields an increase in the signal-to-noise ratio, improving the limits of detection [92]. Since its introduction at the beginning of the 1990s by Liu and Phillips [93], the number of applications of GC × GC in the environmental and food fields has grown exponentially thanks to the advances in the instrument setup, such as more robust interfaces (modulators) between the first and second capillary columns and the possibility of coupling to a number of detection systems. The combination of GC × GC with microelectron detection (GC × GC-mECD) has been regarded as a promising technique for the determination of PCDD/Fs. Besides providing sufficient selectivity and sensitivity for their reliable determination at low levels in complex matrices, it would be a more cost-efficient option than GC-HRMS, the confirmatory method for the official control
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of dioxins in food [43]. The selection of the stationary phase of the first and second dimension columns is a critical step. A number of column combinations have been tested for the complete separation of the 17 priority PCDD/Fs isomers and the 12 WHO-PCBs (that have assigned a toxic equivalency factor [TEF] value) from each other and from other compounds potentially present in the extracts. Korytár et al. [94] found that the combination of a nonpolar stationary phase such as DB-XLB, in the first dimension, and the liquid-crystalline LC-50 (J&K Environmental, Milton, ONT, Canada), as second dimension, provided the complete separation of the 29 priority congeners, as well as from matrix constituents. In a further study, Danielsson et al. [47] used the same column combination (DB-XLB × LC-50) for the analysis of food samples, including fish, and compared the results with those obtained by GC-HRMS. The TEQ data correlated well between the two methods, pointing that although a more intensive validation should be performed to propose GC × GC-mECD as complementary/confirmatory method, it has a great potential as screening method, providing not only the TEQs’ value but also a profile of the congener distribution in the samples [95]. Certainly, the coupling of GC × GC with MS has additional advantages to mECD, including the possibility of using isotope dilution for quantification, although increasing the costs [96]. Up to now most of the studies have been carried out using ToF-MS. Focant et al. [48] explored the possibilities of GC × GC-ToF-MS with the column combination Rtx-500 × BPX-50. The TEQ results obtained compared favorably to those obtained by GC-HRMS for seafood samples, although lower limits of detection would be desirable. On the other hand, the introduction in the market of rapid-scanning quadrupole MS instruments that have a lower cost than ToF-MS systems is promising, enabling also the possibility of using electron-capture negative ionization (ECNI) instead of electronic impact (EI) that would, in many cases, enhance the analyte detectability [97]. Improvements in the general setup of GC × GC system and advances in the MS detectors will lead this technique to a full establishment in routine analysis laboratories for dioxins analysis, if more user-friendly software for visualization and data treatment are available.
40.7.2 GC Detectors As noted earlier, HRMS is mandatory for dioxin analysis in foodstuffs (including seafood and seafood products); however, due to the high cost of acquisition and maintenance, there has been research into suitable alternatives to HRMS over the last few years. HRMS was first used for TCDD determination in seafood samples in 1973 with detection limits in excess of 3 ppt [98]. Since then, improvements in mass spectrometers have made it possible to quantify all PCDDs and PCDFs in the sub-parts per trillion range. Almost all recently investigated MS alternatives are based on tandem mass spectrometry (MS/ MS) because this mode of operation theoretically provides higher signal to noise ratios than lowerresolution MS working in selective ion monitoring (SIM) mode. Of the different instruments capable of performing MS/MS experiments, ITD are the most widely studied. However, ITD instruments are considerably less sensitive than HRMS instruments, and therefore this instrumentation is difficult to use for dioxin analysis in seafood samples, which usually present low dioxin concentrations. During the last decade, GC-ITD working in tandem operation mode (MS/MS) has been successfully used for the analysis of PCDD/Fs and related compounds in environmental samples (with relatively high concentrations of dioxins) such as sewage effluents [99,100], atmospheric aerosols [101], and fly ashes [100]. However, until now very few papers dealing with the
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analysis of PCDD/Fs in food samples have been published, and almost all of them analyzed fatty foods that present concentrations above 2 pg/g [102]. In the case of seafood and seafood products, GC-ITD(MS/MS) has produced comparable results to that of HRMS for PCDD/F determinations in seafood presenting total PCDD/F concentrations higher than 3 pg/g (fresh weight) [103,104]. In other works, Malavia et al. [44,45], compared GC-HRMS with GC-ITD (MS/MS) for the determination of dioxins and furans in vegetable and seafood oils and seafood tissue samples. The study was done within the framework of the European research project DIFFERENCE [63], and the results obtained with both instruments were comparable and within the consensus values. Nevertheless, the limits of detection obtained for GC-ITD(MS/MS) were between 0.07 and 0.20 pg/g of oil, whereas modern HRMS is two or three orders of magnitude lower. The main advantages of using ITD(MS/MS) included rapid determinations and low cost, simplicity of operation (once the method is developed) and maintenance, and high selectivity for dioxin isomers. The main disadvantage of ITD reported by other authors [105], such as the low reproducibility of quantification due to excessive ions coexisting with dioxins in the trap, have been solved in the modern and more recent GC-ITD(MS/MS) instrumentation with the ion source outside the trap, as has been demonstrated by Malavia et al. [46]. In their paper, the authors concluded that it is only possible to achieve reliable results for PCDD/F determinations at concentrations close to the maximum residue levels established by the EU for food by using external ionization. Other systems capable of performing MS/MS experiments, such as triple-quadrupole and hybrid MS instruments have also been proposed as suitable alternatives for dioxin analysis in foods. However, now they are not being used at laboratories for routine analysis of PCDD/Fs due to the high cost of acquisition, not being a real low-cost alternative to HRMS for dioxins analysis. At the same time, some authors have explored the possibilities of the ToFMS analyzer for dioxin analysis [106]. Although the ToFMS analyzer allows simultaneous sampling and measurement of all ions across the mass range and full-spectrum sensitivity is comparable to a quadrupole instrument in the SIM mode, its limits of detection (in the pg range) make dioxin analysis in seafood samples very difficult. In fact the literature records a large variety of environmental applications such as screening for PCBs, pesticides, and brominated flame retardants in biological sample [107,108], but applications to dioxins are still rare. In addition, as mentioned earlier, at present GC × GC–ToF-MS are neither cheaper nor easier to use than HRMS, and so they are still not a real alternative to HRMS.
40.8 Bioanalytical Screening Methods Several dioxin food incidents [8–10], and also new EU regulations [12], highlight the need for screening methods for food and feed materials. Th is need is even more acute during an incident, first to rapidly locate the source and second to reserve often-limited GC-HRMS capacity for confirmation of suspect samples. Several bioanalytical detection methods (BDMs) for measuring dioxin-like activity have been developed since the early 1990s. These methods are based on the ability of key biological molecules to recognize a unique structural property of dioxins or to respond to dioxins in a specific way. Most bioassays are based on the assumption that dioxin compounds act through the aryl hydrocarbon receptor (AhR) signal transduction pathway. The biological methods include biomarkers (e.g., wildlife/human effects) [109], whole animal exposures (in vivo, laboratory exposure) [3], cell- or organ-based bioassays (e.g., EROD, in vitro luciferase) [110], and protein binding assays (e.g., ligand binding as well as immunoassays) [111,112].
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All these methods entail an estimation of the TEQs present in the sample, so that unless an interference-free dioxin fraction is obtained, the TEQs calculated in this way may be dioxin-like PCBs or any other compound that responds to dioxins in a similar way. Of all the methods mentioned, the one that has achieved most popularity and is accepted by the new EU regulations, as a screening method, is the one called DR CALUX bioassay. This method uses genetically modified rat or mouse hepatoma cells which respond to chemicals that activate the AhR. The recombinant CALUX cells contain a stably transfected AhR responsive firefly luciferase reported gene, which responds to dioxins and also to dioxin-like chemicals. At present, the different cell lines are commercialized and sold as the DR CALUX assay; one is based on modified rat H4IIE hepatoma cells (GudLuc1.1), and the other is based on modified H1L6.1 mouse hepatoma cells. The rat cells appear to be more sensitive, showing a response at TCDD concentrations below 1 pM [113]. Recently, there have been a number of international validation studies, such as ring trials on different foodstuffs (including seafood) under the EU-sponsored DIFFERENCE project [114]. The results for both methodologies were comparable and within the consensus values. Bovee et al. [115] were the first to show their utility for screening milk fat around the existing limit of 6 pg TEQ/fat. Based on this work, the test was validated for other food matrices, including seafood and seafood products [116]. However, all of them stress that exhaustive cleanup (similar to that necessary for GC-HRMS) is essential to assure accurate results, which means that one of the advantages of using biological analysis—rapidity—is lost. Clearly therefore, there are some issues that still need improvement, chiefly relating to cleanup procedure.
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12. Council Regulation (EC) No. 199/2006/EC amending Regulation (EC) No. 466/2001 setting maximum levels for certain contaminants in foodstuffs as regards dioxins and dioxin-like PCBs, Official J. Eur. Commun., L 32/34, February 2006. 13. Liem, A.-K.D. et al., Dietary intake of dioxins and dioxin-like PCBs by the general population of ten European countries. Results of EU-SCOOP Task 3.2.5. (Dioxins), Organohalogen Compd., 48, 13, 2000. 14. Kiviranta, H. et al., Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans and polychlorinated biphenyls in Finland, Food Addit. Contam., 18, 945, 2001. 15. Kiviranta, H., Ovaskainen, M.-L., and Vartiainen, T., Market basket study on dietary intake of PCDD/Fs, PCBs, and PBDEs in Finland, Environ. Intern. 30, 923, 2004. 16. Focant, J.-F. et al., Levels and congener distribution of PCDDs, PCDFs and non-ortho PCBs in Belgian foodstuff. Assessment of dietary intake, Chemosphere, 48, 167, 2002. 17. Abad, E. et al., Study on PCDDs/PCDFs and co-PCBs content in food samples from Catalonia (Spain), Chemosphere, 46, 1435, 2002. 18. Karl, H., Ruoff, U., and Blüthgen, A., Levels of dioxins in fish and fishery products on the German market, Chemosphere, 49, 765, 2002. 19. Llobet, J.M. et al., Human exposure to dioxins through the diet in Catalonia, Spain: Carcinogenic and non-carcinogenic risk, Chemosphere, 50, 1193, 2003. 20. Bordajandi, L.R. et al., Study on PCBs, PCDD/Fs, organochlorine pesticides, heavy metals and arsenic content in freshwater fish species from the River Turia (Spain), Chemosphere, 53, 163, 2003. 21. Bordajandi, L.R. et al., Survey of persistent organic pollutants (PCBs, PCDD/Fs, PAHs), heavy metals (Cu, Cd, Zn, Pb, Hg) and arsenic in food samples from Huelva (Spain): Levels, congener distribution and health implications, J. Agric. Food Chem., 52, 992, 2004. 22. Knutzen, J. et al., Polychlorinated dibenzofurans/dibenzo-p-dioxins (PCDF/PCDDs) and other dioxin-like substances in marine organisms from the Grenland fjords, S. Norway, 1975–2001: Present contamination levels, trends and species specific accumulation of PCDF/PCDD congeners, Chemosphere, 52, 745, 2003. 23. Fernández, M.A. et al., Temporal trends in PCDD, PCDF and non-ortho PCB concentrations in Spanish commercial dairy products from 1993 to 2001, Organohalogen Compd., 56, 485, 2002. 24. Fernández, M.A. et al. Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls in Spain, Food Addit. Contam., 21, 983, 2004. 25. Baars, A.J. et al., Dioxins, dioxin-like PCBs and non-dioxin-like PCBs in foodstuff s: Occurrence and dietary intake in The Netherlands, Toxicol. Lett., 151, 51, 2004. 26. Ahmed, F.E., Analysis of polychlorinated biphenyls in food products, Trends Anal. Chem., 22, 170, 2003. 27. Choi, D. et al., Determining dioxin-like compounds in selected Korean food, Chemosphere, 46, 1423, 2002. 28. Abad, E. et al., Evidence for a specific pattern of polycholrinated dibenzo-p-dioxins and dibenzofurans in bivalves, Environ. Sci. Technol., 37, 5090, 2003. 29. Chang, R.R., Jarman, W.M., and Hennings, J.A., Sample cleanup by solid phase extraction for the ultratrace determination of polychlorinated dibenzo-p-dioxins and dibenzofurans in biological samples, Anal. Chem., 65, 2420, 1993. 30. Krokos, F. et al., congener-specific method for the determination of ortho and non-ortho polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in foods by carbon-column fractionation and gas chromatography-isotope dilution mass spectrometry, Fresen. J. Anal. Chem., 357, 732, 1997. 31. van Babel, B. et al., Development of a solid phase carbon trap for simultaneous determination of PCDDs, PCDFs, PCBs and pesticides in environmental samples using SFE-LC, Anal. Chem., 68, 1279, 1996. 32. Miyawaki, T., Kawashima, A., and Honda, K. Development of supercritical carbon dioxide extraction with a solid phase trap for dioxins in soils and sediments, Chemosphere, 70, 648, 2008.
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33. Camel, V., Microwave-assisted solvent extraction of environmental samples, Trends Anal. Chem., 19, 229, 2000. 34. Björklund, E. et al., New strategies for extraction and clean up of persistent organic pollutants from food and feed samples using selective pressurized liquid extraction, Trends Anal. Chem., 25, 318, 2006. 35. Wiberg, K. et al., Pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls, from food and feed samples, J. Chromatogr. A, 1138, 55, 2007. 36. Hess, P. et al., Critical review of the analysis of non- and mono-ortho-chlorobiphenyls, J. Chromatogr. A, 703, 417, 1995. 37. Ramos, L., Hernández, L.M., and González, M.J., Elution pattern of pattern of planar CBs and 2,3,7,8-PCDD/Fs on chromatographic adsorbents and factors affecting the mechanism of retention, possibilities of selective separation of both families, J. Chromatogr. A, 759, 127, 1997. 38. Molina, L. et al., Separation of non-ortho polychlorinated biphenyls congeners on pre-packed carbon tubes. Application to analysis in sewage sludge and soils samples, Chemosphere, 40, 921, 2000. 39. Liu, H.X. et al., Separation of polybrominated diphenyl ethers, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzo-furans in environmental samples using silica gel and florisil fractionation chromatography, Anal. Chem. Acta, 557, 314, 2006. 40. Pirard, C., Focant J.-F., and Pauw, E.D., An improved clean-up strategy for simultaneous analysis of polychlorinated dibenzo-p-dioxins (PCDD), and polychlorinated dibenzofurans (PDF), and polychlorinated biphenyls (PCB) in fatty food samples, Anal. Bioanal. Chem., 372, 373, 2002. 41. US EPA Method 1613 Revision B, Tetra-through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS, US EPA, Washington, DC, April 2002. 42. US EPA Method 1668, Toxic polychlorinated biphenyls by isotope dilution high resolution gas chromatography/high resolution mass spectrometry, US EPA, Washington, DC, 1999. 43. Commission Regulation (EC) No. 1883/2006/EC laying down methods of sampling and analysis for the official control of the levels of nitrates in certain foodstuffs, Official J. Eur. Commun., L 364/32, December 2006. 44. Malavia, J. et al., Analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in vegetable oil samples by gas chromatography-ion-trap tandem mass spectrometry, J. Chromatogr. A, 1149, 321, 2007. 45. Malavia, J. et al., Ion-trap tandem mass spectrometry for the analysis of polychlorinated dibenzo-pdioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in food, J. Agric. Food Chem., 55, 10531, 2007. 46. Malavia, J., Santos, F.J., and Galceran, M.T., Comparison of gas chromatography–ion-trap tandem mass spectrometry systems for the determination of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls, J. Chromatogr. A, 1186, 302, 2008. 47. Danielsson, C. et al., Trace analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans and WHO polychlorinated biphenyls in food using comprehensive two-dimensional gas chromatography with electron-capture detection, J. Chromatogr. A, 1086, 61, 2005. 48. Focant, J.-F. et al., Comprehensive two-dimensional gas chromatography with isotope dilution time-of-flight mass spectrometry for the measurement of dioxins and polychlorinated biphenyls in foodstuffs: Comparison with other methods, J. Chromatogr. A, 1086, 45, 2005. 49. Hoogenboom, L. et al., The CALUX bioassay: Current status of its application to screening food and feed, Trends Anal. Chem., 25, 410, 2006. 50. Eskilsson, C.S. and Björklung, E., Analytical-scale microwave-assisted extraction. J. Chromatogr. A, 902, 227, 2000. 51. Hess, P. and Wells, D.E., Evaluation of dialysis as a technique for the removal of lipids prior to the GC determination of ortho and non-ortho chlorobiphenyls, using 14C-labelled congeners, Analyst, 126, 829, 2001.
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52. Otaka, H. and Hashimoto, S., Fast matrix digestion with ethanolic alkali plus pyrogallol for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and coplanar polychlorinated biphenyls analysis in biological samples, Anal. Chem Acta, 509, 21, 2004. 53. Ryan, J.J. et al., The effect of strong alkali on the determination of polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs), Chemosphere, 18, 135, 1989. 54. Lanbropoulou, D.Q., Konstantinou, I.K., and Albanis, T.A., Sample pretreatment method for the determination of polychlorinated biphenyls in bird livers using ultrasonic extraction followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry, J. Chromatogr. A, 1124, 97, 2006. 55. Smith R.M., Supercritical fluids in separation science-the dreams, the reality and the future, J. Chromatogr. A, 856, 83, 1999. 56. van der Velde, E.G. et al., SFE as clean-up technique for ppt-levels of PCBs in fatty samples, Organohalogen Compd., 27, 247, 1996. 57. Suchan, P. et al., Pressurized liquid extraction in determination of polychlorinated biphenyls and organochlorinated pesticides in fish samples, Anal. Chim. Acta, 520, 193, 2004. 58. Björklund, E., von Holst, C., and Anklam, E., Fast extraction, clean up and detection methods for rapid analysis and screening of seven indicator PCBs in food matrices, Trends Anal. Chem., 21, 39, 2002. 59. Sporring, S. and Björklund, E., Selective accelerated solvent extraction of PCBs from food and feed samples, Organohalogen Compd., 60, 1, 2003. 60. Sporring, S. and Björklund E., Selective pressurized liquid extraction of polychlorinated biphenyls from fat-containing food and feed samples: Influence of cell dimensions, solvent type, temperature and flush volume, J. Chromatogr. A, 1040, 155, 2004. 61. Haglund, P. et al., Hyphenated techniques for dioxin analysis: LC-LC-GC-ECD, GCXGC-ECD, and selective PLE with GC-HRMS or bioanalytical detection, Organohalogen Compd., 66, 376, 2004. 62. Bernsmann, T. and Fürst, P., Comparison of accelerated solvent extraction (ASE) with integrated sulphuric acid clean up and soxhlet extraction for determination of PCDD/PCDF, dioxin-like PCB and indicator PCB in feeding stuffs, Organohalogen Compd., 66, 159, 2004. 63. European Commission DIFFERENCE. Project G6RD-CT-2001-00623. www.dioxins.nl 64. Nording, M. et al., Monitoring dioxins in food and feedstuffs using accelerated solvent extraction with a novel integrated carbon fractionation cell in combination with CAFLUX bioassay, Anal. Bioanal. Chem., 381, 1472, 2005. 65. Focant, J.-F. et al., Integrated PLE-multi step automated clean up and fractionation for the measurement of dioxins and PCBs in food and feed, Organohalogen Compd., 67, 261, 2005. 66. Focant, J.-F., Pirard, C., and De Pauw, E., Automated sample preparation-fractionation for the measurement of dioxins and related compounds in biological matrices: A review, Talanta, 63, 1101, 2004. 67. de Boer, J., Trends in chorobiphenyl contents in livers of Atlantic cod (Gadus morhua) from the North Sea, 1979–1987, Chemosphere, 17, 1811, 1988. 68. De Boer, J. and Lau, R.J., Developments in the use of chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons, J. Chromatogr. A, 1000, 223, 2003. 69. Strandberg, B., Bergqvist, P.A., and Rappe, C., Dyalisis with semipermeable membrane as an efficient lipid removal method in the analysis of bioaccumulative chemicals, Anal. Chem., 70, 528, 1998. 70. Ahmed, F.E., Analysis of pesticides and their metabolites in foods and drinks, Trends Anal. Chem., 20, 649, 2001. 71. Smith, L.M., Stalling, D.L., and Johnson, J.L., Determination of part-per-trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples, Anal. Chem., 56, 1839, 1984. 72. Lamparski, L.L., Nestrick, T.J., and Stehl, R.H., Determination of part-per-trillion concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxins in fish, Anal. Chem., 51, 1453, 1979.
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73. Creaser, C.S., Krokos, F., and Startin, J.R., Analytical methods for the determination of non-ortho substituted chlorobiphenyls: A review, Chemosphere, 24, 1981, 1992. 74. Harrad, S.J. et al., A method for the determination of PCB congeners 77, 126 and 169 in biotic and abiotic matrices, Chemosphere, 24, 1147, 1992. 75. Kannan, N. et al., A comparison between activated charcoals and multidimensional GC in the separation and determination of (non-ortho Cl substituted) toxic chlorobiphenyls, Chemosphere, 23, 1055, 1991. 76. van der Velde, E.G. et al., Analysis and occurrence of toxic planar PCBs, PCDDs and PCDFs in milk by use of carbosphere activated carbon, Chemosphere, 28, 693, 1994. 77. Creaser, C.S. and Al-Haddad, A., Fractionation of polychlorinated-biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans on porous graphitic carbon, Anal. Chem., 61, 1300, 1989. 78. de Boer, J. et al., Non-ortho and mono-ortho substituted chlorobiphenyls and chlorinated dibenzo-pdioxins and dibenzofurans in marine freshwater fish and shellfish from the Netherlands, Chemosphere, 26, 1823, 1993. 79. Ramos, L., Hernández, L.M., and González, M.J., Simultaneous separation of coplanar and chiral polychlorinated biphenyls by off-line pyrenil-silica HPLC/HRGC. Enantiomeric ratios of chiral PCBs by HRGC/LRMS (SIM), Anal. Chem., 71, 70, 1999. 80. Diaz-Ferrero et al., Study of dioxins, furans and polychlorinated biphenyl fractionation on HPLC using a pyrenil column for their analysis in meat and fish samples, Afinidad, 62, 433, 2005. 81. Concejero, M.A. et al., Suitability of several carbon sorbents for the fractionation of various subgroups of toxic polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, J. Chromatogr A, 917, 227, 2001. 82. Concejero, M.A. et al., Different retention of dioxin-like compounds and organochlorinated insecticides on an immunochromatographic column. Interpretation and applicability, J. Sep. Sci., 27, 1101, 2004. 83. Jiménez, B. et al., Levels of PCDDs and PCDFs in oil components of the Spanish diet, Chemosphere, 32, 461, 1996. 84. Serrano, R. et al., Congener-specific determination of polychlorinated biphenyls in shark and grouper livers from the northwest African Atlantic Ocean, Arch. Environ. Contam. Toxicol., 38, 217, 2000. 85. Eljarrat, E. et al., Evaluation of an automated clean-up system for the isotope-dilution high resolution mass spectrometry analysis of PCB, PCDD and PCDF in food, Fresen. J. Anal. Chem., 371, 983, 2001. 86. Focant, J.-F. et al., Fast clean-up for polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls analysis of high-fat-content biological samples, J. Chromatogr. A, 925, 20, 2001. 87. Ryan, J.J. et al., Gas chromatographic separations of all 136 tetra- to octapolychlorinated dibenzo-pdioxins and polychlorinated dibenzofurans on nine different stationary phases, J. Chromatogr. A, 541, 131, 1991. 88. Abad E., Caixach, J., and Rivera, J., Application of DB-5ms gas chromatography column for the complete assignment of 2,3,7,8-substituted polychlorodibenzo-p-dioxins and polychlorodibenzofurans in samples from municipal waste incinerator emissions, J. Chromatogr. A, 786, 125, 1997. 89. Fishman, V.N., Martin, G.D., and Lamparski, L.L., Comparison of a variety of gas chromatographic columns with different polarities for the separation of chlorinated dibenzo-p-dioxins and dibenzofurans by high-resolution mass spectrometry, J Chromatogr A, 1139, 285, 2007. 90. Cochram, J. et al., Retention time profiling for all 136 tetra-through octa-chlorinated dioxins and furans on a unique, low-bleed, thermally-stable gas chromatography column, Organohalogen Compd., 69, 115, 2007. 91. Schomburg, G., Husmann, H., and Hubinger, E., Multidimensional separation of isomeric species of chlorinated hydrocarbons such as PCB, PCDD, and PCDF, J. High Res. Chromatogr., 5, 395, 1985.
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92. Dallüge, J., Beens, J., and Brinkman, U.A.Th., Comprehensive two-dimensional gas chromatography: A powerful and versatile analytical tool, J. Chromatogr. A, 1000, 69, 2003. 93. Liu, Z. and Phillips, J.B., Comprehensive two-dimensional gas chromatography using an on-column thermal modulator interface, J. Chromatogr. Sci., 29, 227, 1991. 94. Korytár, P. et al., Separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls by comprehensive two-dimensional gas chromatography with electron-capture detection, J. Chromatogr. A, 1038, 189, 2004. 95. Haglund, P. et al., GC×GC-ECD: A promising method for the determination of dioxins and dioxinlike PCBs in food and feed, Anal. Bioanal. Chem., 390, 1815, 2008. 96. Mondello, L. et al., Comprehensive two-dimensional gas chromatography-mass spectrometry: A review, Mass Spectrom. Rev., 27, 101, 2008. 97. Korytár P. et al., Quadrupole mass spectrometer operating in the electron-capture negative ion mode as detector for comprehensive two-dimensional gas chromatography, J. Chromatogr. A, 1067, 255, 2005. 98. Baughman, R. and Meselson, M., An analytical method for detecting TCDD (dioxin): Levels of TCDD in samples from Vietnam, Environ. Health Perspect., 5, 27, 1973. 99. Küchler, T. and Brzezinski, H., A comparison of GC-MS/MS for the analysis of PCDD/Fs in sewage effluents, Chemosphere, 40, 213, 2000. 100. Fabrellas, B. et al., Analysis of dioxins and furans in environmental samples, Chemosphere, 55, 1469, 2004. 101. Mandalakis, M., Tsapakis, M., and Stephanou, E.G., Optimization and application of high-resolution gas chromatography with ion trap tandem mass spectrometry to the determination of polychlorinated biphenyls in atmospheric aerosols, J. Chromatogr. A, 925, 183, 2001. 102. Eppe, G. et al., PTV-LV-GC/MS/MS as screening and complementary method to HRMS for the monitoring of dioxin levels in food and feed, Talanta, 63, 1135, 2004. 103. Grabic, R., Noväk, J., and Pacáková V., Optimization of a GC-MS/MS method for the analysis of PCDDs and PCDFs in human and fish tissue, J. High Resolut. Chromatgr., 23, 595, 2000. 104. Haywar, D.G. et al., Quadrupolo ion storage tandem mass spectrometry and high resolution mass spectrometry: Complementary application in the measurements of 2,3,7,8-chlorine substituted polychlorinated dibenzo-p-dioxins, dibenzofurans, in US Foods, Chemosphere, 43, 407, 2001. 105. Eljarrat, E. and Barcelo´, D., Congener-specific determination of dioxins and related compounds by gas chromatography coupled to LRMS, HRMS, MS/MS and ToFMS, J. Mass Spectrom., 37(11), 1105, 2002. 106. Focant, J.-F. et al., Recent advances in mass spectrometric measurement of dioxins. J. Chromatogr. A, 1067, 265, 2005c. 107. van Babel, B. et al., Fast screening for PCBs, pesticides and brominated flame retardants in biological samples by SFE-LC in combination with GC-ToF, Organohalogen Compd., 40, 293, 1999. 108. Dallüge, J., Roose, P., and Brinkman, U.A.Th., Evaluation of a high-resolution time of flight mass spectrometer for gas chromatography determination of selected environmental contaminants, J. Chromatogr. A, 965, 207, 2002. 109. Scheter, A., Dioxins and Health, Plenum, New York, 1994. 110. Jones, J.M., Anderson, J.W., and Tukey, R.H., Using the metabolism of PAHs in a human cell line to characterize environmental samples, Environ. Toxicol. Pharmacol., 8, 119, 2000. 111. Diaz-Ferrero, J. et al., Bioanalytical methods applied to endocrine disrupting polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. A review, Trends Anal. Chem., 16, 563, 1997. 112. Seidel, S.D. et al., Ah receptor-based chemical screening bioassays: Application and limitations for the detection of Ah receptors agonists, Toxicol. Sci., 55, 107, 2005. 113. Goeyens, L. et al., Comparison of the rat and mouse cell lines commercially available for CALUX bioassays, Organohalogen Compd., 66, 608, 2004.
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114. van Loco, J. et al., The international validation of bio- and chemical-analytical screening methods for dioxins and dioxin-like PCBs: The DIFFERENCE project rounds 1 and 2, Talanta, 63, 1169, 2004. 115. Bovee, T.F.H. et al., Validation and use of the CALUX-bioassay for the determination of dioxins and PCBs in bovine milk, Food Addit. Contam., 15, 863, 1998. 116. van Leeuwen, S.P.J. et al., Polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in fish from the Netherlands: Concentrations, profiles and comparison with DR CALUX® bioassay results, Anal. Bioanal. Chem., 389, 321, 2007.
Chapter 41
Environmental Contaminants: Persistent Organic Pollutants Monia Perugini Contents 41.1 Introduction ..................................................................................................................818 41.2 Characterization of PAHs..............................................................................................818 41.2.1 PAHs Methods of Extraction ...........................................................................819 41.2.1.1 Saponification ..................................................................................819 41.2.1.2 Soxhlet Extraction .......................................................................... 820 41.2.1.3 Sonication Method ......................................................................... 820 41.2.1.4 Pressurized Liquid Extraction ......................................................... 820 41.2.1.5 Supercritical Fluid Extraction ..........................................................821 41.2.2 Clean-Up of Extracts........................................................................................821 41.2.2.1 Solid-Phase Extraction .................................................................... 822 41.2.2.2 Gel Permeation Chromatography.................................................... 822 41.2.3 Chromatographic Analysis .............................................................................. 823 41.2.3.1 High-Performance Liquid Chromatography ................................... 823 41.2.3.2 Gas Chromatography ...................................................................... 824 41.3 Characterization of PCBs ............................................................................................. 824 41.3.1 PCBs Methods of Extraction ...........................................................................825 41.3.1.1 Soxhlet Extraction ...........................................................................825 41.3.1.2 Sonication Method and Liquid–Liquid Partitioning ....................... 826 41.3.1.3 Pressurized Liquid Extraction ......................................................... 826 41.3.1.4 Supercritical Fluid Extraction ......................................................... 827 41.3.2 Clean-Up of Extracts....................................................................................... 827 817
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41.3.3 Chromatographic Analysis .............................................................................. 828 41.3.3.1 Gas Chromatography with Electron Capture Detector and Gas Chromatography–Mass Spectrometry............................................. 828 41.3.3.2 Gas Chromatography–High-Resolution Mass Spectrometry .......... 829 References ............................................................................................................................... 830
41.1 Introduction Persistent organic pollutants (POPs) are organic compounds of natural or anthropogenic origin characterized by low water and high lipid solubility, resulting in bioaccumulation in fatty tissues of living organisms. They are widespread contaminants of the marine ecosystem mainly because of their depositions from the atmosphere, able to be transported for long distances from their point of origin, or as result of river wastewater transport, surface runoff, industrial development, or agricultural activities. It is important to highlight that all pollutants, whether in air or on land tend to end up in the ocean [1]; furthermore, closed or semienclosed seas are particularly exposed to the pollution risk. In recent years there has been a growing interest in these pollutants, in particular for their impact on human health. The risks posed by POPs for human health have become of increasing concern and are actually object of a worldwide agreement among several governments, including measures to reduce or eliminate their release in the environment. Their environmental presence is of particular gravity because of their toxicity, bioavailability, and persistence. The seas and costal areas are, generally, final recipients for terrestrial wastewaters containing both anthropological and natural origin pollutants; furthermore, at the same time they are very important economic and aquatic resources. POPs’ presence poses serious adverse effects on the marine ecosystem because they affect all organisms from primary to secondary producer levels up until the top levels of the seafood chain. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) have been listed as priority pollutants by the United Nations Environment Programme (UNEP) because of their potential carcinogenicity, mutagenicity, and toxicity to aquatic organisms and humans. This chapter will focus primarily on the sample preparation and analytical methods of determination of PAHs and PCBs that represent two classes of pollutants very often detected in fish and shellfish. The intent of this chapter is to provide an exhaustive summary of analytical methods, carefully for the sampling, preparation, and analysis techniques.
41.2 Characterization of PAHs Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds consisting of at least two or more fused aromatic rings of carbon and hydrogen atoms. The chemical properties of PAHs depend on their number of rings and molecular mass. In general they are solids having high melting (60°C–450°C) and boiling (200°C–600°C) points, showing low degrees of volatility, and are rather inert lipophilic compounds, which easily dissolve in organic solvents. There have been identified over 100 PAHs in the environment that occur as complex mixtures, of which the composition may differ by source. Of these 100 PAHs 16 were classified as “priority pollutants” according to the U.S. Environmental Protection Agency (EPA). PAHs are produced by natural and anthropogenic activities as products of incomplete pyrolysis from organic materials [2]. Forest fires, domestic heating, combustion of fossil fuels as gasoline, coal, and diesel fuel, industrial activities as petroleum, refining processes and catalytic cracking, rural and urban sewage sludge, smoking
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food processes, and tobacco and cigarette smoke represent only a few PAHs sources. Aquatic organisms that metabolize PAHs to little or no extent, such as algae, mollusks, and the more primitive invertebrates (protozoans, porifers, and cnidaria) accumulate high concentrations of PAHs, whereas fish and higher invertebrates, which metabolize PAHs, accumulate little or no PAHs. Biomagnification of PAHs has not been observed in aquatic systems and would not be expected to occur because most organisms have a high biotransformation potential for PAHs. Organisms at higher trophic levels in food chains show the highest potential for biotransformation. The general concern for this class of compounds is due to their mutagenic and carcinogenic activity. Dihydrodiols and epoxide derivatives, products of the liver by PAHs metabolism, form covalent adducts with DNA and proteins that begin a mutagenic process in the cells. The analytical choice for determining PAHs depends on the purpose of the measurement: carcinogenic PAHs are of interest in studies of human health, but those widespread in the environment may be of interest in ecotoxicological studies. The quantification of PAHs is particularly advantageous when their profiles can be correlated with sources and effects. Many extraction, purification techniques, and combinations have been described and validated, but no single scheme is commonly recognized as “the best” for seafood samples, because all of these display advantages and disadvantages. Chromatographic techniques, such as highperformance liquid chromatography (HPLC) and gas chromatography (GC) are common methods of PAHs detection. The intent of this section is both to provide an exhaustive list of analytical methods to detect PAHs in marine organisms and to compare their efficiency.
41.2.1
PAHs Methods of Extraction
Generally PAH levels are lower in fish musculature than in the liver or in the soft tissues of mollusks, because they have the ability to metabolize and excrete PAHs to water-soluble compounds. All solid samples require homogenization before their extraction. Shells of bivalves are washed, using distilled water, to remove external impurities, in order to avoid the contamination of edible parts. Instead, in fish, the skin and bone are removed before homogenization. The efficiency of PAHs extraction depends both on sample preparations and the polarity of the solvents used [3]. Generally when samples are totally soluble in the organic solvents the recovery of PAHs is high. Furthermore, it is rational to prefer the use of certified reference materials (CRM) rather than add PAHs standards to the samples prior to extraction, because they remain unbound and are easier to extract.
41.2.1.1
Saponification
Saponification is the classical isolation method of PAHs from lipophilic matrices and proteinrich foods [4–7]. The possibility of alkaline saponification in handling wet samples directly assists the recovery of the more volatile PAHs. In marine organisms, due to the presence of insoluble fats and proteins, alkaline digestion, using aqueous, methanolic, or ethanolic potassium hydroxide solutions (KOH) for the hydrolysis of lipids, is necessary [8]. However, methanol in the presence of acid or basic catalyst can convert the fats in fatty acid methyl esters which are difficult to remove from a PAH fraction. The normality of the solution can range from 0.5 to 6 N and the length of saponification varies from 2 to 24 h, depending on the characteristics of the sample. Lean tissues take less time than adipose tissues. Reflux on a water bath, can improve the PAHs recovery and speed the length of extraction. Cyclohexane, hexane, pentane,
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and isooctane are the solvents of election for the following liquid–liquid partition. To protect PAHs from light photodegradation the samples can be covered with aluminum foils. Saponification is an easy method not requiring sophisticated instruments but a long period of analysis, about 3–5 h. Moreover, the presence of alcohol in the hydrolytic solution can interfere with the alkylated PAHs derivatives, and harsh alkaline digestive treatment could have a partial effect of decomposition on the more labile PAHs [5,9].
41.2.1.2
Soxhlet Extraction
Soxhlet extraction represents a common method in routine laboratories [10–14]. Generally extraction is performed using cellulose extraction thimbles filled with fresh homogenized samples and anhydrous sodium sulfate and covered with glass wool. It is possible to use dried samples, but the drying process can determine loss of low-molecular weight PAHs. Physicochemical properties and toxicity are facts considered when choosing extraction solvents. For the extraction, it is possible to use several organic solvents such as acetone, hexane, or methanol, but mixtures of hexane–acetone (1:1, v/v) or chloroform–methanol (2:1, v/v) are the most suitable. The total time of extraction is about 6–8 h. It is possible to cover the Soxhlet apparatus with aluminum foil to avoid access of daylight. The Soxhlet extraction often uses large volumes of organic solvents but its efficiency, considering recoveries of analytes and repeatability, is still the method of choice for many studies [15]. For the high molecular PAHs extraction this method achieves the best recovery.
41.2.1.3 Sonication Method Ultrasonication with solvents, as an alternative to Soxhlet extraction, has advantages in terms of reduced time of extraction. In fact this process lasts only 20–30 min. The reproducibility and the recovery efficiency, above all for the lower PAHs, is practically equivalent to all the other techniques, particularly for solid samples. Homogenized samples, dried with anhydrous sodium sulfate, can be extracted using several extraction solvents including chloroform, hexane–dichloromethane (1:1, v/v), or hexane–acetone (1:1, v/v). As reported in literature this last mixture is the most efficient isolation solvent [16,17]. The ultrasonic procedure can be repeated to improve the efficiency of the extraction. To support the best performance and avoid a decrease of efficiency of the ultrasonic extraction the probe requires frequent replacement.
41.2.1.4
Pressurized Liquid Extraction
Pressurized liquid extraction (PLE) represents a modern and alternative extraction technique, at elevated pressures and temperatures, enabling the reduction of solvent quantities and the time of analysis, the improvement in precision of the analyte recovery, and avoids the contamination of samples. Accelerated solvent extraction system (ASE) is the Dionex trade name for the instrument that uses this technique. Many different solvents or mixtures can be employed for extraction. Hexane, toluene, hexane–acetone (1:1, v/v), and dichloromethane–acetone (1:1, v/v) are the most widely used organic solvents. Considering the hydrophobicity of the majority of PAHs one would expect higher extraction efficiency for less polar mixtures as hexane–acetone (3:1 or 4:1, v/v) but, by using these solvents the recovery for hydrophobic compounds is lower, because the
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solvent is immiscible with water and enables penetrating into the wet sample [18]. The use of the hexane–acetone (1:1, v/v) mixture is recommended by EPA method 3545A for extraction of semivolatile organics, OCPs, and PCBs [19]. The extraction can also be performed at several temperatures within a range of 60°C–200°C, but usually an oven temperature of 100°C is considered the optimal condition. Lower temperatures enable the extraction of low molecular PAHs and the use of temperatures higher than 140°C increases the recovery of single analytes but decreases the selectivity of extraction, and the waxes and pigments matrix can interfere with analytical determination. With ASE it is also possible to select the number of static cycles. One extraction cycle of 5–7 min or two of these cycles are the best choice to achieve maximum efficiency of extraction. Using more or longer cycles increase the risk of the waxes and pigments extraction making more difficult the handling of extracts. Extraction is performed using homogenized fresh fish samples mixed with drying agents, such as sodium sulfate anhydrous or diatomaceous earth. A cellulose paper is placed at the bottom of extraction cells before the sample homogenates are loaded. The final step is the setup of instrument conditions such as heating-up time, number of cycles, oven temperature, flush volume, and purge time. The length of this last parameter does not influence the PAHs recovery. Although the repeatability of PLE extraction is comparable with the classic techniques like the Soxhlet and both methods are able to give good recoveries, however, only the PLE displays a low solvent consumption, a short time of analysis, and a higher sample number. The main disadvantages are represented in the high cost of ASE as compared to equipment used for the Soxhlet or the extraction enhanced by sonication.
41.2.1.5
Supercritical Fluid Extraction
Supercritical fluid extraction (SFE) is an expanding analytical technique that has gained attention as a rapid alternative to conventional liquid extraction. The main advantages of SFE include nontoxicity, cost-effectiveness, high separation efficiencies, and short analysis time. Carbon dioxide (CO2) is the most used supercritical material for its ease of manipulation, good solvent strength, compatibility with solutes, lack of toxicity, and also because it is nonflammable, noncorrosive, odorless, and inexpensive. This technique can also be directly coupled with on-column GC. SFE is often used for PAH extraction from sediments, while its use for processing biotic samples is limited, probably because the sample composition (fat and moisture) influences the robustness of this technique. Compared with the Soxhlet extraction, SFE gives the same results in terms of accuracy and precision but reduces the use of organic solvents and extraction time.
41.2.2
Clean-Up of Extracts
Especially in marine organisms, the PAHs are associated with substances that interfere with their separation and identification, and extracts may necessitate additional clean-up before analysis and quantification, above all, if the alkaline saponification has not been used as an extraction step. Lipids and pigments may be the main interfering substances in the analysis of PAHs in biological samples. The object of the clean-up is to remove these coextracted materials in order to extend the column lifetime and to improve detection and quantification limits. Solid-phase extraction (SPE) and gel permeation chromatography (GPC) are two nondestructive techniques very often applied to purify the samples. Some authors [20] carry out the clean-up using concentrated sulfuric acid, but this method is advised against PAHs because they are compounds of low chemical stability and can be partially destroyed by this treatment.
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Handbook of Seafood and Seafood Products Analysis
Solid-Phase Extraction
This method is a sample treatment technique which passes a liquid sample through a sorbent. For determining PAHs in seafoods or solid samples solid-phase extraction (SPE) is used after the extraction processes. It is a technique of common use in many laboratories because it does not require large quantities of solvents, has a short period of analysis, can be automated, and several kinds of commercial sorbents are available. Recently SPE is carried out in online mode, coupled with HPLC or GC, but usually is used in offline mode. At present, conventional chromatographic columns are substituted by prepacked commercial cartridges, which have advantages in terms of time, solvents consumed, and reproducibility. Disks are also available, but cartridges are more commonly used because the commercial availability of disks is reduced. The classical sorbents, alumina, Florisil, silica gel, or C18-bonded silica are widely used for PAH purification, but the choice of the sorbent depends on the selectivity of the final detection step. The solvents widely used for the elution of PAHs are acetone, acetonitrile, methanol, toluene, dichloromethane, tetrahydrofurane, or mixtures of these. The mixtures represent a better combination to gain best recoveries as the high molecular PAH recovery is generally higher with nonpolar solvents, while less polar solvents ensure higher recovery for low molecular PAHs. An other critical parameter to consider is the solvent for reconstituting the extracts before the SPE and its concentration. The hydrophobicity of PAHs may lead to adsorption problems, and if the organic solvent is weak or low concentrations are used it is difficult to get a full solubilization of the high molecular PAHs. The best method, to gain good recoveries for all compounds before the SPE should be the optimization of these parameters. Some authors suggest to avoid the complete evaporation of extracts after SPE because this procedure may lead to a loss of more volatile PAHs. At last, conditioning of SPE cartridge, flow-rate elution, drying of SPE cartridge, and PAH concentration in the samples are other factors affecting the recovery of these pollutants.
41.2.2.2
Gel Permeation Chromatography
This technique is a chromatographic process of the separation of molecules in which impurities and target contaminants are separated based on their hydrodynamic volume when a solution flows through a packed bed of porous gels. The underlying principle of GPC is that particles of different sizes will elute through a stationary phase at different rates. The collected fractions are examined by chromatographic instruments to determine the concentration of the particles eluted. The GPC procedure may employ several types of gels for the purification such as polystyrene divinylbenzene copolymer gels Bio-Beads S-X3, S-X12, XAD-2, Envirogel, and Phenogel. The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue. Proper column packing is important to maximize resolution: an overpacked column can collapse the pores in the beads, resulting in a loss of resolution. An underpacked column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. The optimization of GPC mobile phase is an other important parameter to consider. The classical mobile phases used for the elution of samples are organic solvents such as chloroform, toluene, benzene, or dichloromethane. Considering workplace hazards as well as ecological aspects for the chloroform several authors prefer to employ safer solvent mixtures as dichloromethane–cyclohexane or acetate–cyclohexane. The GPC provides good purification but when the sample is a rich tissue it is important to inject a small amount of sample extract to avoid the column saturation.
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◾ 823
Chromatographic Analysis
PAHs are now routinely identified and quantified by HPLC or GC. Each technique is rather expensive, and requires qualified operating personnel but both have a number of relative advantages. They are considered necessary in order to analyze “real” samples for a large number of PAHs with accuracy and precision.
41.2.3.1
High-Performance Liquid Chromatography
Liquid chromatography is a very sensitive technique and an excellent detection method for PAHs. Usually the analysis is carried out at ambient temperature, avoiding a thermal decomposition of heat-sensitive compounds and using a guard precolumn in order to require less clean-up than GC. Furthermore, HPLC is more suited to the analysis of high molecular PAHs than GC. The main advantages of HPLC derive from the capabilities of the detectors. Those most widely used for PAHs are ultraviolet (UV), diode array detectors (DAD), and fluorescence detectors (FLD). Especially the latter provides very high selectivity and sensitivity, showing detection limits at least one order of magnitude lower than those obtained with ultraviolet detectors. Furthermore, UV and DAD can be employed but more clean-up is required. DAD can be also used to confirm peaks and moreover, additional information on isomeric structure can be obtained from the spectra seen during the elution phase [21]. The specificity of FLD allows the determination of individual PAHs in the presence of other nonfluorescing impurities, but the solvents have to be oxygen-free to avoid the quenching of fluorescence of some PAHs, e.g., pyrene [7]. In addition, since different PAHs have different absorptivity or different fluorescence spectral characteristics at given wavelengths, the detector can be optimized for maximal response to specific compounds. In particular, wavelength-programmed fluorescence detection, to measure changes in excitation and emission wavelengths during a chromatographic run is being used for the analysis of marine samples. Due to the ring differences among PAHs the selection of an appropriate detection wavelength is very important. Acenaphthylene is not detected by the fluorescence detector because it does not emit any fluorescence [22]. In recent years, several selective HPLC columns for PAH separation are available on the market. The packing material considered most suitable for separating PAHs consists of reversed-phase columns, of silica particles chemically bonded to linear C8 or C18 hydrocarbon chains. With these columns, the mobile phase should be more polar than the stationary phase. Typically, the elution program is in the gradient elution technique, and the mobile phase consists of mixtures of acetonitrile and water or methanol and water. The use of an isocratic separation technique is also possible, but separation time is generally too long and some peaks, like acenaphthene and fluorene can overlap. As the efficiency of separation that can be achieved with HPLC columns is much lower than that with capillary GC, HPLC is generally less suitable for separating samples containing complex PAH mixtures, but more suitable for the separation of isomeric compounds. Several isomers including the chrysene–triphenylene and benzo[b]fluoranthene–benzo[k]fluorathene pairs are difficult to separate efficiently using the usual capillary gas chromatographic columns and can be identified by HPLC because this instrument offers a higher column selectivity [23]. Furthermore, the analysis of PAHs in complex matrices can be carried out using HPLC–mass spectrometry (LC–MS), which is also a very helpful detector for the characterization of thermally unstable compounds and begins to be used in routine analysis. According to the U.S. Environmental Protection Agency, HPLC is suitable, in particular, for lower molecular mass compounds like naphthalene, acenaphthene, and acenaphthylene, for which the detection limits can be relatively high.
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Handbook of Seafood and Seafood Products Analysis
Gas Chromatography
PAHs from aquatic organisms can also be analyzed by GC, that is considered an excellent method for the analysis of complex matrices. The signal is related linearly to the carbon mass of PAHs, and the chromatogram shows the quantitative composition of the sample directly. The greatest separation of these compounds can be obtained using columns with high efficiency in the order of 50,000–70,000 height equivalent to a theoretical plate (HETP). Fused silica capillary columns often with nonpolar phases, nowadays commercially available, are making it possible to analyze very complex mixtures containing more than 100 PAHs. The most widely used stationary phases are the methylpolylsiloxanes: especially SE-54 (5% phenyl-, 1% vinyl-substituted) and SE-52 (5% phenyl-substituted), but SE-30 and OV-101 (unsubstituted), OV-17 (50% phenyl-substituted), Dexsil 300 (carborane-substituted), and their equivalent phases are also used. Chemically bonded phases are used increasingly because they can be rinsed to restore column performance and undergo little “bleeding” at high temperatures of analysis (about 300°C) that are required for determining high-boiling-point compounds. Splitless or cold on-column injection is necessary to gain sensitivity in trace analysis. The latter is preferred as it allows better reproducibility and reduces discrimination against the high molecular PAHs which is difficult to avoid entirely when using splitless injection [7]. Although the flame ionization detector (FID) was the most commonly applied detector in GC in the 1980s and was used because of its excellent linearity, sensitivity, and reliability, a range of more selective and sensitive detectors as the electron capture detector and MS have led to its replacement. Because FIDs are nonselective and are subject to background interferences from other carbonaceous sources the samples must be highly purified. GC–MS, conducted in electron impact (EI) mode, has gained wide acceptance and represents the analytical method of choice for identifying minor as well as major PAHs. By using the selected ion mode method (SIM) the singular compounds can be identified at concentrations of at least 100 times lower than is possible by HPLC and it is possible to simplify the time-consuming clean-up process. Identification of single PAHs in SIM mode is helped by means of available libraries of reference spectra that can be used to match the spectra obtained and control the purity of a compound. As isomeric compounds often have indistinguishable spectra, however, the final assignment must also be based on retention. Capillary GC is an excellent technique to separate and determine PAHs in complex mixtures: it presents a high column efficiency in terms of plate number but is not more suitable for the separation of isomeric compounds because it does not show a great selectivity. A number of unconventional instruments and techniques based on spectroscopic principles have been developed as possible alternatives to the chromatographic methods for PAHs. Most of them are, however, expensive, require skilled personnel, and are not yet considered useful for the practicing analyst [24].
41.3 Characterization of PCBs PCBs constitute a family of environmental persistent pollutants of synthetic organic compounds that have mainly been used in electrical equipment as dielectric insulating media. The use of these compounds is now restricted, but because of their wide usage in the past and of their high stability in the environment they are so widely distributed that detectable levels can be found in marine organisms, from mollusks to fish. PCBs have been linked with subtle subchronic effects such as reduced male fertility and long-term behavioral and non-ortho and mono-ortho PCBs have been assessed as having dioxin-like effects. PCBs are extremely persistent in the environment and
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possess the ability to accumulate in the food chain. These compounds are highly insoluble in water and tend to accumulate in body fat. Human exposure is probably dominated by the accumulation through the food chain of the PCBs present in environmental reservoirs. Determination of PCBs in marine organisms generally consists of three steps: sample extraction, purification, and chromatographic separation, identification, and quantification. The extraction methods are generally set up to maximize the extraction of all analytes and the clean-up is performed to improve the selectivity of the extraction removing lipids and interfering compounds. Low selectivity of extraction method yields considerable amounts of undesirable coextractives. For a correct evaluation of data it is important to report the PCB concentrations in an adequate manner or on wet weight or on fat weight. In the case of marine organisms, since an equilibrium partitioning of PCBs between fats in the organisms and water is established, lipid normalized data is used because it allows to compare the PCBs concentrations in the several species of fish, independently of their lipid content. In general, in biotic samples, the lipids are usually extracted together with PCBs. PCBs can be determined using GC techniques with electron capture detection, though more sophisticated methods, such as GC coupled with mass spectrometry (GC–MS), can be used to identify the individual congeners and to improve the comparability of the analytical data from different sources. Higher specificity than ECD and higher sensitivity of conventional GC–MS techniques can be performed using the high-resolution mass spectrometry (HRMS). This powerful system is the reference method for the determination of trace level of non-ortho and mono-ortho PCBs in various environmental matrices [25]. The accuracy in determining PCB levels is highly variable and matrix dependent. Many factors including the water solubility, volatility, and biodegradability of individual PCBs, will alter the composition of a commercial PCB preparation introduced as a pollutant into the environment. Thus, the composition of PCB extracts from environmental matrices will vary widely and often do not resemble any commercial mixture.
41.3.1
PCBs Methods of Extraction
Sample preparation methods for PCB determination involve several steps for exhaustive extraction of the analytes and subsequent clean-up steps for removal of the lipids. PCB levels in fish or shellfish are very high above all in those organisms located at the top of the food chain, because of their ability to accumulate these compounds. Like PAHs the biotic samples require washing with distilled water and homogenization process before the extraction.
41.3.1.1
Soxhlet Extraction
This method is a traditional procedure, largely used in the laboratory and reported also by EPA [26]. The fresh samples, with high moisture content, have to be dried before the extraction to perform a better penetration of solvent into the sample matrix. All samples have to be mixed with anhydrous sodium sulfate to form a flowing powder. The nature of the extraction solvent influences the efficiency of the procedure. Nonpolar or semipolar solvents as pentane, hexane, dichloromethane, acetone, toluene, diethyl ether, or polar–apolar solvent mixtures are usually selected as extraction solvents because they lead to better extraction of most of the PCB congeners present in marine organisms. For fish and shellfish the hexane–acetone (4:1, v/v or 1:1, v/v), methylene chloride–acetone (1:1), and hexane–dichloromethane (1:1, v/v) mixtures are the most employed
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and give the best recovery for PCBs and lipid content. Use of hexane–acetone mixture generally reduces the amount of interferences that are extracted and improves signal-to-noise ratio. Soxhlet extraction method achieves very high recoveries, superior to those reported in the samples extracted with organic solvents, but requires large volumes of highly purified organic solvents and consumes long time. To perform an efficient extraction of PCBs using the Soxhlet technique the total extraction time requires approximately 6–8 h. In addition, some volatile compounds may be lost unless efficient condensers are used [27].
41.3.1.2 Sonication Method and Liquid–Liquid Partitioning Both methods allow a very simple and fast PCBs extraction, but the main disadvantage is that the extracts are very dirty because they contain a lot of coextracted components and require a more accurate clean-up. Hexane or acetone–hexane mixture [28] (1:1, v/v) are commonly used to extract PCBs by sonication, however this can yield the formation of emulsions which may cause a loss of compounds. The time of extraction is about 15–20 min per cycle but the process is normally repeated two more times with fresh solvent.
41.3.1.3
Pressurized Liquid Extraction
In recent years this technique has been applied to marine matrices and has shown high recovery in the extraction of PCBs, when compared with conventional methods. Furthermore, it provides cleaner extracts in shorter time and smaller solvent consumption. The most important variables affecting the efficiency of the PLE process are the nature of the solvent, the temperature of the extraction, and the extraction time. Most applications dealing with extraction of PCBs make use of hexane because it provides a quantitative extraction of most PCB congeners, also this is a more toxic solvent than other linear alkanes [29–32]. N-Heptane, n-pentane, toluene, or hexane–acetone (1:1, v/v), and hexane– dichloromethane (1:1, v/v or 4:1, v/v) mixtures are further extraction solvents often used for the isolation of PCBs from abiotic or biotic samples like mussel and oyster [33]. The best efficiency of PCBs extraction in fish samples is achieved by mixtures of low-polar and high-polar solvents as boiling point, polarity, and specific density influence the penetration into the sample matrix allowing a more efficient extraction of analytes than single solvents and the complete extraction of lipids. This fact is very important because the PCB concentrations, in fish and shellfish, should be standardized to the lipid content. An other important aspect to consider is the influence of extraction temperature. Higher temperatures decrease the viscosity of solvents allowing their better penetration into the sample and enhance the extraction efficiency [34]. Using a temperature range of 90°C–100°C it is possible to achieve elevated recoveries but it needs to consider that the extraction efficiency for lower chlorinated PCBs can be increased with higher temperatures, in the range 90°C–120°C. The selection of the number of static cycles is another important parameter for achieving quantitative extraction. Two static cycles (5–7 min) offer the practical solution to achieve maximum efficiency of extraction, as reported in literature [35,36]. Using three or more cycles increases the total extraction time and the solvent consumption but does not improve the efficiency of PCBs extraction. Although the repeatability of PLE extraction is comparable with the Soxhlet and both methods are able to give good recoveries, however, only the PLE displays a low solvent consumption, a short time of analysis, and a better efficiency of extraction for lower chlorinated PCBs. The cost of this equipment is the main disadvantage if compared to Soxhlet or batch extraction enhanced by
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sonication. Nevertheless, PLE shows the best performances for extraction of PCBs from fish and shellfish, better than those of classic extraction methods or Soxhlet procedure. With the PLE system it is also possible to perform an online clean-up, introducing the suitable fat retainer directly into the extraction cell. This technique is used for the extraction of PCBs from fatty samples in order to avoid further sample treatment before the GC analyses and to save the time spent on sample handling. Florisil, basic alumina, neutral alumina, acidic alumina, and sulfuric acid-impregnated silica are the main employed sorbent retaining lipids. Silica gel impregnated with sulfuric acid is considered the best choice for fat removal because of the clearness of the extracts and because it is much less sensitive to high temperatures [29,37].
41.3.1.4
Supercritical Fluid Extraction
SFE has received increasing attention and popularity as a technique used in the extraction and clean-up procedures for the determination of organic pollutants in complex matrices. The application of SFE to the isolation of PCBs from fish tissues is documented, also if the high lipid contents of these organisms, when coextracted may block SFE restrictors. The introduction of heated, adjustable restrictors have ameliorated this problem [38]. The use of a single fluid, generally carbon dioxide, the selectivity in the extraction of different classes of compounds, the possibility of extraction at high temperatures, the consumption of very limited volumes of organic solvents and a very limited contamination from the laboratory environment, for the use of a sealed system are the main advantages of SFE. Nevertheless, this technique requires two different sequential extraction steps when, on the same sample, the compounds investigated present a different polarity. Sample size is an other critical parameter when low concentrations of analytes are present. However, lyophilization of the tissues prior to extraction conserves considerable vessel volume by eliminating the need for inclusion of drying agents, such as sodium sulfate anhydrous or diatomaceous earth. The extraction conditions have to be generally set up to maximize the extraction of the analytes. Bøwadt et al. [39] found that, in SFE analysis of lyophilized fish tissue, high extraction temperatures yield scarcely better extraction and also imply a less pure extract, with more lipids and interfering compounds. Mild temperatures (60°C–70°C) avoid this problem, however when these are used on fish species with fat content higher than 8%–10% yielded an extract that is injectable only on a split–splitless injector because of the presence of lipids. Inclusion of alumina in the SFE extraction vessel eliminates the need for any additional offline lipid purification since the combination of selective SFE extraction and alumina retains more than 99% of the lipids. SFE extracts generated could be collected in autosampler vials and injected directly onto a gas chromatograph. Compared with the many hours necessary with the conventional procedures this clean-up procedure takes only 30 min and greatly reduces manipulations. In addition to the parameters discussed above, the selection of one extraction technique in preference to another is usually made on the basis of initial capital cost, operating costs, amount of organic solvent, and sample weight.
41.3.2
Clean-Up of Extracts
The procedures followed to purify sample extracts are common to all regardless of matrix. The first purification treatment consists of the elimination of coextracted materials, as lipids and pigments, which can be performed by either destructive or nondestructive methods. In the first case the lipids are removed by oxidation reactions with concentrated sulfuric acid that is very efficient for the
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most unreactive chemical groups like PCBs. The sulfuric acid can be directly added to the extracts or immobilized on silica layers allowing an on-column removal. When it is added to the extracts the solution has to be centrifuged. This method cannot be used to clean-up extracts for other target analytes, as it will destroy most organic chemicals including the pesticides aldrin, dieldrin, endrin, endosulfan (I and II), and endosulfan sulfate. Adsorption chromatography on Florisil, alumina, or silica columns is a nondestructive method that allows the removal of coextracted materials and the elimination of polar interferences. This approach is performed in order to separate PCBs from other organochlorine compounds. It is possible to use columns with 1–2 cm of anhydrous sodium sulfate to the top, but before the elution the sodium sulfate and the cartridge have to be wet and rinsed by adding hexane. The first fraction eluted, generally with hexane, contains PCBs and some DDTs, whereas the second fraction, eluted with ethylether in hexane, contains the remaining DDTs and other organochlorine compounds. The ease of separation appears to depend on the characteristics of the absorbent, of the eluting solvent, and of the sample extract. The option of using standard column chromatography techniques or solidphase extraction cartridges depends on the amount of interferences in the sample extract and the degree of clean-up required. The cartridges require less elution solvent and less time, however, their clean-up capacity is drastically reduced in comparison with standard chromatographic columns. As a nondestructive clean-up method the GPC was used for removal of lipid from fish sample extracts. Using a column of Bio-Beads S-X3 with dichloromethane/hexane (1:1) as the mobile phase, the GPC clean-up gives an excellent efficiency for the lipid removal. Furthermore, it is capable of separating high boiling material from the sample analytes avoiding the contamination of injection ports and column heads, prolonging column life, stabilizing the instrument, and reducing column reactivity.
41.3.3 Chromatographic Analysis Presently the most widely practiced technique for the PCB determination is capillary GC–ECD. Although ECD has many advantages it is unable, using a single column, to differentiate between coeluting PCBs and interferences, and cannot resolve PCB congener pairs like 77/110 [40,41]. Furthermore, its ability to identify individual PCBs relies on retention time alone and may suffer from limited selectivity in cases of very complicated samples. The use of mass spectrometric detectors not only enhances selectivity but, by the use of selected ion monitoring (SIM) and isotope ratios, provides qualitative information to supplement that supplied by GC retention time. Although the two techniques provide very low detection limits, quantification is complicated with both because detector responses vary significantly with molecular structures. Thus, individual calibration for each compound of interest is required in order to achieve quantitative data of acceptable accuracy. GC–HRMS is an other technique generally employed to solve some specific problems in different GC–MS applications.
41.3.3.1 Gas Chromatography with Electron Capture Detector and Gas Chromatography–Mass Spectrometry GC–ECD is the most popular technique owing to the relatively low costs, whereas the high selectivity of GC–MS is superior in the presence of abundant electron-capturing coextractives. Selection of appropriate chromatographic columns is of the major importance for correctly identifying and quantifying PCBs. Capillary GC columns, currently in use, are made of fused silica, chemically
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bonded with various stationary phases, to achieve a range of different selectivities toward complex samples. In general, packed columns have been replaced by capillary columns, because of their far superior efficiency, but the use of a single column does not allow the separation of all congeners in a single chromatographic run. The use of two columns with different polarities provides different elution patterns enabling separation of coeluting congeners. Furthermore, the approach of using a second column is important to confirm the identity of the compounds. Identification of PCB congeners in the sample is performed by comparing the retention times of the peaks with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be based upon measurements of actual retention time variations of standards over the course of a day and should be carefully established to minimize the occurrence of both false positive and false negative results. If the response for a peak exceeds the working range of the system, a dilution of the extract is required. If the measurement of the peak response is prevented by the presence of interferences, further clean-up is required. The quality and utility of the analytical data depend critically on the validity of the sample and the adequacy of the sampling. For PCB determination an extensive quality assurance program is required and, furthermore intercalibration studies are recommended. If the internal standard calibration procedure is being used, the internal standard must be added to the sample extract and mixed thoroughly before injection into the gas chromatograph. When capillary columns are used with temperature programming, almost all PCB isomers and congeners normally present in samples can be identified. The injector temperature set from 250°C to 280°C and the injections made in the splitless mode are the more suitable conditions for PCB analysis. GC oven temperature can be programmed in order to elute all PCBs during the temperature gradient, but the ramp conditions change depending on the column used. Using a GC–MS the transfer line temperature of the GC–MS interface the ion source temperature can be set at 280°C and 260°C, respectively, and a selected ion monitoring can be performed. GC–MS plays an important role in the identification and quantification of PCBs in complex samples and is one of the most attractive and powerful techniques for routine analysis due to its good sensitivity, selectivity, and versatility. The GC separation usually provides isomer selectivity, while the MS shows compound class and homologue specificity. The MS fragmentation pattern provides unambiguous identification by comparing an unknown electron ionization MS spectrum with library spectra. For the identification of unknown peaks the MS conditions at which spectra have been obtained must be similar and the GC separation must be sufficiently efficient to obtain a clean mass spectrum [42].
41.3.3.2
Gas Chromatography–High-Resolution Mass Spectrometry
Non-ortho and mono-ortho PCBs are specific compounds whose determination is mainly performed by GC–HRMS, to provide the required sensitivity and selectivity for analysis. The use of HRMS is based on enhancing the selectivity of the MS as a detector by increasing resolution. HRMS presents a very high capacity to remove the contribution of matrix interfering compounds in the determination of the analytes. Using SIM at a mass resolution of 10,000, the presence of matrix components in the extracts does not interfere and detection at a high level of mass accuracy can be performed. Very high sensitivity and powerful identification capability of HRMS have made this technique the reference method for determination of many POPs at sub pg/g concentrations. However, HRMS systems are relatively expensive and require specialized laboratory infrastructure to run effectively [25].
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References 1. Williams, C., Combatting marine pollution from land-based activities: Australian initiatives, Ocean Coast. Manage., 33, 87, 1996. 2. Bye, T., Romundstad, P.R., Ronneberg, A., and Hilt, B., Health survey of former workers in a Norwegian coke plant: Part 2. Cancer incidence and cause specific mortality, Occup. Environ. Med., 55, 622, 1998. 3. Moret, S., Conte, L., and Dean, D., Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method, J. Agric. Food Chem., 47, 1367, 1999. 4. Dafflon, O. et al., Le dosage des hydrocarbures aromatiques polycycliques dans le poisson, les produites carnès et le fromage par chromatographie liquide à haute performance, Trav. Chim. Aliment. Hyg., 86, 534, 1995. 5. Vassilaros, D.L. et al., Capillary gas chromatographic determination of polycyclic aromatic compounds in vertebrate fish tissue, Anal. Chem., 54, 106, 1982. 6. Chen, B.H. and Lin, Y.S., Formation of polycyclic aromatic hydrocarbons during processing of duck meat, J. Agric. Food Chem. 45, 1394, 1997. 7. De Boer, J. and Law, R.J., Developments in the use of chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons, J. Chromatogr., A, 1000, 223, 2003. 8. Bartle, K.D. Analysis and occurrence of PAHs in food, in Food Contaminants: Sources and Surveillance, Creaser, C.S. and Purchase R., Eds., Royal Society of Chemistry, Cambridge, U.K., 1991, pp. 41–60. 9. Lebo, J.A. et al., Determination of monocyclic and polycyclic aromatic hydrocarbons in fish tissue, J. Assoc. Off. Anal. Chem., 74, 538, 1991. 10. Chen, B.H., Wang, C.Y., and Chiu, C.P., Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography, J. Agric. Food Chem., 44, 2244, 1996. 11. Jaouen-Madoulet, A.J. et al., Validation of the analytical procedure for polychlorinated biphenyls, coplanar polychlorinated biphenyls and polycyclic aromatic hydrocarbons in environmental samples, J. Chromatogr., A, 886, 153, 2000. 12. Anyakora, C. et al., Determination of polynuclear aromatic hydrocarbons in marine samples of Siokolo fishing settlement, J. Chromatogr., A, 1073, 323, 2005. 13. Vives, I. and Grimalt, J.O., Method for integrated analysis of polycyclic aromatic hydrocarbons and organochlorine compounds in fish liver, J. Chromatogr., B, 768, 247, 2002. 14. Vives, I. et al., Polycyclic aromatic hydrocarbons in fish from remote and high mountain lakes in Europe and Greenland, Sci. Total Environ., 324, 67, 2004. 15. Pensado, L. et al., Application of matrix solid-phase dispersion in the analysis of priority polycyclic aromatic hydrocarbons in fish samples, J. Chromatogr. A, 1077, 1103, 2005. 16. Shu, Y.Y. et al., Analysis of polycyclic aromatic hydrocarbons in sediment reference materials by microwave-assisted extraction, Chemosphere, 41, 1709, 2000. 17. Lopez-Avila, V., Young, R., and Teplitsky, N.L., Microwave-assisted extraction as an alternative to Soxhlet, sonication, and supercritical fluid extraction, J. Assoc. Off. Anal. Chem., 79, 142, 1995. 18. Janská, M. et al., Appraisal of “classic” and “novel” extraction procedure efficiencies for the isolation of polycyclic aromatic hydrocarbons and their derivatives from biotic matrices, Anal. Chim. Acta, 520, 93, 2004. 19. U.S. EPA SW-846, Update III: Test Methods for Evaluating Solid Waste, Method 3545: Fed. Reg. Vol. 62, 114:32451 U.S. GPO, Washington, D.C., 1997. 20. Guangdi, W. et al., Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples, J. Agric. Food Chem., 47, 1062, 1999.
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21. Dong, M.W. and Greenberg, A., Liquid chromatographic analysis of polynuclear aromatic hydrocarbons with diode array detection, J. Liq. Chromatogr. Relat. Technol., 11, 1887, 1988. 22. Stołyhwo, A. and Sikorski, Z.E., Polycyclic aromatic hydrocarbons in smoked fish—a critical review, Food Chem., 91, 303, 2005. 23. Wise, S.A., Bonnett, W.J., and May, W.E., Normal- and reverse-phase liquid chromatographic separations of polycyclic aromatic hydrocarbons, in Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Eff ects, Bjorseth, A. and Dennis, A.J., Eds., Battelle Press, Columbus, OH, 1980, pp. 791–806. 24. Vo-Dinh, T., Significance of chemical analysis of polycyclic aromatic compounds and related biological systems, in Chemical Analysis of Polycyclic Aromatic Compounds, Vo-Dinh, T., Ed., Wiley, New York, 1989, pp. 1–30. 25. Verenitch, S.S. et al., Ion-trap tandem mass spectrometry-based analytical methodology for the determination of polychlorinated biphenyls in fish and shellfish. Performance comparison against electroncapture detection and high-resolution mass spectrometry detection, J. Chromatogr. A, 1142, 199, 2007. 26. Schantz, M.M. et al., Comparison of supercritical fluid extraction and Soxhlet extraction for the determination of polychlorinated biphenyls in environmental matrix standard reference materials, J. Chromatogr. A, 816, 213, 1998. 27. Hess, P. et al., Critical review of the analysis of non- and mono-ortho-chlorobiphenyls, J. Chromatogr., A, 703, 417, 1995. 28. Kitamura, K. et al., Effective extraction method for dioxin analysis from lipid-rich biological matrices using a combination of pressurized liquid extraction and dimethyl sulfoxide/acetonitrile/hexane partitioning, Anal. Chim. Acta, 512, 27, 2004. 29. Sporring, S. and Bjorklund, E., Selective pressurized liquid extraction of polychlorinated biphenyls from fat-containing food and feed samples. Influence of cell dimensions, solvent type, temperature and flush volume, J. Chromatogr. A, 1040, 155, 2004. 30. Focant, J.F. et al., Fast clean-up for polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls analysis of high-fat-content biological samples, J. Chromatogr., A, 925, 207, 2001. 31. Bjorklund, E. et al., New strategies for extraction and clean-up of persistent organic pollutants from food and feed samples using selective pressurized liquid extraction, Trends Anal. Chem. 25, 318, 2006. 32. Ramos, J.J. et al., Miniaturised selective pressurised liquid extraction of polychlorinated biphenyls from foodstuffs, J. Chromatogr. A, 1152, 254, 2007. 33. Hubert, A. et al., Accelerated solvent extraction—more efficient extraction of POPs and PAHs from real contaminated plant and soil samples, Rev. Anal. Chem., 20, 101, 2001. 34. Richter, B.E. et al., Accelerated solvent extraction: A technique for sample preparation, Anal. Chem., 68, 1033, 1996. 35. Schantz, M.M., Nichols, J.J., and Wise, S.A., Evaluation of pressurized fluid extraction for the extraction of environmental matrix reference materials, Anal. Chem., 69, 4210, 1997. 36. Bjorklund, J. et al., Pressurized fluid extraction of polychlorinated biphenyls in solid environmental samples, J. Chromatogr. A, 836, 285, 1999. 37. Björklund, E., Muller, A., and von Holst, C., Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples, Anal. Chem., 73, 4050, 2001. 38. Hale, R.C. and Gaylor, M.O., Determination of PCBs in fish tissues using supercritical fluid extraction, Environ. Sci. Technol., 29, 1043, 1995. 39. Bø´ wadt, S. et al., Supercritical fluid extraction of polychlorinated biphenyls from lyophilized fish tissue, J. Chromatogr., A, 675, 189, 1994.
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40. Harrad, S.J. et al., A method for the determination of PCB congeners 77, 126 and 169 in biotic and abiotic matrices, Chemosphere 24, 1147, 1992. 41. Ayris, S. et al., GC/MS procedures for the determination of PCBs in environmental matrices, Chemosphere 35, 905, 1997. 42. Santos, F.J. and Galceran, M.T., Modern developments in gas chromatography–mass spectrometry based-environmental analysis, J. Chromatogr. A, 1000, 125, 2003.
Chapter 42
Biogenic Amines in Seafood Products Claudia Ruiz-Capillas and Francisco Jiménez-Colmenero Contents 42.1 42.2 42.3 42.4 42.5
Introduction ..................................................................................................................833 Toxicity of Biogenic Amines in Seafood ....................................................................... 834 Biogenic Amines as Quality Index in Seafood .............................................................. 836 Legal Limits of Biogenic Amines in Seafood .................................................................837 Factors Influencing the Formation of Biogenic Amines in Seafood .............................. 838 42.5.1 Raw Material................................................................................................... 838 42.5.2 Microorganisms ...............................................................................................839 42.5.3 Processing and the Storage Conditions of Seafood .......................................... 840 42.6 Determination of Biogenic Amines in Seafood ............................................................ 843 42.6.1 Extraction Process ........................................................................................... 843 42.6.2 Determination Process .................................................................................... 843 Acknowledgments ................................................................................................................... 846 References ............................................................................................................................... 846
42.1 Introduction Biogenic amines (BA) are biologically active low-molecular-weight basic nitrogenous compounds (Figure 42.1) which are present in the great majority of foods, including fish and fishery products. According to their chemical structure, they can be classified as aromatic amines (histamine, tyramine, serotonin, b-phenylalanine, and tryptamine), aliphatic diamines (putrescine and cadaverine), or aliphatic polyamines (agmatine, spermidine, and spermine) [1]. Animal, plant, and microorganism 833
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Amino acids precursors CH2
Histidine
Biogenic amines
Amino acid decarboxylase COOH
CH
N
HN
N
Histidine-decarboxylase
Histamine
NH2 –
N H
COO +
H 3N
CH2CH2NH2
H
C
Tyramine
Tyrosine-decarboxylase
CH2
Tyrosine
CH2CH2NH2
OH
Tryptophan
CH
N H
H CH2
CH2
Lysine-decarboxylase
CH2
CH
CH2
NH2
COOH
H2N
NH2
NH2
CH2
CH
Cadaverine
CH2CH2NH2
Phenylalanine-decarboxylase
Phenylalanine
Tryptamine
COOH
NH2
N
Lysine
CH2CH2NH2
Triptophan-decarboxylase
OH CH2
Phenylethylamine
COOH
NH2
Arginine-decarboxylase
Arginine
H
N
CH2
C
NH
CH2
CH2
CH
COOH
H 2N
N
NH2
Agmatine
NH2
NH2
NH3 O
OH C
Ornithine
H
C NH2
Ornithine-decarboxylase C H2
C H2
H2N
H2N
N H
NH2
Spermidine
Putrescine
NH2
C NH2 H2
H2N
H N N H
NH2
Spermine
Figure 42.1 Amino acid precursors and amino acid decarboxylase enzymes in the formation of BA in seafood.
metabolism form some of them (putrescine, spermidine, and spermine) naturally. In the case of animals or plants, cadaverine and agmatine are also produced naturally. Some of these amines play important roles in many human and animal physiological functions. They are necessary for normal cell growth and play an important role in nucleic acid regulation and protein synthesis, and possibly also in the stabilization of membranes [1,2]. On the other hand, BA are produced by decarboxylation of free amino acids (FAA) from the action of microbial amino acid decarboxylase enzymes (Figures 42.1 and 42.2) [1,3–6], which is of particular interest in fish and fishery products as these are extremely perishable. Biogenic amines are important for two reasons. Firstly, the intake of foods containing high concentrations of BA can present a health hazard [4,6,7,8]; and secondly, they may have a role as indicators of quality and/or acceptability in some foods such as seafood [5,9–13].
42.2
Toxicity of Biogenic Amines in Seafood
The consumption of food containing high concentrations of BA has been associated with toxic effects and constitutes a potential health hazard. The compounds mainly implicated in these toxic effects are histamine and tyramine. Histamine is the most significant biogenic amine in fish and fish products. It is the main component in “scombroid poisoning” or “histamine poisoning” caused by consumption of fish containing high levels of histamine and/or other BA. This foodborne intoxication was originally called “scombroid poisoning” because it was primarily associated with the
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835
Procesing and storage conditions
Raw material Composition FAAs Protein content pH, etc. Microorganisms Enterobacteriaceae Pseudomonadaceae Vibrio Photobacterium Clostridium Lactic acid bacteria, etc.
Handing Canned Salted Fermented Smoked Packaging atmosphere High pressure Irradiation Time/temperature, etc.
Toxicity Free amino acids
+
Amino acid decarboxylases
Biogenic amines
Quality index
Figure 42.2
Factors affecting the formation of BA in seafood.
consumption of fish of the Scombridae and Scomberesocidae families such as tuna, mackerel, bonito, bluefish, and the like. These species contain high levels of free histidine in their muscle that is decarboxylased to histamine [7,8]. However, the term is misleading in that fish from several species of nonscombroid fish such as bluefish, herring, sardine, anchovy, pink salmon, redfish yellowtail, marlin, sailfish, amberjack, mahi-mahi, and the like, have often been implicated in cases of scombroid poisoning in different countries such as Japan, Great Britain, Australia, USA, Taiwan, and the like [7,8,14]. Non scombroid species also contain high levels of free histamine in their muscle tissue [15], hence this illness came to be called “histamine poisoning.” However, histamine does not appear to be the only causative agent of scombroid poisoning, since it is not itself toxic when taken orally [7,8]. Other amines such as putrescine and cadaverine are also implicated in this illness as they enhance the toxicity of histamine [3,16]. The most common symptoms of histamine poisoning involve the cardiovascular system; histamine has a vasodilating effect, producing low blood pressure, reddening of the skin, headaches, edemas, and rashes and a burning or peppery taste in the mouth typical of allergic reactions, diarrhea, and abdominal cramps [4,7,8,]. These symptoms usually disappear within several hours without medical intervention. More severe symptoms (e.g., respiratory distress, swelling of the tongue and throat, and blurred vision) can occur and require medical treatment with antihistamines. In many cases, these symptoms disappear too quickly for the poisoning to be naturally associated with the consumption of food containing BA, and in some cases they are difficult to distinguish from symptoms of other illnesses; for example, the symptoms of food allergy and histamine poisoning are similar. However, the toxicity of BA depends on the ability of individuals to metabolize normal dietary intakes of BAs [4,16]. This
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detoxification system includes specific enzymes such as monoamine oxidase (MAO; EC 1.4.3.4), diamine oxidase (DAO; EC 1.4.3.6), and polyamine oxidase (PAO; EC 1.5.3.11). However, the system is susceptible to some risk factors involving these enzymes, such as dietary consumption of foods containing high levels of BA (as in the case of spoiled or fermented fish), alcohol, gastrointestinal diseases, or in case of amino oxidase activity due to intake of certain drugs that can act as monoamine oxidase inhibitors (MAOI). It is important to note that large quantities of these drugs are consumed in Europe as antidepressants [17]. Alcohol and acetaldehyde seem to promote the transportation of these amines through the intestinal wall and augment their toxic potential [4,8]. The presence of other amines such as putrescine and cadaverine or spermidine, phenylethylamine, agmatine, and spermine, can also contribute to histamine toxicity. Phenylethylamine is a known inhibitor of the enzymes diamine oxidase and histamine methyl-transferase. Putrescine and cadaverine can inhibit the intestinal enzyme MAO, thus promoting the absorption and/or reducing the catabolism of this amine and enhancing its toxicity even when the histamine concentration is not so high [4]. Therefore, the effect of a given amount of histamine in seafood may be greater or lesser depending on the amount of enhancers that are present and on the efficiency of the individual’s detoxification system [7]. Moreover, certain BA, essentially putrescine and cadaverine, can react with nitrites to form toxic or carcinogenic compounds such as nitrosamines [5]. However, this is much less important in seafood products than in meat products.
42.3 Biogenic Amines as Quality Index in Seafood Although the control of BA in seafood has frequently been undertaken because of their involvement in food poisoning [4,8], the determination of these BA has also been proposed as a quality index to detect fish spoilage and/or defective preparation (Table 42.1) [9–13,18–25]. BAs are used as quality control indices because they undergo change during fish processing and storage. They are found at very low levels in fresh fish; their formation tends to increase during storage and is associated with bacterial spoilage [3,19,26]. The biogenic amine index of Mietz and Karmas [9] is the most widely used criterion for assessing spoilage of fish on the basis of biogenic amine (histamine, cadaverine, putrescine, spermidine, and spermine) contents (Table 42.1). The limit of fish acceptability for this quality index was set at 10. Later on, other authors [22] proposed an index calculated from the sum of the contents of histamine, tyramine, cadaverine, and putrescine, which showed good correlations with both time in storage and sensory evaluation, for assessment of tuna. Other individual BAs have also been used as quality indices in fish (Table 42.1). The Food and Drug Administration (FDA) [25] recommended not only histamine to indicate defect action levels, but also other scientific data to judge fish freshness, such as the presence of other BAs associated with fish decomposition. Some BAs such as agmatine and cadaverine have been used as fish freshness indicators (Table 42.1), reflecting the changes taking place in fish at the outset of storage prior to the point of onset of appreciable microbial spoilage [10,12,18,19]. Both agmatine and cadaverine have been associated with the autolytic changes responsible for loss of freshness that take place in fish muscle before the onset of microbial spoilage. Some authors suggest that this be defined as the beginning of the formation of some BAs and other spoilage compounds such as total volatile basic nitrogen or trimethylamine nitrogen, which have traditionally been used as indices to assess the spoilage of refrigerated fish [10,27]. In addition, BA can be useful as indicators of poor-quality raw material in preserved fish products, e.g., canned fish, because they are thermally stable compounds [21]. A good correlation has
Biogenic Amines in Seafood Products Table 42.1
◾
837
Quality Index of Biogenic Amines in Seafood
Seafood
Biogenic Amines
References
Rockfish, salmon, lobster, and shrimp
Quality index = (histamine + putrescine + cadaverine)/(1 + spermidine + spermine)
[9]
Hake
Cadaverine and agmatine
[10]
Hake in protective atmospheres
Cadaverine and agmatine
[11]
Squid
Agmatine
[12,19]
Salmonoid
Cadaverine
[13]
Trout
Putrescine, cadaverine, and histamine
[18]
Skipjack tuna
Putrescine and cadaverine
[20]
Canned tuna
Histamine
[21]
Tuna
Quality index = (histamine + tyramine + putrescine + cadaverine)
[22]
Cold smoked salmon
Cadaverine, histamine, putrescine, and tyramine
[23]
Skipjack and bigeye tuna
Cadaverine
[24]
Cadaverine + histamine Scombridae and Clupeidae families
Histamine
[25]
been found between sensory evaluation and levels of putrescine and cadaverine in canned skipjack tuna [20]. Jorgensen et al. [23] found that the BAs correlated well with sensory analysis in coldsmoked salmon, but they thought that the BAs are not necessarily the causal agents of spoilage off-flavors. BA have also been used as a quality index in fish and fish products treated with other technologies such as protective atmospheres (vacuum, modified atmosphere) [11]. Cadaverine and agmatine have been proposed as a control index for whole chilled hake, gutted and stored in controlled atmospheres [11].
42.4 Legal Limits of Biogenic Amines in Seafood The ingestion of BA can pose a risk to consumers, and therefore there have been various attempts to set limits for safe human consumption, particularly in the case of histamine. The European Union has set [25] a legal limit for histamine in certain fish species taking into account the technology used on them. The maximum permitted average concentration of histamine in fresh fish products of the Scombridae and Clupeidae families is 100 mg/kg and up to 400 mg/kg in cured products from the same families [25]. The FDA [15,28] has set this histamine level at 50 mg/ kg, above which it is considered a potential health hazard. In Australia the legal limit of histamine concentration within which it is regarded as safe for health is 200 mg/kg [29], and in
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South Africa the limit is 100 mg/kg [29]. However, as noted earlier, the relationship between the level of histamine and the toxicity of a fish sample is not clear and the presence of other enhancers of histamine toxicity needs to be taken into account.
42.5 Factors Influencing the Formation of Biogenic Amines in Seafood The formation of BA in seafood is caused mainly by microbial enzymatic decarboxylation of certain FAA (Figure 42.1). Such formation is affected by the substrate and the enzyme, as well as by factors influencing these such as the raw material, microorganisms affecting the amount and type of enzymes, and technological processing and storage conditions which directly affect the enzyme, the substrate, and the reaction medium (Figure 42.2) [3,5].
42.5.1 Raw Material Biogenic amines in the muscle. The raw material is decisive in determining the level and formation of BA in the final product. Initially, the muscle of freshly caught fish contains certain concentrations of BA, mainly polyamines (spermidine and spermine and in some cases putrescine and agmatine), which are characteristic of the fish species and type of muscle concerned [10,11,30]. In the case of tuna, for instance, initial concentrations of BA are generally higher in white than in red muscle. The exceptions are putrescine, levels of which are similar in both muscle types, and spermidine, levels of which are higher in red than in white muscle [30–34]. Wendakoon et al. [31] found higher biogenic amine concentrations in red than in white muscle of herring. Agmatine has only been found in trace amounts in freshly caught squid [19,35]. The BA with the highest concentrations reported in raw hake are putrescine and spermidine, with levels of 3.08 and 2.71 mg/kg, respectively. Initial concentrations of histamine, cadaverine, and agmatine are very low (less than 1 mg/kg) and tyramine concentrations are less than 1 mg/kg, or in some cases not detectable in hake [10,36]. Free amino acids in muscle. Fish raw material is the natural source of FAA from which BA are produced (Figures 42.1 and 42.2). The FAA levels in fish are high when compared to terrestrial animals since the primary function of these compounds in aquatic organisms is to serve as osmoregulators. The level and the type of these FAA in fish depend on the fish (family, species, etc.) and the muscle type (red or white). Fish contain varying proportions of red and white muscle, which perform different physiological functions in the live animal [21]. Members of the Scombridae and Scomberosocidae families contain higher concentrations of free histidine (which is decarboxylated to histamine) (Figure 42.1) than other fish species [30–34,36–40]. The two muscle types in tuna contain different levels of histidine, with higher concentrations in white than in red muscle [30]. In the case of other FAA biogenic amine precursors in tuna muscle, there are no significant differences in concentration between white and red muscle; in the case of lysine, red muscle contains higher concentrations than white muscle [30]. Differences in muscle composition affect the chemical changes taking place during storage, and consequently also in the formation of BA [30,38]. FAA in general are formed in fish as a result of muscle proteolysis, and decreases in FAA concentrations are associated with greater consumption of these amino acids by the microorganisms that use them as a growth substrate, and hence with greater spoilage [21], giving rise to a variety of compounds including BA [30,38]. Initial free
Biogenic Amines in Seafood Products
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amino acid contents have generally been observed to decrease during fish storage. Ruiz-Capillas and Moral [30] observed that histidine concentrations decreased significantly in white muscle but remained stable in red muscle. A correlation has been observed between the amino acid histidine and the biogenic amine histamine during storage of tuna, but only in the white muscle. Other authors have likewise found no correlation between FAA and BA [30,31,40,41]. However, Stede and Stockemer [42] found a relationship between the formation of BA and FAA in cod and haddock. Muscle as a reaction medium. The muscle is also the medium where decarboxylation takes place and is therefore necessary in determining essential factor in enzymatic activity: pH, ionic strength, substrate concentration, inhibitors, and the like [5]. The relatively low pH of sardine muscle possibly favors histidine decarboxylation [41].
42.5.2
Microorganisms
Biogenic amines are mainly produced by the decarboxylation of certain FAA by microbial enzymes [3,4,21,43]. It is important to identify which bacteria possess amino acid decarboxylase activity in order to estimate the risk of biogenic amine production in seafood and to prevent its build up in seafood products. Several Gram positive and Gram negative bacteria are implicated in the formation of BA. The most widely studied descarboxylase bacteria in the case of fish are the ones involved in histamine formation because of their implication in foodborne intoxication. In the case of fish, histamine decarboxylase enzymes are generally found in bacteria belonging to the Enterobacteriaceae family and to genera of Pseudomonas, Clostridium, Vibrio, and Photobacterium spp. [19,21,26,29,35,43–50]. Acinetobacter, Aeromonas, Staphylococcus, and Bacillus spp. have also been reported to have the potential to produce histamine by decarboxylase activity [51–54]. While histamine formation in raw fish products is caused mostly by Gram negative enteric bacteria, histamine in fermented products such as fish sauces is produced by Gram positive lactic acid bacteria (LAB). Tyrosine decarboxylase activity is more common among Gram positive bacteria [55]. Thapa et al. [56] showed that the LAB isolated from traditional processed fish products from the Eastern Himalayas did not produce BA. However, other authors have identified LAB as responsible for the production of some BA in squid kept in ice and in sterile cold-smoked salmon [35,57]. The variation observed in the decarboxylase ability of different species is extremely wide. Proteus vulgaris, P. mirabilis, Enterobacter aerogenes, Enterobacter cloacae, Serratia fonticola, S. liquefaciens, Citrobacter freundii, Vibrio alginolyticus, Acinetobacter lowffi, Plesiomonas shigelloides, Pseudomonas putida, P. fluorescens, Photobacterium phosphoreum, Photobacterium damselae, and Raoultella planticola have all been identified as histamine-formers in fish [35,47,49,50,51,58–65]. Differences in the ability to produce BA have even also been observed between strains of the same species. Morganella (Proteus) morganii, Klebsiella pneumoniae, and Hafnia alvei have all been isolated from fish blamed for scombroid poisoning [47,50]. Most of them are also involved in the production of other BA besides histamine, such as P. phosphoreum, which is also capable of producing agmatine and cadaverine [35,61]. The microorganism with the strongest histidine decarboxylase activity is M. morganii. The next most active appears to be K. pneumoniae, H. alvei, and some strains of E. cloacae and E. aerogenes, which produce more than 500 mg/kg of histamine, always depending on the storage conditions, especially temperature [3,41,47]. These biogenic amine-forming microorganisms may constitute part of the endogenous microbiota associated with the microflora of the fish or may be introduced by contamination during processing and storage of these fish. In freshly caught fish, bacterial contamination is located
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initially on the skin and gills; from there, these microorganisms invade the fish muscle and grow rapidly in response to a number of factors relating to processing and the storage conditions such as temperature, time, and the like.
42.5.3
Processing and the Storage Conditions of Seafood
In general, levels of BA increase during processing and storage of seafood, and this increase depends on many factors associated with processing and storage conditions. These factors affect the main elements implicated in biogenic amine formation, such as FAA or microbial enzyme decarboxylase (Figures 42.1 and 42.2) [4,21]. Therefore, in order to limit the formation of BA in seafood it is not enough to have suitable raw material; it is also necessary to optimize the processing and storage conditions. Biogenic amine formation in seafood products has been studied with reference to the processing and technological practices. Handling. The handling of fish is decisive for the formation of BA, and the result clearly depends on the kind of fish [40,66,67]. Haaland et al. [66] reported that the formation of cadaverine and putrescine was higher in ungutted mackerel than in fillets during the storage. FernándezSalguero and Mackei [67] reported that histamine, cadaverine, and putrescine were produced more rapidly in haddock fillets than in the whole gutted fish, and ungutted fish spoiled more rapidly than fillets. Refrigeration. Storage time and temperature are decisive factors in the production of BA. These factors affect microorganism growth and hence production of the amino acid descarboxylase enzyme necessary for the production of BA. Generally the levels of BA, except the polyamines spermidine and spermine (which usually remain constant), increase progressively throughout fish storage in ice. The increase is smaller if the fish had been efficiently cooled on ice to 0°C–2°C [9–13,31,32]. The behavior in each case is dependent on the fish species and the type of muscle. In tuna, pronounced increases of histamine have been recorded at days 18–25 in white muscle, but only on the last day of storage in the case of red muscle. In the case of cadaverine and agmatine, changes again occur later in red than in white tuna muscle [30]. Other authors [19,12] also observed that agmatine was the biogenic amine in the highest concentration in squid stored in ice. In high quality lean fish such as redfish, haddock, plaice, or hake stored in ice at 0°C–4°C, the levels of BA (histamine, putrescine, cadaverine, and tyramine) remained below 10 mg/kg. However, when storage time is prolonged, BA concentrations increase. Hake kept at 0°C–2°C for 33 days attained peak cadaverine concentrations (72.14 mg/kg) at the end of storage as compared with 13.47 mg/kg of agmatine and 7.12 mg/kg of putrescine, or even with levels of histamine and tyramine, which were less than 3.5 mg/kg by the end of storage [10]. These low levels of BA are the result of very low microbial growth and decarboxylase activity at low storage temperatures [21,42]. Increasing temperature during storage has also been studied as a factor in the formation of BA. Frank et al. [45] reported that biogenic amine formation is more closely related to the activity of mesophilic than of psychrotrophic bacteria, which could explain the fact that formation of those amines was more extensive at 20°C. Klausen and Huss [68] reported that M. morganii grows well at 15°C or above and that growth is greatly reduced at below 10°C. These authors confirmed that M. morganii could produce large amounts of histamine (600–1400 mg/kg) in mackerel stored at low temperature (0°C–5°C) following storage at higher temperatures (10°C–25°C). However, Vibrio and Photobacterium spp. are probably primarily responsible for histamine production at lower temperatures [26,54,64] since the optimum temperature for these bacteria is below 10°C. Ababouch et al. [41] showed that the rate of histamine formation in sardines was greater at ambient temperature than in ice storage. After 24 h of storage at ambient temperature, histamine, cadaverine, and
Biogenic Amines in Seafood Products
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841
putrescine reached levels of 2350, 1050, and 300 mg/kg, respectively. Similar results have been reported by other authors in various fish species [11,13,22,31,51,67,69,70]. Veciana-Nogués et al. [22] found similar profiles for evolution of BA in tuna at three storage temperatures (0°C, 8°C, and 20°C). Levels of the polyamines spermidine and spermine generally remain constant during storage and are independent of temperature. This is because the formation of these amines is scarcely affected by spoilage [22,10]. On the other hand, decreases in histamine in advanced fish spoilage have been reported in association with the growth of microorganisms presenting histaminolytic activity [8,22,41]. Veciana-Nogués et al. [22] reported considerable decreases in histamine and cadaverine after 3 days of storage at 20°C, at which time of spoilage was far advanced, whereas no decreases in histamine, putrescine, cadaverine, and tyramine were observed when samples were stored at 0°C or 8°C. Ababouch et al. [41] observed that for longer periods of storage of sardine at ambient temperature (over 24 h), histamine levels began to decrease, indicating proliferation of bacteria presenting histaminase activity. Therefore, the most effective way to prevent BA formation in fish is to keep it at a low temperature throughout processing and storage. It is commonly accepted that storage at or near 0°C limits the formation of amines by substantially retarding bacterial growth and the activity of decarboxylating enzymes [21]. In general, frozen storage does not affect microbial growth or enzymatic activity and therefore does not affect the production of biogenic amine. However, some authors [47,40] have isolated histamine-producing bacteria from frozen fish. The presence of amines in frozen fish is indicative of the characteristics of the raw material prior to thawing [21]. Canning. Biogenic amines are stable to thermal processing, and therefore the presence of BA in canned products indicates that the fish has been microbially spoiled before heating [21,6]. Fernández-Salguero and Mackie [71] reported very low levels (less than 2 mg/kg) of BA in canned tuna, mackerel, and sardine. Fermentation. Fermented fish products are particularly rich in histamine, followed by phenylethylamine [72]. Stages in fermentation are sometimes carried out without adequate temperature control; and higher fermentation temperatures increase biogenic amine concentrations, especially histamine. A considerable increase in putrescine, histamine, and tyramine contents of fermented sardines has been associated with increased concentrations of halotolerant and halophilic histamine-forming bacteria (Staphylococcus, Micrococcus, Vibrio, and Pseudomonas). Salting. Biogenic amine contents of salted fish generally vary considerably [73]. The average concentration of each biogenic amine in salted mackerel sold in retail markets and supermarkets in Taiwan was less than 3 mg/100 g [74]. Higher levels of histamine have also been detected in some samples, and minute amounts of spermidine, phenylethylamine, agmatine, and spermine have been detected in some tested mackerel samples. Rodríguez-Jerez et al. [54] assessed histidine decarboxylase activity and production of putrescine and cadaverine bacterial isolates from ripened semipreserved Spanish anchovies. They found the highest levels of histidine decarboxylase activity in M. morganii, which is also a producer of putrescine, and cadaverine. Other species such as S. epidermidis, S. xylosus, K. oxytoca, E. cloacae, Pseudomonas cepaciae, and Bacillus spp. were also implicated in the production of these BA. Other authors have identified halotolerant Staphylococcus spp., Vibrio spp., and Pseudomonas III/IV-NH as histamine-formers in salted fish [52–54, 75, 76]. Tsai et al. [74] identified Pantoea sp., Pantoea agglomerans, and E. cloacae as histamine-producing bacteria in salted mackerel. Smoking. Biogenic amines have also been studied in smoked fish. The smoking process usually commences with a drying phase. This phase should be kept short, as prolonged exposure to
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ambient temperature may lead to unwanted microbiological growth and to formation of histamine in susceptible species. Shalaby [6] observed levels of putrescine, cadaverine, and spermine between 1–16 mg/kg and low levels of tyramine, spermidine, and histamine (1–8 mg/kg) in smoked herring. Tryptamine and phenylethylamine were not detected in any of the samples analyzed. Protective atmospheres. This technology, used as a coadjuvant to chilled storage, affects biogenic amine formation in various fish species (tuna, hake cod, sardine, salmon, etc.) [11,26,30, 33,36,39,77,78]. In general, the application of protective atmospheres reduces the production of BA except spermidine and spermine. This effect is mainly due to the way that the mixture of gases in the atmosphere acts on microbial growth and hence on the amino acid descarboxylase enzyme (Figure 42.2). The effect of this technology also depends on the kind of atmosphere used (controlled, modified, etc.), the type of biogenic amine and the fish species [11,26,30,33,36,39,77–79]. Ruiz-Capillas and Moral [30] showed that a controlled atmosphere with a gas mix containing a high concentration of CO2 was most effective in reducing BA in bigeye tuna. However, high levels of CO2 (60%) in the controlled atmosphere were not sufficient to inhibit the production of BA in gutted hake kept in refrigeration, while the high O2 concentration (40%) in the controlled atmosphere had an inhibiting effect on the production of BA in hake [11]. The combination of different protective atmospheres has also been found to be effective in reducing BA levels. Hake bulk-stored in a controlled atmosphere (40% CO2:40% O2:20% N2) for the first 12 days and then packed in trays with modified atmospheres and the same mixture of gases also exhibited lower levels of BA, except for agmatine, throughout storage [36]. However, these protective atmospheres are only effective if the product is kept in refrigerated storage. An appropriate combination of low temperature and atmosphere, then, potentates the inhibiting effect of CO2, retarding the growth of spoilage microorganisms in this fish [11,26,33,77,80]. Emborg et al. [26] showed that the spoilage of modified atmosphere packaged (MAP) cod is caused by growth and metabolism of the CO2-resistant bacterium P. phosphoreum. This specific spoilage organism grows to high levels in different MAP fish but is inactivated by freezing at—20°C, and the shelf life of thawed MAP cod can be substantially prolonged in this way [64]. Jorgensen et al. [23] also found P. phosphoreum to be primarily responsible for the production of BA in vacuum-packed cold-smoked salmon, where agmatine, cadaverine, histamine, and tyramine were formed at 5°C. High-pressure treatment. There has hardly been any research into the effect of high-pressure treatment on the formation and evolution of BA in seafoods. Paarup et al. [35] observed that the onset of formation of agmatine and other BA was delayed by increasing pressure in vacuum-packed squid. The application of moderate pressures (150–200 MPa) reduces the rate of agmatine formation, whereas higher pressures (300 and 400 MPa) delay the onset of production of this amine. Pressurization at 400 MPa inhibits histamine formation and keeps putrescine formation low, while higher concentrations of tyramine have been detected in squid pressurized at 300 and 400 MPa. Fujii et al. [81] also reported absence of histamine in minced mackerel meat pressurized at 200 MPa during chilled storage. It has been suggested that P. phosphoreum is responsible for biogenic amine production, mainly agmatine and histamine, in pressurized squid, while Carnobacterium sp. has been identified as responsible for the production of tyramine [35]. Irradiation. The effect of irradiation on the formation of BA has been studied in Atlantic horse mackerel during chilled storage [82]. Histamine in the irradiated mackerel (even at 1 kg) was undetectable at the end of 23 days when the fish had spoiled. This effect was associated with a majority of Gram negative anaerobic bacteria, since around 10%–18% was Gram positive bacteria in the irradiated samples.
Biogenic Amines in Seafood Products
42.6
◾
843
Determination of Biogenic Amines in Seafood
Considering the importance of BA in fish and fish products for legal, toxicological, and quality purposes, it is essential to have accurate analytical methods. Biogenic amines in different foods, including fish and fish products, have traditionally been determined by means of standard chromatographic techniques such as thin layer chromatography, gas chromatography, capillary electrophoresis, flow injection analysis, and high-performance liquid chromatography (HPLC) [83,84]. Positive confirmation using mass spectrometry after either HPLC [85] or gas chromatographic separation [86] has also been reported for other food. Enzyme-based amperometric biosensors using histamine oxidase have also been developed for the determination of histamine [87]. The determination of BA from the fish matrix frequently presents problems for a variety of reasons. The major BA in seafood (normally not less than nine), which are present in a wide range of concentrations, are usually determined simultaneously. Moreover, this kind of sample is very complex, containing high protein levels and a wide variety of fat contents (0.5%–30%). For these reasons, most methods for determining BA frequently involve preliminary steps to extract these compounds from the fish matrix and subsequent separation and quantification steps.
42.6.1
Extraction Process
Sample preparation, or extraction of BA from the fish matrix, is a crucial step in the analysis. Many different solvents have been used to extract BAs from fish and fish products, including hydrochloric acid, trichloroacetic acid (TCA), perchloric acid (PCA), and other organic solvents such as methanol, dichloromethane, acetone, and acetonitrile [88,89]. Of these solvents the most commonly used are PCA and TCA because of their effect on protein precipitation, which makes them highly effective biogenic amine extractors for fish and fish products. Extraction commonly involves a first step where 5–15 g of fish muscle is homogenized with 10–50 mL of the acids at different concentrations (5%, 6%, and 7.5%); this homogenate is then centrifuged and the extract filtered [10,22]. The precipitate is washed with more acid, centrifuged, and filtered again. The acid extract made with TCA can also be used for other chemical determinations such as trimethylamine, volatile base nitrogen, ammonia, urea, and some FAA [10,90]. In some cases, this first step in the determination of BA in fish and fish products may include purification or cleanup of the final extract based on ion exchange resins and a solid phase. HLPC normally have a small clean-separation ion exchange (guard) column which is set up online prior to the separation column used to determine BA [10,91].
42.6.2
Determination Process
Of the available methods for determination of BA in seafood, the most widely used and frequently reported for the separation and quantification of BA in seafood are chromatographic procedures, especially HPLC with an ion exchange column or a reverse-phase column using ion pairs to separate BA. This procedure offers high resolution, sensitivity, and versatility. Moreover, the sample treatments are generally simple. It is well known that BAs respond poorly to detection systems due to low volatility and lack of chromophores. Many BA occurring in food exhibit neither satisfactory absorption nor significant fluorescence properties. A chemical derivatization is therefore usually performed to increase their sensitivity. Covalent labeling with chromophores or fluorophores normally greatly improves detection sensitivity and detection limits. Thanks to UV–VIS and fluorescence detection [84].
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There are many known derivatization reagents. Among them dansyl and dabsyl chloride, benzoyl chloride, fluoresceine, 9-fluorenylmethyl chloroformate, o-phthalaldehyde (OPA), and naphthalene-2,3-dicarboxaldehyde [84]. Of these, OPA and dansyl chloride are the most widely used. Dansyl chloride forms stable compounds after reaction with both primary and secondary amino groups and the products are more stable than those formed using OPA. This last reagent reacts rapidly with primary amines in the presence of a reducing agent such as N-acetylcysteine, 2-mercaptoethanol, or thiofluor, which is a stable solid substitute for 2-mercaptoethanol during the preparation of the OPA reagent. It forms a more stable and longer-lasting fluorophore with OPA than does 2-mercaptoethanol while possessing the same fluorescence properties. Under basic conditions (pH > 9) and at ambient temperature, the reaction is generally complete in 1–30 s. The products of this reaction, 1-alkyl-2-alkylthio-substituted isoindoles, exhibit optimal excitation at 330 nm and maximum emission at 465 nm [10,91,92]. Moreover, OPAs are faster and much simpler for purposes of sample pretreatment, which can be fully automated using an autosampler and is more sensitive because florescence detection is used rather than spectrophotometric detection as in the case of dansyl chloride [91]. On the other hand, the OPA derivative is unstable and the fluorescence intensity diminishes quickly, especially in alkaline media. This problem requires strict control of reaction times, but it can be solved using postcolumn derivatization or automatic precolumn derivatization. Biogenic amine derivatization may be performed before (precolumn), during (on-column) or after (postcolumn) chromatographic separation [84,85]. Automatic online postcolumn derivatization is the most common procedure as it offers a number of advantages: it entails less handling, thus reducing the likelihood of interferences or artefacts in the sample, the analysis time is shorter and derivatization occurs at the same time, thus enhancing the reproducibility and sensitivity of the analysis [91]. However prederivatization involves more sample preparation steps, which can produce problems in the analysis later on. In addition, postderivatization is usually performed with an internal standard. Postderivatization for the quantification of BA entails comparison with an external calibration standard composed of the different BA. Dansyl chloride is normally used for precolumn derivatization with reverse-phase separation coupled with UV detection [33,80] and OPAs are used in ion-pair reverse-phase HPLC with post and precolumn derivatization coupled with fluorescence detection [10,22,70,91,92]. Flow injection analysis (FIA) is a new, fast, and simple method with low operating costs for the determination of BAs in seafood. FIA coupled with automated OPA derivatization has been used for histamine determination in canned tuna [93,94]. In the FIA method, all reagents are added automatically. Flow rates and OPA reactor volumes afford the required pH and reaction timing. This system employs three channels, using an anion-exchange column to eliminate sample matrix interferences. Selectivity for histamine versus interfering compounds appears to be based on differences in the reaction rates with OPA since histamine reacts quicker than the remainder compounds. Interfering substances such as ascorbate are fully electrooxidized, and the signals are removed upstream of the detector. This system is based on the AOAC method for determining histamine in seafood [95]. Most recently, an FIA method has been reported which uses an enzyme electrode to detect histamine. Takagi and Shikata [96] developed a new FIA method using a histamine dehydrogenase-based electrode, which they used to determine histamine in fish samples. Histamine dehydrogenase is immobilized in an osmium-derivatized redox polymer, poly(1-vinylimidazole) complexed with Os(4,4¢-dimethylbipyridine)2Cl2 (PVI-dmeOs) film on a glassy carbon electrode. This electrode exhibits high selectivity to histamine and is not sensitive to other primary amines including common BA, putrescine, cadaverine, and tyramine. This is an effective method for rapid
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and efficient laboratory histamine testing, particularly in laboratories analyzing large numbers of samples. Not only does this allow more rapid analysis without sample cleanup, but also operator dependence is reduced by automation and the instrument completes each determination step in <1 min [96]. An enzymatic method has also been developed for the determination of histamine in fish. This one is based on the reaction of diaminoxidase (DAO) with histamine to yield hydrogen peroxide which, coupled with horseradish peroxidase, converts a reduced dye to its oxidized form. The color change is used to quantify the histamine in the sample [97]. However, this technique has a drawback in the low specificity of the enzyme, which can react with other BA such as putrescine and tyramine, and as in the case of FIA, only one biogenic amine can be determined at a time. More recently, this enzymatic reaction has been used to develop a biosensor with integrated pulsed amperometric detection for determination of BA in salted anchovy samples. The probe is based on a platinum electrode, which senses the hydrogen peroxide produced by the reaction catalyzed by the enzyme diamine oxidase (DAO). This is obtained from different sources (microorganisms, plants, and animal tissue) with different enzymatic activities such as seeds of cicer, porcine kidney, pea lentil, and the like. The DAO is immobilized on the electrode surface. The conditions selected were immobilization of the enzyme on a nylon-net membrane using glutaraldehyde as cross-linking agent and phosphate buffer at pH 8.0. Carelli et al. [98] also developed an amperometric biosensor for determination of total biogenic amine content using commercial diamino oxidase (from porcine kidney) as the biocomponent, entrapped by glutaraldehyde onto an electrosynthesized bilayer film. In order to minimize both fouling and interference caused by direct electrochemical oxidation of both the analytes (i.e., BA) and the common interferents usually present in food products, the performances of Pt and Au electrodes and of several electroproduced antiinterferent mono- and bilayer films were tested. Although the commercial DAO presented very low activity, the biosensor displayed high response sensitivity in flow experiments, short response times, a good linear response, and low detection limits. The antiinterference characteristics are so good that the biosensor can be used in screening analysis of seafood products [98]. A capillary electrophoresis method with conductometric detection of BA has been reported [99]. Clear separation of six BAs (cadaverine, putrescine, agmatine, histamine, tryptamine, and tyramine) from other components of acidic sample extract was achieved within 10 min. The advantages of this capillary electrophoresis method include low laboriousness (no derivatization step or sample cleaning, dilution or acidic extraction, and filtration only), adequate sensitivity, low running costs (no separation column, only an empty capillary), and speed of analysis and environmental friendliness (small amounts of water-based diluted electrolyte for analysis). The disadvantages are nonselectivity of conductometric detection and higher detection limits in the case of salty samples [99]. Interest in “portable” procedures for analysis of BA that would be capable of rapidly screening fishery products has led to the development of commercial test kits, which have been proposed for histamine determination and for hazard analysis and critical control point (HACCP) applications. A number of these commercial test kits have been compared with the AOAC method by analyzing samples of tuna and mahi-mahi. These kits are based on an enzymatic immunoassay [100,101]. Take for example the ALERT® kit and the Veratox histamine kit. Both kits are direct competitive enzyme-linked immunosorbent assays (ELISA). They are used for quantitative analysis of histamine in scombroid fish species such as tuna, bluefish, and mahi-mahi. Histamine is extracted from a sample using a quick water extraction process. Free histamine in the sample and controls competes with enzyme-labeled histamine (conjugate) for the antibody-binding sites. After a wash step, the substrate reacts with the bound enzyme conjugate to produce a blue color. A microwell
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reader is used to yield optical densities. Control optical densities are used to form a standard curve, and sample optical densities are plotted against the curve to calculate the exact concentration of histamine. The results are read using a microwell reader at 450–650 nm. The method offers simplicity, rapidity, and relatively low cost in comparison with other methodologies such as HPLC, and no previous derivatization is required [101].
Acknowledgments This research was also supported under projects AGL2003-00454, AGL2007-61038/ALI of the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I), the Consolider CSD2007–00016, Ministerio de Ciencia y Tecnología.
References 1. Smith, T.A. Amines in food, Food Chem., 6, 169, 1980. 2. Bardócz, S. et al. The importance of dietary polyamines in cell regeneration and growth, Brit. J. Nutr., 73, 819, 1995. 3. Halász, A. et al. Biogenic amines and their production by microorganisms in food, Trends Food Sci. Tech., 5, 42, 1994. 4. Bardócz, S. Polyamines in food and their consequences for food quality and human health, Trends Food Sci. Tech., 6, 341, 1995. 5. Ruiz-Capillas, C. and Jiménez-Colmenero, F. Biogenic amines in meat and meat products, Crit. Rev. Food Sci., 44, 489, 2004. 6. Shalaby, A.R. Significance of biogenic amines to food safety and human health, Food Res. Int., 29, 675, 1996. 7. Lehane, L. and Olley, J. Histamine fish poisoning revisited, Int. Food. Microbiol., 58, 1, 2000. 8. Taylor, S.L. Histamine food poisoning: Toxicology and clinical aspects, Crit. Rev. Toxicol., 17, 91, 1986. 9. Mietz, J.L. and Karmas, E. Polyamine and histamine content of rockfish, salmon, lobster and shrimp as an indicator of decomposition, J. Assoc. Offic. Anal. Chem., 61, 139, 1977. 10. Ruiz-Capillas, C. and Moral, A. Production of biogenic amines and their potential use as quality control indices for hake (Merluccius merluccius L.) stored in ice, J. Food Sci., 66, 1030, 2001. 11. Ruiz-Capillas, C. and Moral, A. Effect of controlled atmospheres enriched with O2 in formation of biogenic amines in chilled hake (Merluccius merluccius L.), Eur. Food Res. Technol., 212, 546, 2001. 12. Yamanaka, H., Shiomi, K., and Kikuchi, T. Agmatine as a potential index for freshness of common squid, J. Food Sci., 52, 936, 1987. 13. Yamanaka, H., Shiomi, K., and Kikuchi, T. Cadaverine as a potential index for decomposition of salmonoid fishes, J. Food Hyg. Soc. Jpn., 30, 170, 1989. 14. Gessner, B., Hokama, Y., and Isto, S. Scombrotoxicosis-like illness following the ingestion of smoked salmon that demonstrated low histamine levels and high toxicity on mouse bioassay, Clin. Infect. Dis., 23, 1316, 1996. 15. Food and Drug Administration. Fish and fisheries products hazards and controls guidance. 3rd ed. Scombrotoxin (histamine) formation: A chemical hazard, Available at http://www.cfsan.fda. gov/~comm/haccp4g.html (accessed 5 November, 2008). 16. Rice, S.L., Eitenmiller, R.R., and Koehler, P.E. Biologically active amines in food: A review, J. Milk Food Technol., 39, 353, 1976. 17. Sattler, J. et al. Food induced histaminosis as an epdemiological problem: Plasma histamine elevation and haemodynamic alterations after oral histamine administration and blockade of diamine oxidase (DAO), Agents Actions, 23, 361, 1988.
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18. Dawood, A.A. et al. The occurrence of non-volatile amines in chilled-stored rainbow trout (Salmo irideus), Food Chem., 27, 33, 1988. 19. Paarup, T. et al. Sensory, chemical and bacteriological changes during storage of iced squid (Todaropsis eblanae), J. Appl. Microbiol., 92, 941, 2002. 20. Sims, G.G., Farn, G., and York, R.Y. Quality index for tuna correlation of sensory attributes with chemical indices, J. Food Sci., 57, 1112, 1992. 21. Huss, H.H. Quality and quality changes in fresh fish, FAO. Fisheries Tecnical., Paper. 348, 1995. 22. Veciana-Nogués, M.T., Mariné Font, A., and Vidal Carou, M.C. Biogenic amines as hygienic quality indicators of tuna. Relationships with microbial counts, ATPrelated compounds, volatile and organoleptic changes, J. Agric. Food Chem., 45, 2036, 1997. 23. Jorgensen, L.V., Dalgaard, P., and Huss, H.H. Multiple compound quality index for cold-smoked salmon (Salmo salar) developed by multivariate regression of biogenic amines and pH, J. Agric. Food Chem., 48, 2448, 2000. 24. Rossi, S. et al. Biogenic amines formation in bigeye tuna steaks and whole skipjack tuna, J. Food Sci., 67, 2056, 2002. 25. Commission Regulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs, 2005. 26. Emborg, J. et al. Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere packed salmon (Salmo salar) at 2°C, J. Appl. Microbiol., 92, 790, 2002. 27. Ruiz-Capillas, C. and Moral, A. Correlation between biochemical and sensory quality indices in hake stored in ice. Food Res. Int., 34, 441, 2001. 28. Food and Drug Administration (FDA). 2001, In Fish and Fishery Products Hazards and Controls Guidance, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC, 83–102. 29. Auerswald, L., Morren, C., and Lopata, AL. Histamine levels in seventeen species of fresh and processed South African seafood, Food Chem., 98, 231, 2006. 30. Ruiz-Capillas, C. and Moral, A. Free amino acids and biogenic amines in red and white muscle of tuna stored in controlled atmospheres, Amino Acids., 26, 125, 2003. 31. Wendakoon, C.N., Murata, M., and Sakaguchi, M. Comparison of non-volatile amine formation between the dark and white muscles of mackerel during storage, Nippon Suisan Gakk., 56, 809, 1990. 32. Watanabe, H., Yamanaka, H., and Yamanakawa, H. Post mortem biochemical changes in the muscle of disk abalone during storage, Nippon Suisan Gakk., 58, 2081, 1992. 33. López-Gálvez, D., De la Hoz, L., and Ordóñez, J.A. Effect of carbon dioxide and oxygen enriched atmospheres on microbiological and chemical changes in refrigerated tuna (Thunnus alalunga) steaks, J. Agric. Food Chem., 30, 435, 1995. 34. Du, W.X. et al. Development of biogenic amines in yellowfin tuna (Thunnus albacore): Effect of storage and correlation with decarboxylase-positive bacterial flora, J. Food Sci., 67, 292, 2002. 35. Paarup, T. et al. Sensory, chemical and bacteriological changes in vacuum-packed pressurised squid mantle (Todaropsis eblanae) stored at 4°C, Int. J. Food Microbiol., 74, 1, 2002. 36. Ruiz-Capillas, C., and Moral, A. Formation of biogenic amines in bulk stored chilled hake (Merluccius merluccius, L.) packed under atmospheres, J. Food Prot., 64, 1045, 2001. 37. Wei, C.I. et al. Bacterial growth and histmamine production on vacuum packaged tuna, J. Food Sci., 55, 59, 1990. 38. Ochiai, Y., Aleman-Polo, J.M., and Hashimoto, K. Postmortem diffusion of taurine and free histidine between ordinary and dark muscle of mackerel during ice storage, Nippon Suisan Gakk., 56, 1017, 1990. 39. Ruiz-Capillas, C., and Moral, A. Effect of controlled and modified atmospheres on the production of biogenic amines and free amino acids during storage of hake, Eur. Food Res. Technol. A., 214, 476, 2002. 40. Mendes, R., Goncalves, A., and Nunes, M.L. Changes in free amino acids and biogenic amines during ripening of fresh and frozen sardine, J. Food Biochem., 23, 295, 1999.
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41. Ababouch, L.H. et al. Quality changes in sardines (Sardina pilchardus) stored in ice and at ambient temperatures, Food Microbiol., 13, 123, 1996. 42. Stede, M. and Stockemer, J. Biogenic amines in marine fish, Lebensm Wiss. Technol., 19, 283, 1986. 43. Silla, M.H. Biogenic amines: Their importance in foods, Int. J. Food Microbiol., 29, 213, 1996. 44. Gram, L. Fish spoilage bacteria–problems and solutions, Curr. Opin. Biotechnol., 13, 262, 2002. 45. Frank, H.A. et al. Identification and decarboxylase activities of bacteria isolated from decomposed mahimahi (Coryphaena hippurus) after incubation at 0 and 32°C, Int. J. Food Microbiol., 2, 331, 1985. 46. Taylor, S.L. and Sumner, S.S. (eds.), Determination of histamine, putrescine, and cadaverine, In Seafood Quality Determination, Kramer & Liston, Amsterdam, the Netherlands, 1986, 235. 47. Taylor, S.L. and Speckhard, M.W. Inhibition of bacteria histamine production by sorbate and other antimicrobial agents, J. Food Prot., 47, 508, 1984. 48. Arnold, S.H., Price, R.J., and Browen, W.D. Histamine formation by bacteria isolated from skipjack tuna Katsuwonus plamis, Bull. Jpn. Soc. Sci. Fish., 46, 991, 1980. 49. López-Sabater, E.I. et al. Evaluation of histidine decarboxylase activity of bacteria isolated from sardine (Sardina pilchardus) by an enzymatic method, Lett. Appl. Microbiol., 19, 70, 1994. 50. Eitenmiller, R.R. et al. Production of histidine decarboxylase and histamine by Proteus morganii, J. Food Prot., 44, 815, 1981. 51. Middlebrooks, B.L. et al. Effects of storage time and temperature on the microflora and amine development in Spanish mackerel (Scomberomorus maculatus), J. Food Sci., 53, 1024, 1988. 52. Yatsunami, K. and Echigo, T. Changes in the number of halotolerant histamine-forming bacteria and contents of non-volatile amines in sardine meat with addition of NaCl, Bull. Jpn. Soc. Sci. Fish., 59, 123, 1993. 53. Hernández-Herrero, M.M. et al. Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies, J. Food Prot., 62, 509, 1999. 54. Rodríguez-Jerez, J.J. et al. Histamine, cadaverine and putrescine forming bacteria from ripened Spanish semipreserved anchovies, J. Food Sci., 59, 998, 1994. 55. Bover-Cid, S. and Holzapfel, W.H. Improved screening procedure for biogenic amine production by lactic acid bacteria, Int. J. Food Microbiol., 53, 33, 1999. 56. Thapa, N., Pal, J., and Tamang, J.P. Phenotypic identification and technological properties of lactic acid bacteria isolated from traditionally processed fish products of the Eastern Himalayas, Int. J. Food Microbiol., 107, 33, 2006. 57. Jorgensen, L.V., Huss, H.H., and Dalgaard, P. The effect of biogenic amine production by single bacterial cultures and metabiosis in cold-smoked salmon, J. Appl. Microbiol., 89, 920, 2000. 58. López-Sabater, E.I. et al. Sensory quality and histamine formation during controlled decomposition of tuna (Thunnus thynnus), J. Food Prot., 59, 167, 1996. 59. Tsai et al. Histamine related hygienic qualities and bacteria found in popular commercial scombroid fish fillets in Taiwan, J. Food Prot., 67, 407, 2004. 60. Yoshinaga, D.H. and Frank, H.A. Histamine-producing bacteria in decomposing skipjack tuna (Katsuwonus pelamia), Appl. Environ. Microbiol., 44, 447, 1982. 61. Okuzumi, M. et al. Photobacterium histaminum sp. nov., a histamine-producing marine bacterium, Int. J. Syst. Bacteriol., 44, 631, 1994. 62. Ryser, E.T., Marth, E.H., and Taylor, S.L. Histamine production by psychrotrophic pseudomonas isolated from tuna fish, J. Food Prot., 47, 378, 1984. 63. Kanki, M. et al. Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers, Appl. Environ. Microbiol., 68, 3462, 2002. 64. Dalgaard, P. et al. Importance of Photobacterium phosphoreum in relation to spoilage of modified atmosphere packed fish products, Lett. Appl. Microbiol., 24, 373, 1997. 65. Kanki, M. et al. Photobacterium phosphoreum caused a histamine fish poisoning incident, Int. J. Food Microbiol., 92, 79, 2004. 66. Haaland, H., Arnesen, E., and Njaa, L.R. Amino-acid-composition of whole mackerel (Scomber scombrus) stored anaerobically at 20°C and at 2°C, Int. J. Food Sci. Technol., 25, 82, 1990.
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67. Fernández-Salguero, J. and Mackei, I.M. Comparative rates of spoilage of fillets and whole fish during storage of haddock (Melanogammus aeglefinus) and herring (Clupea arengus) as determined by the formation of non-volatile and volatile amines, Int. J. Food Sci. Technol., 22, 385, 1987. 68. Klausen, N.K. and Huss, H.H. Rapid method for detection of histamine-producing bacteria, Int. J. Food Microbiol., 5, 137, 1987. 69. Nagayama, T. et al. Non-volatile amines formation and decomposition in abusively stored fishes and shelfishes, J. Food Hyg. Soc. Jpn., 31, 362, 1985. 70. Baixas-Nogueras, S. et al. Volatiles and nonvolatile amines in Mediterranean Hake as a function of their storage temperature, J. Food Sci., 66, 83, 2001. 71. Fernández-Salguero, J. and Mackie, I.M. Technical note: Preliminary survey of the content of histamine and other higher amines in some samples of Spanish canned fish, Int. J. Food Sci. Tech., 22, 409, 1987. 72. Fardiaz, D. and Markakis, P. Amine in fermented fish paste, J. Food Sci., 44, 1562, 1979. 73. Lee, H. et al. Histamine and other biogenic amines and bacterial isolation in retail canned anchovies, J. Food Sci., 70, C145, 2005. 74. Tsai, Y.H. et al. Occurrence of histamine and histamine-forming bacteria in salted mackerel in Taiwan, Food Microbiol., 22, 461, 2005. 75. Yatsunami, K. and Echigo, T. Isolation of salt tolerant histamine-forming bacteria from commercial rice-bran pickle sardine, Bull. Jpn. Soc. Sci. Fish., 57, 1723, 1991. 76. Yatsunami, K. and Echigo, T. Occurrence of halotolerant and halophili histamine-forming bacteria in red meat fish products, Bull. Jpn. Soc. Sci. Fish., 58, 515, 1992. 77. Emborg, J., Laursen, B.G., and Dalgaard, P. Significant histamine formation in tuna (Thunnus albacares) at 2 C—effect of vacuum- and modified atmosphere-packaging on psychrotolerant bacteria, Int. J. Food Microbiol., 101, 263, 2005. 78. Suzuki, S., Noda, J., and Takama, K. Growth and polyamine production of Alteromonas ssp. in fish meat extracts under modified atmosphere, Bull. Fac. Fish. Hokkaido Univ., 41, 213, 1990. 79. Randell, K. et al. Quality of whole gutted salmon in various bulk packages, J. Food Quality, 22, 483, 1999. 80. Lannelongue M. et al. Microbiological and chemical changes during storage of swordfish (Xiphias gladius) steaks in retail packages containing CO2-enriched atmospheres, J. Food Prot., 45, 1197, 1982. 81. Fujii, T. et al. Changes in freshness indexes and bacterial flora during storage of pressurized mackerel, J. Food Hyg. Soc. Jpn., 35, 195, 1994. 82. Mendes, R. et al. Effect of low-dose irradiation and refrigeration on the microflora, sensory characteristics and biogenic amines of Atlantic horse mackerel (Trachurus trachurus), Eur. Food Res. Technol., 221, 329, 2005. 83. Teti, D., Visalli, M., and McNair, H. Analysis of polyamines as markers of (patho) physiological conditions, J. Chromatogr. B., 781, 107, 2002. 84. Önal, A. A review: Current analytical methods for the determination of biogenic amines in foods, Food Chem., 103, 1475, 2007. 85. Gosetti, F. et al. High performance liquid chromatography/tandem mass spectrometry determination of biogenic amines in typical Piedmont cheeses, J. Chromatogr. A., 1149, 151, 2007. 86. Awan, M.A., Fleet, I., and Thomas, C.L.P. Determination of biogenic diamines with a vaporization derivatisation approach using solid-phase microextraction gas chromatography-mass spectrometry, Food Chem., 111, 462, 2008. 87. Draisci, R. et al. Determination of biogenic amines with an electrochemical biosensor and its application to salted anchovies, Food Chem., 62, 225, 1998. 88. Lapa-Guimarães, J. and Pickova, J. New solvent systems for thin-layer chromatographic determination of nine biogenic amines in fish and squid, J. Chrom. A., 1045, 223, 2004. 89. Moret, S. and Conte, L.S. High-performance liquid chromatographic evaluation of biogenic amines in foods—An analysis of different methods of sample preparation in relation to food characteristics, J. Chrom. A., 729, 363, 1996.
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90. Ruiz-Capillas, C. and Horner, W.F.A. Determination of trimethylamine nitrogen and total volatile basic nitrogen in fresh fish by flow injection analysis, J. Sci. Food Agric., 79, 1982, 1999. 91. Tracy, M.L., Pickering M.V., and Verhulst, T. Cation exchange analysis of foods and beverages for biogenic amines, Food Test. Anal., 1, 48, 1995. 92. Tapia-Salazar, M., Smith, T.K., and Harris, A. High performance liquid chromatographic method for detection of biogenic amines in feedstuffs, complete feeds and animal tissues, J. Agric. Food Chem., 48, 1708, 2000. 93. Gutiérrez, C., Rubio, S., Gómez-Hens, A., and Valcárcel, M. Determination of histamine by derivative synchronous fluorescence spectrometry, Anal. Chem., 59, 769, 1987. 94. Hungerford, M., Hollingworth, T.A., and Wekell, M.M. Automated kinetics-enhanced flow-injection method for histamine in regulatory laboratories: Rapid screening and suitability requirements, Anal. Chim. Acta., 438, 123, 2001. 95. Association of Official Analytical Chemists, Histamine in seafood: Fluorometric method [35.1.32 method 977.13], In Official Methods of Analysis, 17th edn., AOAC International, Gaithersburg, MD, 2003. 96. Takagi, K. and Shikata, S. Flow injection determination of histamine with a histamine dehydrogenase-based electrode, Anal. Chim. Acta., 505, 189, 2004. 97. Lerke, P.A., Martina P.N., and Henry, B.C. Screening test for histamine in fi sh, J. Food Sci., 48, 155, 1983. 98. Carelli, D., Centonze, D., Palermo, C., Quinto, M., and Rotunno, T. An interference free amperometric biosensor for the detection of biogenic amines in food products, Biosens. Bioelectron., 23, 640, 2007. 99. Kvasnicka, F. and Voldrich, M. Determination of biogenic amines by capillary zone electrophoresis with conductometric detection, J. Chromatogr., 1103, 145, 2006. 100. Staruszkiewicz, W.F. and Rogers, P.L. Performance of Histamine Test Kits for Applications to Seafood. Presentation on October 26, 2001. 4th World Fish Inspection & Quality Control Congress, Vancouver, BC. 101. Rogers, P.L. and Staruszkiewicz, W.F. http://seafood.ucdavis.edu/pubs/histamine.htm (accessed December, 10th 2008). 102. Neogen, (http://www.neogen.com/FoodSafety/V_Index.html) (accessed December, 10th 2008).
Chapter 43
Residues of Food Contact Materials Emma L. Bradley and Laurence Castle Contents 43.1 Introduction ..................................................................................................................852 43.2 Food Contact Materials and Chemical Migration .........................................................852 43.2.1 The Nature of the FCM .................................................................................. 854 43.2.2 The Nature of the Foodstuff ............................................................................ 854 43.2.3 The Nature of the Migrating Substance .......................................................... 854 43.2.4 The Extent of Contact with the Foodstuff ........................................................856 43.2.5 The Duration of the Contact ............................................................................857 43.2.6 The Temperature of the Contact ......................................................................857 43.3 Why Test for Residues of FCMs? ..................................................................................857 43.4 What Residues Need Testing? .......................................................................................857 43.5 Testing Strategies...........................................................................................................858 43.5.1 Overall Migration and Total Extractables......................................................... 858 43.5.2 Specific Migration Limits ................................................................................ 858 43.5.3 Extraction Tests Followed by Estimation of Migration Levels .......................... 858 43.5.4 Using Food Simulants ..................................................................................... 859 43.5.5 Testing for the Unexpected ............................................................................. 860 43.6 Packaging Formats of Relevance to Seafood and Seafood Products .............................. 860 43.6.1 Plastics .............................................................................................................861 43.6.2 Paper/Cartonboard .......................................................................................... 862 43.6.3 Metal Cans with Polymeric Internal Coatings ................................................ 863 43.6.4 Glass Jars with Lacquered Metal Lids and PVC Gaskets ..................................867 43.6.5 Multilayer Packaging Materials ........................................................................867 851
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43.6.6 Active and Intelligent Packaging ......................................................................867 43.6.7 Surface-Active Biocides ................................................................................... 869 43.7 Conclusion ................................................................................................................... 870 References ............................................................................................................................... 870 Further Reading ...................................................................................................................... 870
43.1 Introduction Th is chapter deals with the topic of the transfer of chemical residues from contact materials into seafood and seafood products. If chemicals transfer to the product they may cause taint or odor problems and if the concentrations are high enough this may even make the product unsafe to eat. So it is important to understand how this can be tested for and kept under control by the proper selection and use of packaging materials. Th is chapter aims to give the reader such an understanding and includes some examples which, although by no means exhaustive, are illustrative of the main scientific and technical issues. It will start fi rst with the chemical and physical processes that underlie this transfer process, which is called chemical migration. Th is is because it is the migration phenomenon that makes testing of seafood and seafood products for residues of food contact materials (FCMs), and testing the FCMs themselves, a special topic. Table 43.1 lists some packaging materials and packaging formats that are used commonly for seafood and seafood products.
43.2
Food Contact Materials and Chemical Migration
The term “food contact material” describes any material that may come into contact with a foodstuff. The most obvious example is food packaging, but the term also encompasses materials (and articles) used in food processing, transport, preparation, and consumption. A distinction is often made by the cognoscenti between materials and articles. Materials being such as films and sheets that require fabrication into their final usable form, and articles being such as boxes and pouches that are the final form. In this chapter we shall refer to both as “materials.” Packaging may be made from plastic, paper/board, rubber, metal, or glass, etc. Chemicals are needed to make these materials with their desirable properties. Any chemical constituents present have the potential to transfer to the foods with which they come into contact. In addition, the chemicals present in any adhesives, coatings, or printing inks applied to these substrates also have the potential to transfer. This transfer is known as chemical migration. Chemical migration is defined as “the mass transfer from an external source into food by submicroscopic processes.” The extent to which any substance migrates into a foodstuff is controlled by diff usion processes which are subject to both kinetic and thermodynamic control. These processes can be described by Fick’s second law, and the extent of any chemical migration is dependent on ◾ ◾ ◾ ◾
The nature of the FCM The nature of the foodstuff The nature of the migrating substance The extent of direct or indirect contact between the FCM and the foodstuff
Residues of Food Contact Materials
◾
853
Table 43.1 Examples of Packaging Formats Used for Seafood and Seafood Products Form
Typical Material Types
Typical Applications
Boxes
High-density polyethylene or polypropylene
Fresh fish, seafood in their shells— mussels, crabs, oysters, lobster, clams, caught and transported on ice to wholesalers and retailers
Insulated boxes
Expanded polystyrene
For chilled fish transport
Trays and pots
Polystyrene, polyethylene terephthalate, polypropylene, and polyvinylchloride
Retail sale of fresh, frozen, or ready meals, including ovenable trays
Film lidding
Polyester or ethylene-vinyl acetate copolymers
As above, but film lidding may be removed before cooking
Bags
Polyethylene
General purpose point of sale and storage of chilled and frozen seafood
Bags
Nylon or polyester, with or without heat-sealable polyethylene layer
Cook-in-bag fish and seafood products, using microwave or boil-in-bag formats
Cans
Tin-plated steel, tin-free steel, or aluminum; coated or uncoated internally; traditional or easy-open ends
Salmon, tuna, anchovies, fish roe (e.g., caviar), other heat-processed (sterilized) seafood products, fish in oil, brine, or sauces, soups, chowders, fish-based babyfoods
Closures on glass jars
Coated and with an integral plastic sealing gasket
Anchovies, other heat-processed (sterilized) seafood products, pickled fish, fish-based babyfoods
Metal tubes
Coated or uncoated
Fish pastes
Sheets and boxes
Plain paper or board, possibly wet-strength treated
Fresh or frozen fish, frozen fish products including breaded, battered, and other coatings
Absorbent pads
Paper or paper/synthetic composites
Fresh fish and other seafoods in trays
Laminates
Paper/plastic or paper/foil/ plastic
Salmon boards, frozen fish products including breaded, battered, and other coatings, carton boxes of fresh or chilled fish, shelf-stable fish soups
Plastics
Metals
Paper or Board
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◾ The duration of the contact ◾ The temperature of the contact The interplay of these different factors is illustrated using salmon as an example in Table 43.2.
43.2.1 The Nature of the FCM Migration from a material occurs at the interface with the food. Chemical migration depends on the concentration of the substance in the FCM and on the diff usion characteristics of the substance within the material and away from the surface of the food into which it migrates. For a material with a low diff usivity the speed with which the surface is replenished with the migrant will be slower than that of a high-diff usivity material. As a result, the rate of the migration will be reduced for the low diff usivity material. Migration from materials such as glass, ceramics, or metal occurs only from the surface of the material; no diff usion of migrants will occur from within these materials to the surface. Plastic materials exhibit diff usivity to different extents depending on the structure, crystallinity, etc. However, in all cases diff usion of migratable substances from within the plastic to the food contact surface can occur. More porous materials such as paper and board provide practically no resistance to the movement of some substances within the matrix. Th is is depicted in Figure 43.1. Multilayer packaging materials are also commonplace where a barrier layer such as aluminum foil is included in the packaging structure. In these cases any migratable substances on the nonfood side of the aluminum foil layer will not be able to pass through this barrier layer and therefore migration of such substances into the foodstuff will not occur by this mechanism. However, if a material has been rolled (reeled) or stacked such that the food contact surface is stored in contact with the nonfood contact surface then transfer of chemicals between the two can occur. In such cases even the presence of a functional barrier such as a layer of aluminum foil is not sufficient to ensure that no migration will occur. Th is transfer process in known as set-off and it is especially important when evaluating inks.
43.2.2
The Nature of the Foodstuff
When considering migration, foodstuffs are conventionally split into five categories: aqueous, acidic, alcoholic, fatty, and dry. The solubility of the migrating substance in the foodstuff will influence the extent of the migration. Lipophilic (“fat-loving”) substances have a greater solubility in fatty foods or foods with free fat on the surface, and so the migration of such substances into these food types will be greater than that into an aqueous foodstuff. Conversely, polar molecules are more soluble in aqueous media and less soluble in fatty foods.
43.2.3
The Nature of the Migrating Substance
Any substance which is incompatible with the FCM type will “bloom” to the surface resulting in it being readily available to transfer to the foodstuff. Conversely, any strong interaction which occurs between a substance and the material containing it will slow down the mass transfer process.
a
0°C–5°C
1 day
1×
Time
Severity indexa
121×
14 days
0°C–5°C
(b) 3040×
(a) 2880×, then;
(b) 1,460 days
(a) 30 min, then;
(b) Store at room temperature
(a) Retort at 121°C, then;
Severity index is for illustration only. It is estimated based on the approximation that migration concentration levels are proportional to the area:mass ratio, proportional to the square root of the duration of contact, and that the migration rate approximately doubles for each 10°C rise in temperature.
14×
5 days
0°C–5°C
0.3 cm2/g for coating #2
1.9 cm2/g for film
Temperature
0.9 cm2/g for coating #1
0.06 cm2/g
Area-to-mass ratio
1.7 cm2/g for board
55 cm2 (can end—coating #2)
185 cm2 with the film
Incidental only with the film 0.36 cm2/g for tray
155 cm2 (can body—coating #1)
171 cm2 with the board
130 cm2 with the tray
3,000 cm2
180 g
Contact area
100 g
Fish in a two-piece metal can with an internal polymeric coating and with a different coating on the easy-open end
360 g
Smoked fish slices on foil-coated paperboard and vacuum-packed in a multilayer plastic film and with a self-adhesive label (removed for picture)
50,000 g
Chilled fish steaks in a deep plastic tray with a plastic film overwrap and with a self-adhesive label (film and label removed for picture)
Food mass
Fresh fish transported on ice in a reusable plastic crate
Table 43.2 The Range of Food Contact Applications Used and the Relative Demands Placed on Packaging with Respect to Chemical Migration
Residues of Food Contact Materials ◾ 855
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Packaging
Foodstuff
Impermeable materials such as glass, ceramics, metals, and alloys
Migration can occur from the food contact surface only. Packaging
Foodstuff
Permeable materials such as plastics and rubber
Migration of substances can occur from within the polymer as well as those at the surface. Packaging
Foodstuff
Porous materials such as paper and board
Migration of substances occurs from the food contact surface, from within the material as well as any substances contained in inks and coating applied to the nonfood contact surface. Porous substrates offer practically no resistance to chemical migration.
Figure 43.1
Depiction of chemical migration.
43.2.4 The Extent of Contact with the Foodstuff Both the nature of the FCM and the nature of the foodstuff will influence the partitioning between the two. If the foodstuff interacts strongly with the FCM it can cause swelling at the surface which increases the rate at which chemicals are released. The greater the surface area of the material in direct contact with the foodstuff the greater the potential for migration. Similarly where intimate contact is made as opposed to point contact, e.g., liquid or semisolid foods including sauces and pastes, compared to solid foods, the potential for migration also increases.
Residues of Food Contact Materials
43.2.5
◾
857
The Duration of the Contact
The longer the material is in contact with the foodstuff the greater the extent of the migration that will occur. Migration kinetics are normally first-order, which means that the extent of any migration increases relative to the square root of the contact time.
43.2.6 The Temperature of the Contact Similarly as migration is a diffusion process that occurs more rapidly at elevated temperature, the extent of the migration increases with increasing contact temperature.
43.3 Why Test for Residues of FCMs? By the diff usion processes described above any substance present in a material placed in contact with a foodstuff has the potential to migrate. This migration can impact on the safety of the food because some substances used to make FCMs may be harmful if consumed in high enough amounts. Migration can also impact on the quality of the food because the transfer of sensorially active substances may impart a taint or odor to a foodstuff such that it is no longer appealing to the consumer. The need to control the effects of FCMs on both of these aspects is considered in legislation. Extensive references to the legislation in Europe, the United States, and other countries, can be found in the book listed under “Suggested further reading.” For example, in the European Union the Framework Regulation (EC) No. 1935/2004 covers all FCMs. It states in the general requirements of Article 3 that Materials and articles, including active and intelligent materials and articles, shall be manufactured in compliance with good manufacturing practice so that, under normal or foreseeable conditions of use, they do not transfer their constituents to food in quantities which could: (a) endanger human health; (b) bring about an unacceptable change in the composition of the food; (c) bring about a deterioration in the organoleptic characteristics thereof. A similar philosophy on the need for controls operates in the United States, Japan, and in other countries although the detailed legislative and technical instruments used differ.
43.4
What Residues Need Testing?
As mentioned previously a range of different chemicals are needed to make materials intended for food contact. There are several thousands of chemicals in inventory lists used by producers, and of these probably several hundred find regular use. They include monomers and other starting substances needed to make plastics, catalysts, and production aids to make plastics and paper, additives to modify the properties of the finished products, ingredients of inks and adhesives, etc. Since chemical migration is a diff usion phenomenon it is the small, low-molecular weight substances that tend to migrate fastest. This is certainly true for the monomers used to make the high-volume plastics and coatings such as vinyl chloride, 1,3-butadiene, acrylonitrile, and styrene. Additives on
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the other hand must remain in the finished material in order to have a technical effect and so they tend to be higher molecular weight substances to reduce their loss. Finally, as producers strive to make materials with lower migration properties, they are turning to so-called polymeric additives of molecular weight of 1000 Da or more. This means that the full range of analytical methods are deployed in testing for these residues; with headspace gas chromatography–mass spectrometry (GC–MS) for the volatiles, GC–MS for the semivolatiles, and increasingly liquid chromatography (LC)–MS for the nonvolatiles and the polar residues. The detection level needed depends on the toxicological or organoleptic properties of the substances but typically it is in the range of a few parts per million (ppm, mg/kg) down to ca. 10 parts per billion (ppb, mg/kg) in the food.
43.5 Testing Strategies The food itself can be tested for undesirable chemical residues. Alternatively, the packaging material can be tested before it is used to ensure that it does not contain residues that can migrate at levels that could cause problems. Finally, uniquely for FCMs, the packaging can be tested for its suitability before use by employing food simulants that are intended to mimic the migration properties of different categories of foods.
43.5.1
Overall Migration and Total Extractables
By way of an example, the EU Plastics Directive imposes an overall migration limit to ensure that materials do not transfer large quantities of substances which, even if they are not unsafe, could bring about an unacceptable change in the food composition—amounting to adulteration. The total amount of all migrating substances is limited to 60 mg/kg of food. This is tested for using food simulants, and a set of test methods is available as European standards. Because a test for overall migration using food simulants is entirely conventional—i.e., the test result depends on the method used—the standard test procedures have to be used and followed exactly. In countries such as the United States and Japan suitability end-tests of materials may use extraction solvents rather than food simulants.
43.5.2 Specific Migration Limits Again by way of an example, the EU Plastics Directive 2002/72/EC as amended, contains a positive list of monomers and additives permitted for use in the manufacture of plastic for food contact. This list contains any limits on the migration of individual substances—limits that have been assigned following the toxicological assessment of these substances. Similar lists exist as National Legislation in European and other countries for the chemical ingredients used to make paper, silicones, inks, adhesives, coatings on metal, etc. The form of any restrictions—as specific migration limits or limits on the extractable substance or on the total content in the material—differs from country to country and for the different material types.
43.5.3 Extraction Tests Followed by Estimation of Migration Levels Compliance of a material with a specific migration limit or some other migration restriction can be tested for by extracting the material to determine the concentration of the substance(s) of interest. Then the migration expected into food can be estimated either by assuming total
Residues of Food Contact Materials
◾
859
mass transfer (worst-case 100% migration scenario) or by using mathematical models. The measured concentration in the packaging (cp,0) may also be available from formulation details provided by the producer. A number of commercial software packages (e.g., Migratest© Lite, SMEWISE, and EXDIF v 1.0) are available to predict the extent of migration from the cp,0 value. They have been validated mainly for plastics. All are based on diff usion theory and a consideration of partitioning effects. The underlying key parameters are the diff usion coefficient of the migrant in the plastic (D P) and the partition coefficient of the migrant between the plastic and the food or food simulant (K P,F). These models have been tuned to provide an overestimation of migration in the majority of cases so that they can be used with confidence in compliance testing.
43.5.4
Using Food Simulants
Food simulants are an important tool for testing the suitability of materials for the food that are intended to be placed in contact with. Again the EU system for plastics is taken as an illustrative example. Simulants intended to mimic the migration from plastics into foods were introduced in the early 1980s (Directive 82/711/EEC, as amended) along with the rules for using simulants (Directive 85/572/EEC, as amended). Simulants are specified for the five food categories described earlier: Food Type
Food Simulant
Aqueous foods of pH ≥ 4.5
A—distilled water
Acidic foods of pH < 4.5
B—3% acetic acid solution
Alcoholic foods
C—10% ethanol solution (or higher)
Fatty foods
D—rectified olive oil or similar
Dry foods and frozen foods
No migration testing is specified
Seafoods and seafood products may be mimicked by using both simulant A (water; to represent the aqueous phase) and/or simulant D (oil; to represent the fatty phase of the food). It is considered that the oil simulant is too severe compared to the foods—that means, it elicits higher migration levels. So for plastic packaging intended to be used for fish (fresh, chilled, salted, or smoked) a reduction factor of 3 is applied to the test result using oil. Clearly this is a conventional approach because a factor of 3 cannot be strictly correct for all these types of fish, for all types of different plastics, for different substances, etc. (e.g., see Section 43.2 describing the influence of these different parameters on the migration process). Similarly, as well as having differences between fatty (e.g., salmon, sardines) and less fatty seafoods (e.g., cod, squid, mussels) the packaged product may come in different “carriers.” Examples would be fish-in-oil, fish-in-brine, pickled fish, and fish in tomato-based sauces in which the oil, brine, vinegar, and the sauce act as “carriers.” In these cases, in which intimate contact exists between the carrier and the packaging, the choice of simulant would depend on the nature of the liquid “carrier” rather than the fish itself. Simulants were introduced at a time when analytical instrumentation and methods were not available to test foods for all the substances of interest at ppm to ppb levels. Simulants also provide a means to test for broad food categories rather than having to test individual food items. However,
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Handbook of Seafood and Seafood Products Analysis
as methodology and instrumentation have advanced it becomes clear that in some circumstances the simulants may not overestimate migration (as designed) but may underestimate migration into foods. For seafood and seafood products the example of retorting conditions is relevant. Under the severe conditions used to sterilize foods in metal cans or glass jars, it may be that migration from polymeric coatings or polymeric sealing gaskets is extensive and not properly described using the conventional reduction factors.
43.5.5 Testing for the Unexpected As well as testing for known ingredients used to make FCMs, a proper safety assessment must go further. For example, the 4th amendment to Directive 2002/72/EC includes the explicit provision that there is a general requirement to assess the safety of all potential migrants. This includes what have become know as the nonintentionally added substances (NIAS) such as impurities, reaction, and breakdown products. The onus is placed on the business operator to make this assessment. Again although this directive is applicable to plastics it can also be used as a guide for other FCMs. To demonstrate their safety, these “nonlisted substances” should be assessed in accordance with international risk assessment procedures. In the authors’ opinion such a risk assessment should have three components: (a) the identification of the substances present in the material, (b) an estimation of their migration level leading to an estimate of possible consumer exposure, and (c) a risk assessment which considers the potential exposure in context with any hazard (nature and potency) posed by the chemical. This requirement to identify substances places emphasis on the information-rich separation techniques using mass spectrometry as the detection system; i.e., GC–MS and LC–MS(MS). Increasingly, testing laboratories will turn to LC–time-of-flight (TOF)-MS to get accurate mass information on molecular ions and fragment ions to gain further confidence in substance identification.
43.6
Packaging Formats of Relevance to Seafood and Seafood Products
Table 43.1 gave some examples of packaging materials used for seafood and seafood products. They include rigid packaging (boxes, trays, cans, plastic containers, paperboard sleeves, and others), flexible packaging (fi lm, bags, pouches, paper, and foil), and packaging accessories (labels, absorbent pads, and others). Specifically the following packaging materials were identified: ◾ ◾ ◾ ◾ ◾
Plastics Paper/cartonboard Metal cans to which a polymer coating has been applied to the food contact surface Glass jars with lacquered metal lids and polyvinyl chloride (PVC) gaskets Multilayer flexible packaging materials including inks and adhesives.
And to a lesser extent currently ◾ Active and intelligent packaging (e.g., absorbent pads, oxygen absorbing films, amine (offflavor) scavengers ◾ Surface-active biocides Testing these packaging formats is described in more detail below using selected examples.
Residues of Food Contact Materials
43.6.1
◾
861
Plastics
Plastics are the most commonly used material type for packaging foodstuffs. Storage boxes may be manufactured from high-density polyethylene, polypropylene, or expanded polystyrene. Examples of plastics used to package seafood and seafood products include trays made of polystyrene, polypropylene, polyethylene terephthalate, or PVC, for fresh fish and fish-based convenience ready meals. The film-lidding materials used with these trays are usually polyester or ethylene-vinyl acetate copolymer. Polyethylene is used for general purpose food bags. Nylon or polyester/polyethylene laminates are used for boil-in-the-bag and microwaveable pouches. The different types of plastics and the typical monomers and additives used in their production have been reviewed elsewhere [1]. Example 1 describes a typical procedure for testing for a volatile migrant—in this case styrene monomer from foamed polystyrene insulation boxes. Example 1:
Testing for a Volatile Migrant Using Headspace-GC–MS
Purpose: Whole salmon transported on ice in an insulated expanded polystyrene foam (EPS) container are tested for any pick-up of styrene (Figure 43.2). Procedure: The fish are rinsed in cold water and the head, tail, fins, and central bone are removed. The remainder of the fish is homogenized using a food blender. An aliquot of the homogenized fish is transferred into a headspace vial (10 mL capacity). The samples are analyzed in three ways: (a) as received (no additions); (b) with d8-strene internal standard added at 200 mg/ kg (duplicates); and (c) with d8-styrene internal standard added at 200 mg/kg and a 100 mg/kg spike of styrene added (duplicates). Calibration standards are prepared by addition of styrene (0–500 mg/kg) to blank salmon (i.e., salmon containing no styrene). The specimens are incubated at 90°C for 30 min and a portion of headspace gas (1 mL) is analyzed by GC–MS. Mass spectrometric detection is conducted in selected ion mode (m/z 78 and 104 monitored for d0-styrene and m/z 84 and 112 monitored for d8-styrene). Results: No styrene was detected in the control fish samples packaged not in EPS but in polyethylene bags. The limit of detection is <1 mg/kg. In individual fish transported in EPS boxes the concentration of styrene found was in the range 10–25 mg/kg. Styrene was confirmed to be present
Styrene
Calibration line for ion 104 (styrene analyte) versus ion 112 (d8-deuterated styrene, internal standard) 3.5
CH2
y = 0.0054x R2 = 0.9997
PAR 104/112
3.0 2.5 2.0 1.5 1.0 0.5 0.0 0
100
200
300
400
Styrene concentration (μg/kg)
Figure 43.2
Styrene—calibration.
500
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on the basis of the GC retention time and by the ratio of the m/z 78 and m/z 104 ions in the four fish received packaged in the polystyrene box. There was good agreement between the duplicate portions and spikes. All results are corrected for analytical recovery which was acceptable, in the range 91%–100%. Interpretation: The relatively low migration level of 10–25 mg/kg found in the salmon is probably due to the facts that; the product is chilled, has only a short contact time in the foam boxes, the contact is not especially extensive given the size-to-mass ratio of the large fish, and that the fish is packed with ice. The between-specimen variability is not untypical for migration into such foods given the variability in the contact made between the food and the packaging.
43.6.2
Paper/Cartonboard
Several standard methods have been published for the testing of paper and board intended to come into contact with foods. These include methods for papermaking chemicals, for contaminants such as those that may be introduced by recycling, and for taint and odor transfer. Paper and board materials are chemically complex systems with special challenges regarding their safety evaluation. Figure 43.3 shows a GC–MS total ion chromatogram of a solvent extract of a food contact paperboard studied in our laboratory. Numerous peaks (individual substances) are detected. Limited peak identification can be performed by comparison of the mass spectra of each peak with those spectra contained in spectroscopic libraries. Typically many of the substances detected remain unidentified. Th is is because many are derived from the woods, rosins, etc., used to make paper, and consequently they are not in standard libraries of spectra. Laboratories expert in paper analysis have over the years built up their own libraries of commonly encountered substances. Given the natural source of the paper, its variability (e.g., different wood species), and the use of recycled fibers with their attendant contaminants that a water-based recycling–papermaking process may not remove completely, the safety evaluation of paper is difficult using Abundance TIC:screen_230606_053.D
2e + 07 1.8e + 07 1.6e + 07 1.4e + 07 1.2e + 07 1e + 07 8,000,000 6,000,000 4,000,000 2,000,000 6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
Time-->
Figure 43.3 GC–MS total ion chromatogram obtained from the analysis of an ethanol extract of a food contact paper/cartonboard.
Residues of Food Contact Materials
◾
863
chemical analysis alone. Therefore, an approach has been proposed that complements chemical analysis. Th is involves the application of a battery of short-term bioassays to extracts of paper and board, to assess the toxicity of the total migrate. Within the BIOSAFEPAPER project (http://www.uku.fi /biosafepaper) a battery of cytotoxicity tests was applied to extracts of paper and board materials. These assays correctly identified a nonfood grade board as being unsuitable for contact with food. Th is approach, i.e., assessing the toxicity of the whole migrate, may also be applicable to other materials particularly in cases where a number of nonintentionally added substances are present in the finished material and for which the toxicity of the individual substances is not known. Direct contact with plain paper/cartonboard only occurs for fresh “wet” fish (e.g., short duration only, sold at a fishmonger) and for frozen fish and seafood products. In other applications the fat and water present in the product means that direct contact with a porous substrate such as paper and board renders it unsuitable for this type of contact. In such cases the cartonboard may be laminated to a polymer film with the cartonboard providing the rigidity required of the packaging and the polymer film protecting the cartonboard from the foodstuff. The barrier properties of the laminated polymer will determine whether or not any migration of chemicals derived from the cartonboard will occur. For frozen foods it is often assumed that migration does not occur at the low contact temperature. However, migration into frozen foods has been reported [2]. Paper and board materials are porous substrates within which the diff usion of low- and medium-molecular weight chemicals readily occurs both originating from the paper/board itself and from any printing inks and coatings applied to the external (nonfood contact) surface. The migration of the printing ink photoinitiator benzophenone through cartonboard substrates into frozen foods has been reported [2,3]. In such cases the migration of volatile substances can transfer by vapor phase diff usion from the cartonboard with adsorption onto the surface of the foodstuff.
43.6.3 Metal Cans with Polymeric Internal Coatings Products such as fish (e.g., salmon, tuna, anchovies), other seafood products (e.g., roe, fish-based soups), and fish-based baby foods are often packed in metal cans with a polymer coating inside. This coating is intended to form a barrier between the food and the metal of the can. In this way the coating protects the food from the metal substrate as well as protecting the metal substrate from the potentially corrosive foodstuff contained within. The major types of can coatings are made from epoxy resins. These coatings exhibit a combination of toughness, adhesion, formability, and chemical resistance under the conditions that the coated metal is subjected to. The most widely used epoxy resins are based on bisphenol A diglycidyl ether (BADGE) itself synthesized by the reaction of bisphenol A with epichlorohydrin. The migration of bisphenol A and BADGE is well documented (e.g., [4] and references therein). Example 2 describes a typical procedure for testing for nonvolatile migrants (BADGE and its regulated derivatives (BADGE · H2O, BADGE · 2H2O, BADGE · HCl, BADGE · 2HCl, and BADGE · HCl · H2O), in these cases using LC fitted with a fluorescence detection (FLD). Although a lot of work has been carried out to determine the migration levels of these substances, there are many other potentially migratable substances in the coatings. Example 2:
Testing for a Nonvolatile Migrant Using HPLC–FLD (Figure 43.4)
Purpose: Tuna packaged in cans is analyzed for BADGE and its regulated derivatives (BADGE · H2O, BADGE · 2H2O, BADGE · HCl, BADGE · 2HCl, and BADGE · HCl · H2O).
Figure 43.4
0
O
5
H3C
10
O
HO
15
BADGE . H2O . HCl
O
BADGE . 2H2O
CH3
20 Time/min
25
BADGE . H2O
OH
O
BADGE. HC1 . H2O CH3
30
O
35
OH
Cl
40
BADGE
BADGE . HCl
BADGE . 2HCl
BADGE isomers
H3C
High-performance liquid chromatography (HPLC)–FLD chromatogram of BADGE and its derivatives.
HPLC–FLD chromatogram
–50
100
250
400
550
700
Normalised signal
O
BADGE
45
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Residues of Food Contact Materials
◾
865
Procedure: Cans are opened and the contents are homogenized prior to subsampling. Duplicate specimens (5 g) are extracted with acetonitrile (5 mL) by shaking for 18 h. The extract is passed through a cotton wool filter plug and then through a 0.45 mm filter. A third specimen of each sample is overspiked with all of the analytes as a recovery check. Solvent standards of the analytes (0.5–25 mg/kg equivalent concentration in the foodstuff) are prepared alongside the samples. Liquid chromatography analysis is conducted using an ODS column and a gradient of water and acetonitrile. The fluorescence detector is set at excitation 275 nm and emission 305 nm. Results: BADGE · 2H2O was detected in all the samples. BADGE · HCl · H2O was detected in some of the samples. The calibration lines were linear, the spike recovery values acceptable, and the results were corrected for recovery. Limits of detection (3× baseline noise) were ≤0.01 mg/kg for all analytes. Interpretation: EU Commission Regulation (EC) No. 1895/2005 on the use of certain epoxy derivatives in materials and articles intended to come into contact with foodstuffs gives the specific migration limit for BADGE and certain of its derivatives as 1. The sum of BADGE, BADGE · H2O, and BADGE · 2H2O shall not exceed 9 mg/kg in foodstuffs or in food simulants. 2. The sum of BADGE · HCl, BADGE · H2O · HCl, and BADGE · 2HCl shall not exceed 1 mg/kg in foodstuffs or in food simulants. In this example the samples easily passed both restrictions (1) and (2).
As well as the epoxy resins, hardeners such as acid anhydrides, aminoplasts, or phenolplasts may also be included in the formulation as well as additives, such as pigments, fillers, wetting and flow aids, defoamers and lubricants, and any reaction/breakdown products formed from these starting materials. As mentioned previously migration is influenced by both contact temperature and time. Most canned foods are sterilized (e.g., at 121°C for 1 h) and also have long shelf lives (up to 3–5 years is not uncommon) so the migration conditions in canning are severe. Consequently, coatings manufacturers are constantly striving to produce “cleaner coatings” with fewer low-molecular weight migratable substances [5]. Example 3 gives a procedure used to identify unknown migrants from a can coating using LC–TOF-MS. Example 3:
Identifying Unknown Migrants Using LC–TOF-MS (Figure 43.5)
Purpose: In investigating the total migration from a fish can coating based on BADGE, several peaks were seen in the FLD analysis that could not be identified and for which no analytical standards exist. Thus TOF-MS was used for identification purposes. Procedure: To give a concentrated extract for identification purposes, a can coating is extracted using acetonitrile solvent. Using the same LC conditions as in Example 2, the peaks with the same retention time as observed in migration tests are located using the FLD. A TOF-MS was placed in tandem with the FLD and used to aid substance identification. The TOF-MS used a nebulizer pressure of 30 psi, the capillary was held at 4000 V, the drying gas was nitrogen at 325°C and 10 L/ min. The MS fragmentor was held at 150 V. Results: As well as identifying the monomer BADGE and its regulated reaction products, the Peaks 1 and 2 were confirmed as isomers of cyclo-di-BADGE. The coeluting peak 3 was identified as the BADGE-BuOH adduct formed by reaction between BADGE and butanol solvent when the wet can coating was dried and cured (stoved) at high temperature. The identity of the other peaks was also established (see [6] for full details). Interpretation: This application illustrates how TOF-MS can be used in the safety evaluation of FCMs. Previously unconfirmed migrants were confidently identified using the accurate mass information provided by the TOF-MS.
0.0
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Figure 43.5 Identifying unknown migrants using LC–TOF-MS.
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Glass Jars with Lacquered Metal Lids and PVC Gaskets
Examples of seafood and seafood products packaged in glass jars include pastes, pickled fish, fish-inoil and sauces, and baby foods based on fish. Whereas the glass containers themselves are generally considered to be inert, they need a metal closure—a lid—that will be coated (see above) and will have an integral plastic sealing gasket. These PVC gaskets contain high levels (typically 40%–45%) of plasticizers to make them soft enough to form an airtight and microbiologically safe seal against the rim of the glass jar. With the high temperatures used to sterilize fish-based products in the jars, there can be extensive migration from the gasket—even though the surface area of gasket exposed to food is generally small. This can be especially marked if the jar is sterilized in a rotating head-overheels retort (rather than a static retort) which brings the gasket into intimate contact with the hot food contents and also fouls the gasket to promote further migration during long-term storage. Plasticizers used in gaskets include phthalic acid esters, adipic acid esters, epoxidized soybean oil, and acetyl tributyl citrate. Some of these are complex mixtures and no standard methods exist for testing either the foods or food simulants, although several research groups have published in this area (e.g., [7,8] and references therein). As well as these plasticizers, other substances used to make the gaskets may migrate. One recent example is semicarbazide (SEM) formed as a breakdown product of azodicarbonamide used as a blowing agent [9]. Azodicarbonamide is added to the gasket formulation and when heated it decomposes to liberate gases, primarily nitrogen and carbon monoxide together with some carbon dioxide and ammonia. These gases turn the PVC into a closed-cell foam which helps it to make an effective seal. SEM was an unexpected and unwanted side product formed at very low yields. Th is example is of special interest because SEM is used as a marker residue for the misuse of nitrofuran veterinary medicines in aquaculture and so the possibility of misleading test results existed.
43.6.5 Multilayer Packaging Materials Many types of packaging materials consist of more than one layer. This is especially true for the flexible packaging films where the combinations of toughness for protection, barrier properties against gasses [e.g., modified atmosphere packaging (MAP)] or odor, printability, heat sealability, economy, etc., can be provided by combining two and sometimes several layers in a multilayer structure. The layers may be joined by coextrusion processes or by lamination using adhesives. As well as the potential migrants derived from the individual materials that make up the different layers the potential also exists for migration of components present in any adhesive used. Typically, reactive adhesive systems are used for this purpose. These include polyurethanes and to a lesser extent epoxy adhesives which are polymerized in situ. A very common multilayer film would be nylon or polyester (for toughness and barrier properties) laminated using reactive polyurethanes to a polyethylene film (for heat sealability) and printed on the outside or reverse printed with inks inside the laminate sandwich (for decoration and consumer information). Any residues of incomplete polymerization of the adhesive or any reaction by-products, may remain in the FCM and may then migrate into a foodstuff on contact.
43.6.6 Active and Intelligent Packaging One of the most innovative developments in food packaging in recent years is the use of active and intelligent packaging. Active packaging materials can be defined as: “ food packaging which has
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an extra function, in addition to that of providing a protective barrier against external influence.” It is intended to change the condition of the packed food, to extend shelf life, or improve sensory properties while maintaining the freshness and the quality of the food. This can be achieved through the removal (scavenging) of substances that have a detrimental effect on food quality. Examples of active absorbers and scavengers include • Oxygen scavengers
• Amine scavengers
• Moisture absorbers
• Sulfide scavengers
• Ethylene and off-flavor scavengers
• Bitter taste removers
• Acetaldehyde scavengers
The use of active packaging should not mislead the consumer. So for example, using scavengers for amines that are released as fish spoils would be unacceptable if it misled the consumer into believing the product was fresh and, more importantly, if it prevented the consumer from detecting if the product had spoiled and therefore could not be consumed safely. Active packaging systems can also aim to emit substances that improve the foodstuff. Examples of active releasing substances include • Carbon dioxide-regulating systems
• Antioxidant releasers
• Antimicrobial-releasing systems
• Sulfur dioxide releasers
• Nitrogen releasers
• Flavor releasers
Intelligent packaging materials can be defined as: “Concepts that monitor to give information about the quality of the packed food.” Examples of monitoring systems used in food contact applications include • Time and/or temperature indicators
• Oxygen indicators
• Freshness and ripening indicators
• Carbon dioxide indicators
Consequently for active packaging, the packaging is intended to influence the food and for intelligent packaging, the food is intended to influence the packaging. As well as using scavenging and releasing systems to maximize the shelf life of fish and seafood products, vacuum packaging, controlled atmosphere packaging (CAP), and MAP can also be used. Oxygen in the air increases the rate of both the chemical breakdown and microbial spoilage of many foods. Vacuum packaging removes air from packages and produces a vacuum inside. MAP and CAP help to preserve foods by replacing some or all of the oxygen in the air inside the package with other gases such as carbon dioxide or nitrogen thereby reducing the oxidative damage. These systems are often used alongside oxygen absorbing, carbon dioxideregulating systems, working together to maximize the product’s shelf life. In most countries, any active substance(s) emitted into the food is considered to be a direct food additive, and food additive rules and regulations apply. So the food should be tested for the
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additive using the available methods—e.g., preservatives (see Chapter 6), flavors (Chapter 8), or colors (Chapter 9). Any chemical migration of other components of the delivery system of the active ingredient (e.g., SO2/sulfite sorbed onto an inorganic reservoir), the holding system for the scavenging ingredient (e.g., a separate sachet of iron oxide as an O2 scavenger), or the intelligent components (e.g., an impregnated plastic time/temperature strip) should be tested for migration of ingredients, reaction products, breakdown products, and impurities, in the normal way for conventional packaging materials.
43.6.7 Surface-Active Biocides A number of products have come onto the market in recent years with surface biocidal properties. These include conveyor belts, cutting boards, the inside linings of commercial and domestic refrigerators, and the plastic parts of complex food-processing machinery such as fish-processing lines. These surface-active biocidal materials should not be confused with active packaging (see earlier text) because there is no intention that the biocidal agent has any preservative effect on the food. Rather, the intention is that the biocide remains in the FCM, perhaps concentrated at the surface, and improves the surface hygiene and cleanability. Surface-active biocidal materials may have benefits especially for food-processing machinery parts that are awkward to clean in place (in situ). A common biocide used for this is silver in a number of chemical forms. It is generally accepted that silver ions are antimicrobial to all microbial species that are likely to be found in a food environment, including against Gram-negative bacteria, Gram-positive bacteria, moulds, and yeast. Another biocide used is 2,4,4′-trichloro-2′-hydroxydiphenyl ether which seems to have a less uniform activity against bacteria, moulds, and yeast. Although these surface-active biocides are not intended to migrate into the food and exert any preservative effect, some level of migration is inevitable and should be tested for as for any other substance used in FCMs. In the two examples given, the inorganic silver compounds may be expected to migrate mostly into aqueous and acidic foods whereas the organic substance 2,4,4′trichloro-2′-hydroxydiphenyl ether is expected to migrate more into fatty foods. The unavoidable migration level should not be high enough to exert any preservative effect on the food. This can be checked by calculation, i.e., by comparing the migration concentration against the minimum inhibitory concentration (MIC) values of common food-related microbes. But these calculations can be difficult to interpret because; first, the MIC values are usually recorded in pure buffer media and may change significantly in the presence of food components. This is especially true for silver ions which can be sequestered. Second, for solid and semisolid foods like fish and processed seafood products, the migration will be concentrated at the food contact surface and of course the surface of the food is most prone to microbiological spoilage. So the migration concentration at the surface could be much higher than when calculated as an average for the whole mass of the food. For these reasons, there is a need to conduct real food trials to ensure that indeed there is no preservative effect exerted. There does seem to be two areas where test methods are lacking. The first is demonstrating whether or not these materials really are effective under the particular conditions of recommended use. Simple film tests looking at surface inhibition against different organisms seem to be the only laboratory tool available at present. The alternative is real-life factory trials with full microbiological audit. Since these materials are intended to complement and not replace normal cleaning and hygiene procedures, hard facts and data are difficult to find. The second, related area, is for how long these biocidal materials retain their efficacy. Again, laboratory tests seem to be inadequate to
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simulate the resistance of the biocidal agent to loss through repeated washing, exposure to caustic cleaning agents, repeated contacts with food, etc., during the service life of the material. These research and development needs for test methods should be addressed.
43.7 Conclusion Chemical residues in seafood and seafood products may occur as a result of chemical migration from FCMs, of which food-packaging materials are the most important example. Analysis of the food for these chemical residues uses basically the same chemical analytical methods that are in the food analysts armory. What makes the topic special is the added dimension of needing to analyze also the food-packaging materials themselves (to indicate what chemical(s) may migrate) and analysis of food simulants used to test materials for their suitability for contact with different types of foods.
References 1. Cooper, I., 2007. Plastics and chemical migration into food. In Chemical Migration and Food Contact Materials. K. A. Barnes, R. Sinclair, and D. Watson (eds). pp. 228–250. Woodhead Publishing, Cambridge, England. 2. Johns, S. M., Jickells, S. M., Read, W. A., and Castle, L., 2000. Studies on functional barriers to migration. 3. Migration of benzophenone and model ink components from cartonboard to food during frozen storage and microwave heating. Packaging Technology and Science, 13, 99–104. 3. FSIS, 2006. Food Survey Information Sheet 18/06: Benzophenone and 4-Hydroxybenzophenone Migration from Food Packaging into Foodstuff s. Food Standards Agency, London (UK). http://www. food.gov.uk/science/surveillance/fsisbranch2006/fsis1806 (accessed 28-6-2009). 4. Goodson, A., Robin, H., Summerfield, W., and Cooper, I., 2004. Migration of bisphenol A from can coatings-effects of damage, storage conditions and heating. Food Additives and Contaminants, 21, 1015–1026. Agilent Technologies (USA), http://www.chem.agilent.com/enus/Search/Library/_ layouts/Agilent/PublicationSummary.aspx?whid=48269 (accessed 28-6-2009). 5. Stocker, J., 2007. New technologies and chemistries for can coatings. New Food, 3, 46–81. 6. Driffield, M., Bradley, E. L., Castle, L., and Zweigenbaum, J., 2006. Agilent Application Note 59895898EN—Identification of unknown reaction by-products and contaminants in epoxyphenolicbased food can coatings by LC-TOF-MS. 7. Suman, M., Tegalo, S., Catellani, D., and Bersellini, U., 2005. Liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of epoxidized soybean oil in food products. Journal of Agricultural and Food Chemistry, 53, 9879–9884. 8. Biedermann, M., Fiselier, K., Marmiroli, G., Avanzini, G., Rutschmann, E., Pfenninger, S., and Grob, K., 2008. Migration from the gaskets of lids into oily foods: First results on polyadipates. European Food Research and Technology, 226, 1399–1407. 9. Stadler, R. H., Mottier, P., Guy, P., Gremaud, E., Varga, N., Lalljie, S., Whitaker, R., et al., L., 2004. Semicarbazide is a minor thermal decomposition product of azodicarbonamide used in the gaskets of certain food jars. Analyst, 129, 276–281.
Further Reading Chemical Migration and Food Contact Materials. K. A. Barnes, R. Sinclair, and D. Watson (eds.). Woodhead Publishing, 2007. ISBN-13: 978-1-84569-029-8.
Chapter 44
Detection of GM Ingredients in Fish Feed Kathy Messens, Nicolas Gryson, Kris Audenaert, and Mia Eeckhout Contents 44.1 Introduction ................................................................................................................. 872 44.1.1 Genetically Modified Organisms .................................................................... 872 44.1.2 GMOs in Fish Feed......................................................................................... 872 44.1.3 International Regulations ................................................................................ 873 44.2 GMO Analysis ..............................................................................................................874 44.2.1 Introduction .....................................................................................................874 44.2.2 DNA-Based Detection .....................................................................................874 44.2.2.1 Extraction of DNA ..........................................................................874 44.2.2.2 Qualitative Conventional PCR ........................................................874 44.2.2.3 Multiplex PCR .................................................................................876 44.2.2.4 Quantitative PCR ............................................................................876 44.2.3 Protein-Based Detection ................................................................................. 877 44.3 Comparison of DNA and Protein Methods.................................................................. 879 44.3.1 Protein-Based Methods ................................................................................... 879 44.3.2 DNA-Based Methods ...................................................................................... 880 44.4 Recent Developments in GMO Detection ....................................................................881 44.4.1 Microarray Technology ....................................................................................881 44.4.2 Biosensors ....................................................................................................... 882 44.5 Conclusions .................................................................................................................. 882 References ............................................................................................................................... 883 871
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44.1 Introduction 44.1.1
Genetically Modified Organisms
Genetically modified organisms (GMOs) can be defined as organisms whose genetic constitution has been altered by gene technology. The genetic material is changed in a way that is not possible by reproduction or natural recombination (transgene, genetically altered, or GMOs). Gene technology makes the insertion of new properties possible in an efficient and direct way. For this purpose, a coding DNA sequence is brought to expression, e.g., in the genome of a plant. This gene transfer is possible through a physical process (injection, gene gun) or a biological process (plasmids, viruses). It is possible to select a property in different kinds of organisms and insert it in the crop of interest enabling the enlargement of genetic variations.1 Gene technology has mainly been used to produce agriculturally improved plant varieties. The majority of plants commercialized are either herbicide tolerant (canola, sugar beet, chicory, soybean, flax, alfalfa, tobacco, rice, wheat, and maize), produce their own insecticide (Bt cotton, potato, maize, and tomato) or both (cotton, maize). Next to this, genes of interest can be related to delayed ripening, altered amino acid and fatty acid composition, starch hydrolysis, male sterility, virus and lepidopteran resistance.2 Genes have also been inserted that speed up the growth rate or lead to the synthesis of new proteins, leading to the development of so-called bio-factories, which produce substantial quantities of pharmaceuticals.3 In May 1994, the first genetically modified (GM) product for food use, the FlavrSavr™ tomato, was approved for commercial sale in the United States. Since then, the amount of GM crops has increased significantly. Nowadays, more than 80 different GM plants are commercially available for food and feed purposes all over the world. Next to the aforementioned GMOs, the list of approved GM crops worldwide also includes carnation, creeping bent grass, lentil, papaya, plum, squash, and sunflower.2 At present 60% of all soybeans on the world market are GM and almost one-fourth of all maize is GM, with numbers increasing every year. However, growth of GM soy and GM cotton in the United States is decreasing due to the growing market for ethanol.4–6 Although genetic engineering has emerged as one of the most powerful transforming technologies, no higher animals have been exploited as GMOs until now.7 To date, the only animals that have a chance of being genetically engineered for commercial purposes are fish species. The possible applications are medical research, increased food/feed production, and development of specific ornamental traits. The main constrain on the commercial use of these GMOs is the absence of laws regulating production, importation, and consumption of transgenic fish.8 The commercial pressure groups focus on developing rapidly growing fish with a highly efficient food conversion, but many questions remain on benefits and risks. Transgenic fish that escape into natural ecosystems could turn into an invasive species eliminating native fish populations since they often possess increased fitness or breeding capacities. The use of sterile GMOs might provide a solution for this problem. In spite of doubts on the commercial use of GMO fish, a number of transgenic fish are being developed on a lab scale for scientific research. Salmon, trout, tilapia, bass, catfish, and flounder have been genetically engineered for faster growth, higher disease resistance, and a better temperature tolerance.8
44.1.2 GMOs in Fish Feed Fish feed formulation will vary according to nutritional requirements of the species. Fish feed ingredients may be derived from GMOs with agronomical desirable traits.9 Soybean meal is
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universally available and has one of the best amino acids profiles of all protein-rich plant feedstuffs to meet most of the essential amino acid requirements of fish. Some fish such as young salmon, find soybean meal unpalatable while others, such as channel catfish, readily consume diets containing up to 50% soybean meal. For Egyptian sole (Solea aegyptiaca) juveniles for example, a diet with 56.2% fish meal, 5% maize gluten meal, and 4% of soy oil has been taken as a reference in replacement trials.9 In this study, growth performance was evaluated when fish meal was replaced by soybean meal (48% protein) up to an amount of 30%, with a subsequent reduction of fish meal down to 37.1%. According to the performance of sole during an experiment of 87 days, the amino acid profile of the soybean meal diets did not seem to influence the protein utilization negatively and this despite to a different content of certain essential amino acid such as methionine. Therefore, the authors suggest that soybean meal and even soy protein concentrates could be good replacers for fish meal.9 This could suggest an increasing use of soybean products in fish feed in the future. Meals from cottonseed and peanut have also been used in fish feeds in the United States, as well as meal from canola seeds for salmonids and lupin flour in feeds for rainbow trout. The use of GMO-derived products in fish feed does not influence the animal performance. Studies performed for catfish, rainbow trout, and salmon show that there is no difference in animal performance or the composition of the end product between animals fed with conventional crops and those fed with grain, silage, or byproducts derived from GM crops.10
44.1.3
International Regulations
GM crops have become part of the global feed and food market. In the United States, three independent authorities are involved in the regulation of the release of GM plants and their use in foodstuffs: Animal and Plant Health Inspection Service (APHIS), Food and Drug Administration (FDA), and Environmental Protection Agency (EPA). The authorization procedure is simple and based on the principle of substantial equivalence, which means that a GM product is in essence not distinct from a its conventional counterpart. The GM risk assessment focuses on human, animal, and environmental safety and there is no requirement for traceability or labeling of approved GMOs. Product tracing should only be considered in cases of food safety concerns, which is obtained through the governmental establishment of food performance standards for food producers and processors.11,12 The current European-based GM legislation is more complex and includes pre-authorization safety assessments13 by the European Food Safety Authority,14 availability of validated detection methods, reference materials, and thresholds for labeling,15,16 post-market monitoring, and postmarketing traceability requirements.17,18 More and more emphasis is also set on the coexistence of GM crops next to the conventional and organic production systems throughout the entire supply chain.19–22 Gene-stacked GMOs, which are the result of the crossbreeding between two GMO lines, require separate authorization in the EU. European Regulations 1829/2003 and 1830/2009/EC mandate the labeling and traceability of GMOs in the EU.15,17 Only in cases where the content of the authorized GMO ingredient is below 0.9% and in cases of accidental of adventitious presence of this GMO in the product, the labeling requirement may be omitted. Seed legislation requires that GM varieties have to be authorized in accordance with EU GMO legislation, in particular with Directive 2001/18/EEC before they are included in the Common Catalogue and marketed in the EU. If the seed is intended for use in food or feed, it can also be authorized in accordance with the GM food and feed Regulation. Similar labeling legislations exist in countries such as Russia (0.9%), Brazil (1%), and Japan (5%), whereas
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in Canada and the Philippines, GMO products require no labeling23 All these legislations, together with the Cartagena protocol,24 which has been established to preserve the worldwide biosafety, enforce the need for the identification and traceability of GMOs worldwide. For this purpose, methods to sample, detect, identify, quantify, and trace GMOs and derived products are necessary. GM crops can be detected either by searching for the altered DNA, by detecting the newly expressed proteins or by assessing the presence of the trait (bioassays). This chapter explores the existing methods based on DNA and protein detection. Advantages and limitations of both detection methods are discussed.
44.2 GMO Analysis 44.2.1 Introduction The manner in which to discriminate between GM versus nonmodified products is in most cases based on the presence of the newly introduced genes. Besides protein- and DNA-based methods, the so-called genetic analyses, biological (phenotypical), and chemical methods also exist. This chapter will focus on the genetic analyses. Methods have been developed either based on the detection of DNA using the polymerase chain reaction (PCR), or based on protein detection using enzyme-linked immunosorbent assays (ELISA). These methods however vary in their reliability, robustness and reproducibility, cost, complexity, and speed. Although PCR-based methods are known to be highly sensitive, the use of protein-based methods is in some cases more obvious.
44.2.2
DNA-Based Detection
44.2.2.1
Extraction of DNA
A first step in the DNA-based detection methods is the extraction of suitable DNA from the sample. In the context of GMO analysis, where GMO quantification is necessary according to legislation, it is of utmost importance to extract DNA from samples, which are homogeneous and representative for an entire lot or batch. DNA extraction methods may be based on the precipitation of DNA in a test tube, on the binding of DNA to a (silica) resin in an extraction column or on a combination of both. The choice for a specific extraction method may depend on the characteristics of the sample. For instance, for the extraction of DNA from soybean oil, a hexane-based extraction method may be used,25 whereas the extraction from particulate material may be performed using CTAB, as proposed by the European Committee for Standardization.26 The evaluation of the efficiency of different DNA extraction methods has shown that, due to the variability and complexity of food and feed products, the choice for a particular method should be done on a case-by-case basis.27–30 The suitability of a method may be tested by a DNA amplification test, which amplifies a target gene specific for the species under investigation (species- or taxon-specific PCR). For example, in the case of soybean the detection of the lectin gene is used as an endogenous control,31 for maize the zein gene32 or the invertase gene31 may be targeted (Figure 44.1).
44.2.2.2
Qualitative Conventional PCR
Depending on the sequences selected for PCR amplification, the detection of GMOs can be categorized into different levels of specificity: screening methods, gene-specific, construct-specific, and line- or event-specific methods (Figure 44.1).
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Gene or construct
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Species specific PCR
Screening PCR Screening PCR Construct-specific PCR Line-specific PCR
Figure 44.1 Novel gene construct integrated into a plant genome and the PCR amplification styles.
PCR screening methods target the regulatory elements that are used in the transformation process, e.g., the cauliflower mosaic virus (CaMV) 35S promotor, the nopalin synthase (nos) terminator, and the kanamycin resistance marker gene (nptII), which are present in many of the transgenic plants that have been developed so far. However, certain limitations of this method have been described. Because the CaMV 35S promotor, 3′-nos terminator, and nptII sequence may occur naturally, their detection does not necessarily prove that a GMO is present.33 Moreover, this screening system does not allow to discriminate between different transgenic lines and to identify them, since only the presence of one specific element is shown. GMO mixtures cannot be detected.34 Construct-specific PCR methods have also been derived for a range of different GMOs. The construct-specific PCR is based on the use of primer pairs that span the boundary of two (or more) adjacent introduced genetic elements (e.g., promotor, target gene(s), and terminator) or that are specific for the altered gene sequence.35 This construct-specific PCR will identify a specific insert and lines transformed with different inserts can be discriminated. However, lines with an identical insert cannot be distinguished and it is impossible to distinguish nonauthorized from authorized GMOs. Line-specific PCR detection methods target the junction at the integration site between the plant genome and the inserted DNA. This method was developed by the use of anchored PCR.35–37 Through the amplification of the left and the right border of the insertion site (i.e., the junction between the insert and the plant DNA), a typical fingerprint can be obtained. In the latter PCR one primer, labeled with a radioactive marker, is complementary to the DNA, which is integrated in the target genome, while the other is complementary to the adapter linked to a restriction site generated by a frequent cutting enzyme. As a result, a specific junction that spans the boundary between plant DNA and inserted DNA will be amplified. This junction fragment will be unique for each transgenic line. After sequencing this junction fragment, the obtained data of the flanking plant DNA makes it possible to design line-specific primers. The created linespecific primer and the anchored primer can then be used to perform line-specific PCR.35,38,39 All the line-specific detection methods for the EU-authorized GMOs have been validated by the Community Reference Laboratory (CRL) of the Joint Research Center (Ispra, Italy) and are available on the net (http://gmo-crl.jrc.it/statusofdoss.htm). In addition, different methods are available to confirm the PCR results: specific cleavage of the amplification product by restriction
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endonuclease digestion; hybridization with a DNA probe specific for the target sequence; direct sequencing of the PCR product; and finally nested PCR, in which two sets of primers bind specifically to the amplified target sequences.37,40
44.2.2.3
Multiplex PCR
The specific requirements for fast, multiple, and high-throughput GMO determination is fed by the increasing amount of transgenic events, the co-occurrence of several GMOs in one given matrix, and the customs formalities of countries with restricted import of GMOs. Each of these are a driving force to accelerate cost-effective methods assessing multiple GMO presence. Multiplex PCR methods based on the simultaneous amplification of different sequences save considerable time and effort by decreasing the number of reactions.23,41 The different size of the amplicons leading to differential migration through agarose or polyacrylamide gels makes it possible to differentiate between the different GMOs present. Capillary electrophoresis in combination with different fluorochromes for each amplicon is a useful alternative for size-based differentiation.42 A plethora of successful examples in which multiplex PCR has been applied successfully in detection of GMO are available for maize, canola, and soybean.43–45 In a study by Hernandez et al.,44 a multiplex PCR able to detect several GM maize lines proved to be 100% event-specific. Although this technique has the advantage of being time-saving, multiplex PCR often involves the amplification of larger amplicons and is therefore sometimes less suitable to assess GMO presence in processed food or feed since DNA degradation precludes the survival of long stretches of intact DNA. Furthermore, multiplex PCR is less sensitive compared to simplex (conventional) PCR methods due to competition between amplicons, depletion of reagents in the PCR tube, and the production of aspecific PCR products. In this context, Morisset et al.46 published a review on alternative DNA amplification methods that could be used in GMO detection to overcome these problems.
44.2.2.4
Quantitative PCR
Because in several countries maximum limits are set for the (accidental) presence of GMOs in food and feed products, a critical aspect of the GMO analysis is the quantification. Initially quantitative competitive PCR was developed. The principle of this method is the co-amplification of internal DNA standards together with target DNA and their subsequent quantification. Therefore, DNA standards were constructed in a similar manner to the construction of the target DNA, but these are distinct from the GMO DNA by specific sequence insertions. The system is calibrated by coamplification of mixtures of GMO DNA and corresponding amounts of conventional DNA. Such standards are commercially available and contain known amounts of standard DNA. Determination of the so-called equivalence point is the basis for quantification.47 The system has been tested on several samples such as soy and maize.48–50 PCR–ELISA uses the strategy of real-time PCR and can be quantitative when the PCR is stopped before a significant decrease in amplification efficiency occurs (i.e., before the plateau phase is reached). Then ELISA can be used to quantify the relatively low amounts of PCR products.51,52 Real-time PCR (qPCR), an online method for determining ratios of transgenic to nonmodified DNA components, is the method of choice at the moment to detect and quantify DNA. This real-time PCR allows the quantification of DNA by measuring the kinetics of the PCR amplification instead of an endpoint measurement. This continuous measurement is based on the accumulation of a fluorescence signal. A signal is created by the binding of a fluorogene compo-
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nent to the amplified DNA and is proportional to the amounts of PCR products generated. There exist different strategies: the detection is either based on the measurement of the accumulation of DNA-binding dyes (e.g., SYBR Green) or the binding of specific labeled probes (e.g., TaqMan probe). During PCR, the accumulation of fluorescence can then be measured online. With the first method, the DNA-binding dye does not discriminate between the target and any co-amplified nontarget sequence, e.g., primer dimers. Still it is possible to verify the identity of the amplified dsDNA by melting curve analysis. The reassociation of the dsDNA will result in the creation of a peak signal and the specific temperature profile of the peak is characteristic of a specific sequence. Nevertheless, in the presence of more than one GMO in a sample, it is difficult to quantify each specific target DNA sequence. With probes, the reliability of the real-time PCR increases significantly. These probes can be divided into different categories: hydrolysis and hybridization probes. Hydrolysis probes emit fluorescence upon degradation and require that the two fluorophores are brought into close proximity by hybridization and/or altered configuration of the probes. Hybridization probes emit fluorescence upon specific hybridization to the target amplicon sequence and require that the two fluorochromes are sufficiently separated in space by hydrolysis and/or altered configuration of the probes.53 The most commonly used real-time probe is the TaqMan probe, a hydrolysis probe where reporter and quencher are located on the same probe within a short distance apart and where the hydrolysis of the probe results in the release of the reporter from the quencher. This results in a fluorescence signal, emitted by the reporter dye. In the field of GMO analysis, the quantification requires simultaneous assessment of the recombinant and of a species- or taxon-specific reference gene. The quantification standards are usually certified reference material or cloned plasmid standards.23,54 The method is well suited for automation and high throughput of samples and can be used for raw, processed, and even mixed products.31,55,56
44.2.3
Protein-Based Detection
The majority of protein detection methods are based on immunoassays, i.e., they rely on the interaction between a specific antibody and its antigen. Due to this strong interaction, immunoassays are highly specific and generally samples only need a simple preparation before analysis. The Western blot technique is based on the separation of total protein on a gel followed by staining of the target protein with a labeled antibody. The method is highly specific, which provides qualitative results for determining whether a sample contains the target protein below or above a predetermined threshold level.57 The ELISA is the most common type of immunoassay. It covers any enzyme immunoassay involving an enzyme-labeled immunoreactant (antigen or antibody) and an immunosorbent (antigen or antibody bound to a solid support). Therefore, several variants of the ELISA-method exist, with the sandwich assay being the most widely used and most flexible type of ELISA. The detections of GM proteins in feed, based on commercially available ELISA kits, are mostly sandwich assays, although some are competitive ELISA assays.58 For the sandwich ELISA, antibodies specific to the target protein are bound to the surface of typically a microtiter well plate. When the solution containing the test material is added, the antibody will work as a capture molecule for the target protein. Following an incubation period, a washing step removes all unbound components. Then a second specific antibody, chemically bound to an enzyme that catalyzes a color reaction, is added. If the target protein is present, the second labeled antibody binds to it and any unbound labeled antibody is washed away. Enzyme substrate
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is then added to yield a colored product. The intensity of the signal produced is proportional to the amount of target protein present.57 In a competitive ELISA assay, the wells of the plate are coated with the target protein. A solution containing a limited number of first antibody together with the test sample is added. A competition for the first antibody will then occur between the target protein in the sample and the target protein coated on the wells. The antibodies that are not bound to the antigens bound to the well will be washed away. A second antibody–enzyme complex is then added which specifically binds the antigen–antibody complex. After a second washing step, an enzyme substrate is added, resulting in color production. For the competitive assay, the intensity of the color is inversely proportional to the concentration of the GM protein in the sample.59 Some commercial ELISA plate kits are supplied with calibrators (known concentrations of the target analyte in solution) and a negative control. These standards are run concurrently with each sample set and allow a standard curve to be set up. By using the spectrophotometer for all samples and all standards at the same time, a quantitative interpretation can be performed. The protein concentration in the sample can then be calculated from the standard curve. A semiquantitative interpretation can be made by comparing the color of the sample against the standards without the use of a spectrophotometer and determining the concentration range of the sample.58 The lateral flow strip is a variation on ELISA, using strips rather than microtiter well plates. The test strip is placed in an Eppendorf vial containing the test solution. The sample migrates up the strip by capillary action. As it moves up the strip, the sample passes through a zone that contains mobile antibodies, usually labeled with colloidal gold. This labeled antibody binds to the target protein, if present in the sample. The antibody–protein complex then continues to move up the strip through a porous membrane that contains two capture zones. The first capture zone of immobilized antibodies binds the antibody–protein complex and the gold becomes visible as a red line. The second capture zone acts as a control zone and contains antibodies specific for untreated antibodies coupled to the color reagent. If there is no target GM protein present, only one single line will appear at the control zone. A result is called positive when both the control line and the line indicating presence of target protein change color.58 The test is simple, cheap, and fast, and does not require any laboratory facilities or technical expertise. Van den Bulcke et al.60 compared the use of this strip test with PCR analysis for the detection of GM soy in feed samples. In most cases, matches were found between the obtained results. However, in cases of low level of GM soy the PCR analysis gave more positive results, due to its higher sensitivity. The strip test however remains a good tool in the traceability of GMOs early in the chain. Some other drawbacks are discussed later in this chapter. Another format of immunoassays uses magnetic beads as a solid surface. The principle is the same as the ELISA-format, with the magnetic particles being coated with capture antibodies and the reaction being performed in a test tube. The target protein, bound to the magnetic particles, can be separated using a magnet. The advantages include superior kinetics, as the particles are free to move in the reaction solution, and increased precision, due to uniformity of the particles.52 A new evolution in protein-based assays is high-throughput technologies that have gained a great deal of attention over the last several years. The high throughput is obtained by miniature and highly sensitive microfluidic devices.61 Although very small volumes are used, the technique remains reliable compared to the traditional immunoassays.62 The device consists of a flat surface, usually glass or silicon, onto which narrow bands of antigen are deposited. Perpendicular to these stripes, a second set of lines is engraved. When diluted proteins flow through this second set of small channels, induced by capillary forces, they bind with their specific antigen, and a mosaic pattern of tiny squares occurs. This can then be analyzed using fluorescence microscopy.62 The
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advantages of this technique are numerous. One sample can be screened for several proteins in one test and several assays can be performed simultaneously. The amount of sample needed is reduced to nanoliter range, thereby reducing the time to perform the analysis. In addition, this technique is highly suitable for automation. However, the equipment needed is expensive and skilled personnel are required for the analysis of results. Therefore, this technique is currently not used for routine analysis of feed samples.
44.3
Comparison of DNA and Protein Methods
44.3.1 Protein-Based Methods GMOs can be detected on basis of the altered genome, thus by DNA analysis, or on basis of the novel protein. The protein-based assays comprise a very large and diverse group of assays and are commercially very successful. The strong interaction between the antibody and the antigen is translated into high sensitivity assays and antibody specificity minimizes sample preparation. Their cost, ease-of-use, and flexible test format have resulted in wide-scale use in highly diverse markets. The cost per test of an immunoassay compared to other analytical methods is low and a number of test formats exist, which require little or no training to perform the analysis. For instance, the one-step “strip” tests can be performed by untrained personnel and give yes/no type results in minutes. The time-to-result may be one of the most critical attributes of a test method in applications where a large lot of material needs to be screened before being pooled with other lots or proceeded to the next stage in a process. For on-site testing, the lateral flow test is generally the most cost-effective solution.63 Immunoassays can yield quantitative results in about an hour or they can be incorporated into fully automated instruments capable of running hundreds of samples an hour. Quantification of proteins is easier than quantification of DNA if one looks at the expression unit of GMO levels. Quantitative results from protein analyses are expressed on a weight/weight basis (molar concentrations), while those from DNA assays represent genome equivalents. The influence of gene copy numbers makes DNA quantification more complex and the results more uncertain.37 Despite the advantages protein-based methods offer, many drawbacks need to be considered when it comes to their application for the detection of GMOs. In those cases, the DNA-based method is preferred. One of the difficulties of an immunoassay system is experienced at the outset. Generating a specific antibody against the antigen of concern can be a slow, difficult, and time-consuming process, and requires many skills and a lot of experience. This is where the use of recombinant antibody technology could offer benefits. Using this technique, it will be easier to select antibodies with rare properties and to manipulate the characteristics of already existing antibodies.64 Once a specific (monoclonal or polyclonal) antibody with high affinity for its target protein is generated, careful standardization and testing for unexpected cross-reactivity must be performed. Nonetheless, once established, the flexibility and turnaround time for immunoassay systems is excellent.65 Since the binding between antibody and antigen is based on the native structure of the target, any conformational change in the epitope of the antigen renders the assay ineffective. Food and feed processing, such as heating or exposure to strong acids or alkalis, can cause this conformational change, by which detection of the protein will no longer be possible. Therefore, protein detection using immunoassays is limited to grains, raw materials, and unprocessed products.66 Furthermore, the accuracy and precision of the assay can be affected by substances present in
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feed matrices such as surfactants, phenolic compounds, fatty acids, endogenous phosphatases, or enzymes that may inhibit the specific antigen–antibody interaction.64–67 Another disadvantage of the immunoassays is due to the fact that they rely on the expression of the newly introduced gene, e.g., genetic modification does after all not always result in the production of a new protein or the expression level of the introduced DNA can be too low for detection. Moreover, the protein levels can vary from tissue to tissue. As a result, the transgenic protein might not be present in the part of the plant that is used in feed production. For example, the endotoxin cry1A of GM maize Bt-176 is expressed in the green tissues and in the pollen of the plant. Since the novel protein is not expressed in the maize kernels, protein analysis of the kernels will not reveal the presence of any foreign protein.68 Expression is furthermore influenced by external factors such as weather, soil, and other cultivation conditions.69 This complicates the quantification of the GMO by proteinbased detection methods. Quantification by the use of immunoassays is already a complex subject, since the assays generate absolute values, such as the total amount of a novel protein present. To comply with GMO labeling legislation however, not the absolute quantity but the relative quantity of the GM trait is important, i.e., the relative ratio of the GM trait to the conventional counterpart of the same ingredient (e.g., percentage of GM soy out of total soy in the feed product). The relative proteinbased quantification is only possible if a species- or taxon-specific protein is measured simultaneously70 or if the sample exists of one single ingredient and an appropriate reference material for the standard dose response curve is available. Since there are no structures common to all GM proteins or groups of proteins and a single antibody will only bind to one particular protein, the immunoassays are less suited for a general GM screening.35 In the future, it is likely that tests will be developed that detect multiple novel proteins using a single lateral flow strip. Moreover, protein-based methods cannot distinguish between GM varieties with a different genetic construct but the same expressed protein.38,71,72
44.3.2 DNA-Based Methods The suitability of the PCR technique to detect genetic modifications in feedstuffs is determined by the quality and quantity of the DNA still present in the final product. Various factors can contribute to the degradation of the DNA such as heat treatments, nuclease activity, and low pH. In a typical feed-pelleting process, the feedstuff is first moisture conditioned, then subjected to pressures, and finally, with the aid of the associated heat generated, forced through die openings. The larger particles or pellets formed are easier to handle, more palatable, and usually result in improved feeding results when compared to the unpelleted feed. The extrusion of cereals aims at the complete rupture of the starch granules by a combination of moisture, heat, pressure, and mechanical shear. Although extensive research has already been performed on the effect of food processing on the stability and detection of DNA,73–76 research on feed products is poor.77,78 Gawienowski et al.74 and Aulrich, Pahlow, and Flachowsky79 have investigated the effect on ensilage of maize, showing that DNA is degraded due to a pH drop with time. Dry and steam heating under laboratory conditions by Forbes et al.80 and Chiter, Forbes, and Blair81 indicated that temperatures above 95°C for at least 5 min completely fragment DNA, whereas physical extrusion highly fragments DNA. Results indicate that DNA of sufficient quality and quantity remained detectable after steam conditioning at 95°C, allowing subsequent GMO analysis (personal communication). However, the extrusion of feed samples, where temperatures up to 135°C were applied, led to the degradation of
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DNA. A lower moisture content even emphasizes the destruction of DNA. A significant decrease in the average DNA fragment length may render the detection of the inserted DNA sequence impossible. Other processing steps may remove DNA from the sample, as during the refining of glucose syrup, soy lecithin, or oil.82,83 In these cases, DNA is depleted to very small quantities, rendering detection impossible with the current official ISO detection methods.75,84,85 Furthermore, potential problems will arise with respect to adequate sampling (homogeneity and representativity) and PCR inhibitors in the feed matrix. Inhibition of the DNA polymerase by co-purification of proteins, fats, polysaccharides, polyphenolics, and other components present in highly processed feedstuffs is a major problem in the preparation of DNA for PCR.86 These circumstances may not only influence GMO screening, construct-, gene-, and linespecific PCRs, but may render the quantification of GMOs inaccurate. Although validated by the Community Reference Laboratory (CRL, Ispra, Italy), these methods are rarely tested and optimized for DNA samples extracted from processed food or feed samples.87,88 Problems encountered with the quantification of GMOs are translated in high limits of detection and quantification. Furthermore, as GMO quantification is based on the amplification of two DNA fragments, different PCR amplification efficiencies for those fragments may divert the calculated GMO content from its true value.88 Another problem encountered with GMO quantification is due to the ploidy of the material under investigation, as GMO percentages should be expressed in terms of haploid genome equivalents. Since the true level of zygosity or ploidy of the material to be analyzed is not always known, a high degree of measurement uncertainty is associated with quantitative analytical estimates.33 All these inconveniences result in a standard deviation of 20%–30% for quantitative GMO analysis.
44.4 Recent Developments in GMO Detection Conventional methods for GMO detection have their own limitations. Qualitative PCR reactions are time-consuming and technically demanding, quantitative PCRs require internal standards and protein-derived methods often suffer from protein denaturation.89 Therefore, new technologies are under development in order to overcome these shortcomings.
44.4.1 Microarray Technology From an economic point of view, microarray technology is very promising by viewing its feature of simultaneous identification of multiple GMOs. In addition compared to multiplex PCR, it has the advantage of being a more flexible tool not being hampered by the range of GMOs, the amplicon size or the number of amplicons in the analysis. This is a direct consequence of the fact that microarrays are two-dimensional compared to classical gel electrophoresis rendering it more discriminative. The principle of microarray analysis is the attachment of nucleic acids to a solid support (often glass), which is subsequently probed by labeled nucleic acid molecules. Although microarray technology has its main applications in transcriptome analysis, some specific arrays were developed for GMO detection and characterization. Several low-density arrays allowing the simultaneous detection of nine GMO events including the GMO screening elements (35S promotor, nos terminator, and nptII) were developed on a laboratory scale.90,91 The enormous data obtained after a microarray analysis however is probably one of the only restraints that has hampered this method being applied routinely. To our knowledge, only a few
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studies resulted in the commercially available microarray device.92 A commercial kit, DualChip® GMO has been developed by Eppendorf array technology by coupling multiplex PCR to microarray hybridization and as such combining the best of both worlds. The systems detects and identifies GM events by screening multiple genetic elements, namely, Bt-176 maize, Mon 810 maize, Bt-11 sweet maize, Mon 531 cotton, GA21 maize, and Roundup Ready™ soja GMO events.89
44.4.2 Biosensors The major restraint on the use of PCR-derived techniques in commercial samples is undoubtedly the highly technical demand. The use of biosensors could be a useful alternative to detect GMOs in the future. The principle of a sensor is that a change in the probe environment results in a specific change in a physical property of the probe, which in turn is converted into an electrical signal. The development of different types of biosensors is in progress. In the work by Mannelli et al.93,94 two different types of sensors were developed. A bulk acoustic wave affinity biosensor for GMO detection was developed. This device consisted of a DNA probe immobilized on the sensor surface while the target sequence was free in solution and available for hybridization.93 Probe immobilization was monitored by a surface plasmon resonance (SPR) system. The same group described a piezoelectric sensor based on the immobilization of single-stranded DNA probes on the surface of a quartz crystal microbalance device.94 Since its development, SPR technology has been successfully used for the detection of Roundup Ready® soybean.95 In this application, household and transgene sequences were mounted on the sensor chips and were shown to detect the transgene in real-time formats. In another approach, biotinylated PCR products containing 0.5% and 2% of Bt-176 sequences were mounted on a chip. After immobilization, the authors succeeded to discriminate between both Bt-176 concentrations making them conclude that biosensor technology has the capacity to be as efficient as real-time PCR. Even though biosensors may not be able to entirely replace the use of PCR for the accurate determination of GMO in products, they can nonetheless be useful in the preliminary stages of control to identify samples to be subjected to successive analyses.23
44.5 Conclusions A wide variety of protein- and DNA-based formats are available to analyze products for the presence of GMOs. Immunoassays are very suitable for the detection of GMOs at critical control points early in the feed production chain, i.e., on raw materials, unprocessed ingredients, and simple food or feed matrices (single ingredients products or products with only one GMO ingredient). Application on processed and complex matrices is limited. Thanks to the higher stability of DNA compared to proteins, PCR-based methods are currently the methods of choice in the field of GMO detection and quantification. In cases where no protein or DNA can be detected in the feed sample, enforcement of the legislation should be supported by traceability measures. It is to be expected that in the near future, an increasing number of GM crops will be released on the market, producing an increased variety of genetic constructs present in food and feed. To challenge the entrance of these new GM products, high throughput technologies will be required. The multiplex amplification method, possibly in combination with microarray format detection,
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will be a very interesting option in order to decrease the number of assays needed for efficient identification and quantification of feedstuffs containing GM material. With respect to the current quantification of GMOs, which is mostly based on real-time PCR quantification, still work has to be done to improve the accuracy of the available methods. Other issues that will need attention in the following years are the detection of unauthorized GMOs, the detection and quantification of stacked genes (because no distinction can be made between the gene-stacked GMO and a mixture of its two parental GMOs), the optimization of extraction methods and sampling procedures, and the development of reliable field tests for all GMOs. This could be obtained by the use of lateral flow test that are able to detect different GMOs at the same time. Some tests are already commercially available.
References 1. Gryson, N., Messens, K., and Dewettinck, K., Genetically modified organisms in food industry, in Handbook of Food Science, Technology and Engineering, Hui, Y.H., Ed., CRC Press, Boca Raton, FL, 2006. 2. Agbios, Agriculture and Biotechnology Strategies, Canada Inc. GMO database, Available from: http:// www.agbios.com. 2009. 3. Ivarie, R., Competitive bioreactor hens on the horizon, Trends Biotechnol., 24, 99, 2006. 4. James, C., Executive Summary of Global Status of Commercialised Biotech/GM Crops, ISAAA Briefs 34, Ithaca, NY, 2005. 5. James, C., Executive Summary of Global Status of Commercialised Biotech/GM crops, ISAAA Briefs 37, Ithaca, NY, 2007. 6. Greiner, R. and Konietzny, U., Presence of genetically modified maize and soy in food products sold commercially in Brazil from 2000 to 2005, Food Control, 19, 499, 2008. 7. Maga, E.A., Genetically engineered livestock: Closer than we think? Trends Biotechnol., 23, 533, 2005. 8. Varzakas, T.H., Arvanitoyannis, I.S., and Baltas, H., The politics and science behind GMO acceptance, Crit. Rev. Food Sci., 47, 335, 2007. 9. Bonaldo, A. et al., Influence of dietary soybean meal levels on growth, feed utilization and gut histology of Egyptian sole (Solea aegyptiaca) juveniles, Aquaculture, 12(2), 580, 2006. 10. Anonymous, Safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials, Food Chem. Toxicol., 46, S1, 2008. 11. Gryson, N., Rotthier, A., Messens, K., and Dewettinck, K., Regulation of genetically-modified crops in the European Union, Lipid Technol., 17(1), 11, 2005. 12. USDA-APHIS (US Department of Agriculture, Animal and Plant Health Inspection Service), Factsheet on Biotechnology Regulatory Services, USDA’s Biotechnology Deregulation Process. Fabruary 2006, Available from http://www.aphis.usda.gov/. 13. European Commission, Directive 2001/18/EC of the European Parliament and of the Council of 17 April 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC, Off. J. Eur. Commun., L106, 1, 2001. 14. European Commission, Regulation (EC) 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety, Off. J. Eur. Commun., L31, 1, 2002. 15. European Commission, Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed, Off. J. Eur. Commun., L268, 1, 2003.
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16. European Commission, Commission Regulation (EC) No 641/2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003 of the European Parliament and of the Council as regards the application for the authorisation of new genetically modified food and feed, the notification of existing products and adventitious or technically unavoidable presence of genetically modified material which has benefited from a favourable risk evaluation, Off. J. Eur. Commun., L102, 14, 2004. 17. European Commission, Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC, Off. J. Eur. Commun., L268, 24, 2003. 18. European Commission, Commission Regulation (EC) No 65/2004 of 14 January 2004 establishing a system for the development and assignment of unique identifiers for genetically modified organisms, Off. J. Eur. Commun., L10, 5, 2004. 19. European Commission, Commission recommendation 2003/556/EC of 23 July 2003 on guidelines for the development of national strategies and best practices to ensure the co-existence of genetically modified crops with conventional and organic farming, Off. J. Eur. Commun., L189, 36, 2003. 20. European Commission, Commission decision 2005/463/EC of 21 June 2005 establishing a network group for the exchange and coordination of information concerning coexistence of genetically modified, conventional and organic crops, Off. J. Eur. Commun., L164, 50, 2005. 21. Gryson, N. et al., Co-existence and traceability of GM and non-GM products in the feed chain, Eur. Food Res. Technol., 226, 81, 2007. 22. Gryson, N. and Eeckhout, M., Co-existence of GM and non-GM crops: Vision of the European Commission anno 2007 (Co-existentie van GGO en non-GGO gewassen: Visie van de Europese Commissie anno 2007), De Molenaar, 1, 24, 2008. 23. Marmiroli, N. et al., Methods for detection of GMOs in food and feed, Anal. Bioanal. Chem., 392, 369, 2008. 24. Cartagena protocol on biosafety to the convention on biological diversity. Montreal, Quebec, Canada. Available from: http://www.biodiv.org/doc/legal/cartagena-protocol-en.pdf. 25. Wurz, A. et al., DNA-Extraktionsmethode für den Nachweis gentechnisch veränderter Soja in Sojalecithin, Deut. Lebensm.-Rundsch., 94(5), 159, 1998. 26. International Organization for Standardization, ISO 21571, Foodstuff s—Methods of Analysis for the Detection of Genetically Modified Organisms and Derived Products—Nucleic Acid Extraction, ISO, Geneva, Switzerland, 2005. 27. Di Pinto, A. et al., A comparison of DNA extraction methods for food analysis, Food Control, 18, 76, 2007. 28. Gryson, N., Messens, K., and Dewettinck, K., Evaluation and optimalisation of five different extraction methods for soya DNA in chocolate and biscuits. Extraction of DNA as a first step in GMO analysis, J. Sci. Food Agr., 84, 1357, 2004. 29. Holden, M.J. et al., Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA, J. Agric. Food Chem, 51, 2468, 2003. 30. Mafra, I., Ferreira, I., and Oliveira, M., Food authentication by PCR-based methods, Eur. Food Res. Technol., 227, 649, 2008. 31. Berdal, K.G. and Holst-Jensen, A., Roundup Ready soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses, Eur. Food Res. Technol., 213, 432, 2001. 32. Peano, C., Samson, M.C., Palmieri, L. Gulli, M., and Marmiroli, N., Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods, J. Agric. Food Chem., 52, 5829, 2004. 33. Miraglia, M. et al., Detection and traceability of genetically modified organisms in the food production chain, Food Chem. Toxicol., 42, 1157, 2004.
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34. Pietsch, K. et al., Screeningsverfahren zur identifizierung gentechnisch veränderter pflantelicher Lebensmittel, D. Lebensm.-Rundsch., 93, 35, 1997. 35. Griffiths, K. et al., Review of Technologies for Detecting Genetically Modified Materials in Commodities and Food. Department of Agriculture, Fisheries and Forestry, Australia, 126, 2002. 36. Windels, P. et al., Characterisation of the Roundup Ready soybean insert. Eur. Food Res. Technol., 213(2), 107, 2001. 37. Anklam, E. and Neumann D.A., Method development in relation to regulatory requirements for detection of GMOs in the food chain,. J. AOAC Int., 85, 754, 2002. 38. Gachet, E. et al., Detection of genetically modified organisms (GMOs) by PCR: A brief review of methodologies available, Trends Food Sci. Technol., 9, 380, 1999. 39. Meyer, R., Development and application of DNA analytical methods for the detection of GMOs in food, Food Control, 10, 391, 1999. 40. Tengs, T. et al., Microarray-based method for detection of unknown genetic modifications, BMC Biotecnol., 7, 91, 2007. 41. Mafra, I. et al., Comparative study of DNA extraction methods for soybean derived food products, Food Control, 19, 1183, 2008. 42. Nadal, A. et al., A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events, Electrophoresis, 27, 3879, 2006. 43. Germini, A. et al., Determination of transgenic material on the Italian food market using a new multiplex PCR method, Ital. J. Food Sci. 17, 371, 2005. 44. Hernandez, M. et al., Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21, J. Agric. Food Chem., 53, 3333, 2005. 45. James, D. et al., Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis, J. Agric. Food Chem., 51, 5829, 2003. 46. Morisset, D. et al., Alternative DNA amplification methods to PCR and their application in GMO detection: A review, Eur. Food Res. Technol., 227, 1287, 2008. 47. Raeymackers, L., Quantitative PCR: Theoretical considerations with practical implications, Anal Biochem., 214, 582, 1993. 48. Wurz, A. et al., Quantitative analysis of genetically modified organisms in processed food by PCRbased methods, Food Control, 10, 385, 1999. 49. Zimmerman, A., Lüthy, J., and Pauli, U. Event specific transgene detection in Bt11 corn by quantitative PCR at the integration site, Lebensm. Wiss. Technol., 33, 210, 2000. 50. Studer, E. et al., Quantitative competitive PCR for the detection of genetically modified soybean and maize, Z. Lebensm. Unters. For. A, 207, 207, 1998. 51. Landgraf, A., Reckmann, B., and Pingoud, A., Direct analysis of polymerase chain reaction products using Enzyme-Linked Immunosorbent Assay techniques, Anal. Biochem., 198, 86, 1991. 52. Ahmed, F.E., Detection of genetically modified organisms in food, Trends Biotechnol., 20, 215, 2002. 53. Holst-Jensen, A., Ronning, S.B., Lovseth, A., and Berdal, K.G., PCR technology for screening and quantification of genetically modified organisms (GMOs), Anal. Bioanal. Chem., 375, 985, 2003. 54. Taverniers, I., Windels, P., Vaitilingom, M., Milcamps, A., Van Bockstaele, E., Van den Eede, G., and De Loose, M., Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola, J. Agric. Food Chem., 53, 3041, 2005. 55. Greiner, R., Konietzny, U., and Villavicencio, A.L.C.H., Qualitative and quantitative detection of genetically modified maize and soy in processed foods sold commercially in Brazil by PCR-based methods, Food Control, 16, 753, 2005. 56. Hernàndez, M. et al., A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard® based on the 3′-transgene integration sequence, Transgenic Res., 12, 179, 2003.
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57. Lipton, C.R. et al., Guidelines for the validation and use of immunoassays for determination of introduced proteins in biotechnology enchanced crops and derived food ingredients, Food Agric. Immunol., 12, 153, 2000. 58. Rotthier, A. et al., Protein-based detection of GM ingredients, in Advances in Food Diagnostics, Nollet, L. and Toldrá, F., Eds., Blackwell Publishing, Ames, IA, 10, 199, 2007. 59. Yeung, J. Enzyme-linked immunosorbent assays (ELISAs) for detecting allergens in foods, in Detecting Allergens in Food, Koppelman, S. and Hefle, S., Eds., Woodhead Publishing in Food Science and Technology, Cambridge, U.K., 109, 2006. 60. Van de Bulcke, M. et al., Detection of genetically modified plant products by protein strip testing: An evaluation of real-life samples, Eur. Food Res. Technol., 225, 49, 2007. 61. Burns, M.A. et al., Microfabricated structures for integrated DNA analysis, Proc. Natl. Acad. Sci. USA, 93(11), 5556, 1996. 62. Bernard, A., Michel, B., and Delamarche, E., Micromosaic immunoassays, Anal. Chem., 73, 8, 2001. 63. Stave, J.W., Detection of new or modified proteins in novel foods derived from GMO - future needs, Food Control, 10, 367, 1999. 64. Brett, G.M. et al., Design and development of immunoassays for detection of proteins, Food Control, 10, 401, 1999. 65. Bindler, G. et al., Report of the CORESTA Task Force Genetically Modified Tobacco: Detection methods, CORESTA, 1, 1998. 66. Anklam, E. et al., Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products, Eur. Food Res. Technol., 214, 3, 2002. 67. Spiegelhalter, F., Lauter, F.R., and Russel, J.M., Detection of genetically modified food products in a commercial laboratory, J. Food Sci., 66, 634, 2001. 68. Popping, B. and Broll, H., Detection of genetically modified foods—Past and future, Actual. Chim., 11, 3, 2001. 69. Wilson, R.F., Hou, C.T., and Hildebrand, D.F., Eds., Dealing with Genetically Modified Crops, AOCS Press, Champaign, IL, 2001. 70. Van Duijn, G. et al., Detection of genetically modified organisms in foods by protein- and DNAbased techniques: Bridging the methods, J. AOAC Int., 85, 787, 2002. 71. Lüthy, J., Detection strategies for food authenticity and genetically modified foods, Food Control, 10, 359, 1999. 72. Kok, E.J. et al., DNA methods: Critical review of innovative approaches, J. AOAC Int., 85, 797, 2002. 73. Chen, Y. et al., Degradation of endogenous and exogenous genes of Roundup-Ready soybean during food processing, J. Agric. Food Chem., 53, 10239, 2005. 74. Gawienowski, M.C. et al., Fate of maize DNA during steeping, wet-milling, and processing, Cereal Chem., 76, 371, 1999. 75. Gryson, N., Stability and detection of genetically modified soy in processed foods, PhD dissertation, 2006. 76. Gryson, N., Messens, K., and Dewettinck, K., PCR detection of soy ingredients in bread, Eur. Food Res. Technol., 227, 345, 2008. 77. Alexander, T.W. et al., A review of the detection and fate of novel plant molecules derived from biotechnology in livestock production, Anim. Feed Sci. Technol., 133, 31, 2007. 78. Alexander, T.W. et al., Impact of feed processing and mixed ruminal culture on the fate of recombinant EPSP synthase and endogenous canola plant DNA, FEMS Microbiol. Lett., 214, 263, 2002. 79. Aulrich, K., Pahlow, G., and Flachowsky, G., Influence of ensiling on the DNA-degradation in isogenic and transgenic corn, Proc. Soc. Nutr. Physiol., 13, 112, 2004. 80. Forbes, J.M. et al., Effect of feed processing conditions on DNA fragmentation, Scientific Report No. 376 to the Ministry of Agriculture, Fisheries and Food, United Kingdom, 1998.
Detection of GM Ingredients in Fish Feed
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887
81. Chiter, A., Forbes, J.M., and Blair, G.E., DNA stability in plant tissues: Implications for the possible transfer of genes from genetically modified food, FEBS Lett., 481, 164, 2000. 82. Gryson, N. et al., Detection of DNA during the refining of soy bean oil, J. Am. Oil Chem. Soc., 79, 171, 2002. 83. Gryson, N., Messens, K., and Dewettinck, K. Influence of different oil refining parameters and sampling size on the detection of GM-DNA in soybean oil, J. Am. Oil Chem. Soc., 81, 231, 2004. 84. International Organization for Standardization, ISO 21569, Foodstuff s—Methods of Analysis for the Detection of Genetically Modified Organisms and Derived Products—Qualitative Nucleic Acid Based Methods, ISO, Geneva, Switzerland, 2005. 85. International Organization for Standardization, ISO 21570, Foodstuff s—Methods of Analysis for the Detection of Genetically Modified Organisms and Derived Products—Quantitative Nucleic Acid Based Methods, ISO, Geneva, Switzerland, 2005. 86. Gryson, N., Dewettinck, K., and Messens K., Influence of cocoa components on the PCR detection of soy lecithin, Eur. Food Res. Technol., 226, 247, 2007. 87. Trapman, S. and Emons, H., Reliable GMO analysis, Anal. Bioanal. Chem., 381, 72, 2005. 88. Weighardt, F., GMO quantification in processed food and feed, Nat. Biotechnol., 25, 1213, 2007. 89. Lasken, R.S. and Egholm, M., Whole genome amplification: Abundant supplies of DNA from precious samples or clinical specimens, Trends Biotechn., 21, 531, 2003. 90. Leimanis, S. et al., A microarray-based detection system for genetically modified (GM) food ingredients, Plant Mol. Biol. 61, 123, 2006. 91. Xu, X.D. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray, J. Agric. Food Chem., 53, 3789, 2005. 92. Birch, L. et al., Evaluation of LabChip™ technology for GMO analysis in food, Food Control, 12, 535, 2001. 93. Mannelli, I. et al., Bulk acoustic wave affinity biosensor for genetically modified organisms detection. IEEE Sens. J., 3, 369, 2003. 94. Mannelli, I. et al., Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection, Biosens. Bioelectron., 18, 129, 2003. 95. Feriotto, G. et al., Biosensor technology and surface plasmon resonance for real-time detection of genetically modified Roundup Ready soybean gene sequences, J. Agric. Food Chem., 50, 955, 2002.
Index A Acanthobrama marmid, 768 Acanthocephalans, in humans, 595–596 Acanthocephalus bufonis, 595 Acanthocephalus rauschi, 595 Acanthopagrus latus, 364 Accelerated solvent extraction system (ASE), 718, 798, 801–802, 820; see also Dioxin analysis, from seafoods Acid hydrolysis, in vitamin B6 determination, 338 Acid-soluble collagen, 14 Acinetobacter lowffi, 839 Acipenser gueldenstaedtii, 364 Acipenser nudiventris, 364 Acipenser persicus, 364 Acipenser stellatus, 364 Active and intelligent packaging, in seafoods, 867–869 Active oxygen method (AOM), 92 1-Adamantylamine (ADAM), 295 Adenosine-derived nucleotides, structure, 60; see also Seafood and seafood products Adenosine monophosphate (AMP), 58 Adulteration of seafood, detection; see also Seafood and seafood products foreign proteins chromatographic techniques, 679 DNA technologies, 679–683 electrophoresis, 676–677 immunological techniques, 677–678 microscopic methods, 678–679 visible and near-infrared spectrometry, 678 seafood sp. identification DNA-based techniques, 692–703 future perspectives, 703–704 protein based methods, 691–692 species replacement, 688–691 Aerobic bacteria plate count (APC), 788 Aerobic plate count (APC), 203 Agar Listeria according to Ottaviani and Agosti (ALOA), 568
Agmatine, as fish freshness indicators, 836–837 Alkali-soluble collagen, 14 2-Alkylcyclobutanones (2-ACBs), analysis, 780, 782–783 Allergic reactions, in seafood, 34–35; see also Seafood analyses, 2DE applications Aluminum, in seafood, 353 American Oil Chemists’ Society (AOCS), 13, 381 American type culture collection (ATCC), 516, 613 Amino acid analysis, for food proteins quantification, 13; see also Seafood and seafood products Amino acid precursors and decarboxylase enzymes, in BA formation, 834 Aminoglycosides, in aquaculture, 726 Aminoquinoline (AMQ), 296 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), 296 Amnesic shellfish poisoning (ASP), 6, 660–662 Amperometric gas sensors, usage, 158; see also Seafood and seafood products Amplified fragment length polymorphism (AFLP), 44, 681, 698–700; see also Adulteration of seafood, detection Analytical atomic spectroscopic technique, usage, 760–761 Anarhichas lups (Wolfish ), 507 Anguilla anguilla (Eel), 354, 798 Anguilla japonica (Japanese eel), 508 Anguilla rostrata (American eel), 765 Animal and Plant Health Inspection Service (APHIS), 873 Anisakid nematodes, in seafoods, 590–595 Anisakidosis; see also Fish and shellfish; Seafood and seafood products clinical manifestations and symptoms, 592 occurrence, 594–595 Anisakis physeteris, 590 Anisakis simplex, 590 Anisidine value (AnV), 90, 203 Anomalocardia brasiliana (Clam), 360 Anthelmintic drugs, in aquaculture, 729–730 Antibacterial drugs, application, 717
889
890 ◾
Index
Antibody detection, diagnosis, 621–622 Antimycotica, in aquaculture, 727–728 Antioxidants, in seafood and seafood products, 311–313 antioxidant enzymes, 313–314 ascorbic acid, 314–315 carotenoids, 317–319 determination, 313 endogenous, 320 ubiquinone, 319–320 vitamin E, 315–317 Antiparasitica, in aquaculture, 728–729 Antisera, types, 617 AOAC Official Method 940.33, 336 AOAC Official Method 942.23, 335 AOAC Official Method 2004.05, 339 Aquaculture and antemortem effects, 33 (see also Seafood analyses, 2DE applications) color measurements, 400–402 (see also Fish and fishery products) and genomics, 49–51 (see also Seafood and seafood products) veterinary drugs, 714–716 analytical techniques for analysis, 717–721 anthelmintic drugs, 729–730 antimycotica, 727–728 bactericidal drugs, 721–727 future perspectives, 730–731 hormones, 730 origin of residues, 716–717 tranquilizers and antiparasitica, 728–729 Aquatic species; see also Seafood and seafood products genomic resources and genome projects, 45–47 limitation, 5–6 variability, 5 Aroma extract dilution analysis (AEDA), 440, 451–452 Aroma extraction methods, in seafood; see also Fish and seafood aroma adsorption, 446–447 distillation/extraction, 443–445 geosmin and 2-Methylisoborneol, analysis, 449 headspace analysis, 446 high vacuum distillation, 445 solvent extraction, 442 SPME, 447–449 steam distillation and vacuum steam distillation, 443 TMA, analysis, 449 Arsenic, in seafood, 353–354 Artificial neural networks (ANNs), 196 Artificial Quality Index (AQI), 400, 432 Aryl hydrocarbon receptor (AhR) signal transduction pathway, 808 Ascorbic acid (AA), in seafoods, 314–315 Assessors, for seafood analysis, 503 Association of Official Analytical Chemists (AOAC), 13, 381, 656 Astaxanthin and salmon flesh color, 318
Atlantic mackerel, variability, 5 At-line sensors device role, 170; see also Seafood and seafood products Atmospheric pressure chemical ionization (APCI), 81, 300, 718 Atmospheric pressure chemical ionization MS (APCI-MS), 453 Atmospheric pressure microwave-induced plasma ionization (AP-MIPI), 300 Atomic absorption spectrometry (AAS) for seafood metals determination, 752–756 in zinc determination, 367 Atomic emission spectrometry (AES), for seafood metals determination, 752, 756–759 Atomic fluorescence spectrometry (AFS), 752 ATP degradation, in fish muscle, 197–198; see also Seafood and seafood products ATP-related compounds, analysis, 59–60; see also Seafood and seafood products nucleotides and nucleosides determination, 61–65 nucleotides and nucleosides, extraction, 60–61 Atwater general factor system, 275 Australia, legal limit of histamine concentration, 837–838 Automatic online postcolumn derivatization, of BA, 844 Autooxidation, of lipids, 88 Avermectins, in aquaculture, 728–729 Azaspiracids, 666; see also Marine toxins
B Bacteria, in seafood spoilage; see also Seafood and seafood products Aeromonas genus, 525 Alteromonas genus, 521 bacterial identity confirmation tests, 528–532 Brochothrix genus, 524 Flavobacterium and Cytophaga genera, 524 genera Moraxella and Acinetobacter, 523 Lactobacillus genus, 525 Photobacterium genus, 524–525 Pseudomonas genus, 518–521 Shewanella genus, 521–523 varius bacterial counts determination, 527–528 Vibrio genus, 525 Bacterial Analytical Manual (BAM), 560 Bacterial artificial chromosomes (BACs), 45 Bactericidal drugs, in aquaculture, 721–727 Benzimidazoles, in aquaculture, 729–730 Benzo[a]pyrene metabolization, 248 Bicinchoninic acid (BCA), 15, 272 Bioanalytical screening methods, in dioxin analysis, 808–809; see also Dioxin analysis, from seafoods Biodiversity management, of seafood, 47–49; see also Seafood and seafood products Bioelectrical impedance analysis (BIA), 174
Index ◾ Biogenic amine index (BAI), 199 Biogenic amines (BA), in seafood, 833–834; see also Seafood and seafood products determination, 843–846 in fish quality measurement, 199 formation microorganisms, 839–840 processing and storage, 840–842 raw material, 838–839 legal limits, 837–838 production, by muscle microbial biota, 542 as quality index, 836–837 quantification, 547–548 toxicity, 834–836 Biological test systems, for veterinary drugs analysis, 717 Biorad method, usage, 15; see also Seafood and seafood products Biosensors, in GMO detection, 882 Biotin, in seafoods, 341–342 Bisphenol A diglycidyl ether (BADGE), 863 Biuret method, in soluble protein analysis, 14 Black Sea fish, metal concentration determination, 767 Bligh & Dyer method (B&D) in lipid extraction, 379–380 usage, 267–268 Bomb calorimeter, usage, 274 Boops boops, 357 Boron trifluoride, usage, 75 Bovine serum albumin (BSA), 16 Bovine spongiform encephalopathy (BSE), 675 Brama raii, 357 Brevetoxin, see Neurotoxic shellfish poisoning (NSP) Brochothrix thermosphacta, 539 Brosme brosme (Cusk), 507 Bruker Professional MOUSE®, usage, 131 Brycon moorei (Dorada), 32 Butylated hydroxytoluene (BHT), 328
C Cadaverine, as fish freshness indicators, 836–837 Cadmium accumulation, in mollusks, 6 Cadmium level in fish, determination, 768 Callinectes sapidus (Atlantic blue crab), 678 Callinectes sapidus (Blue crab), 360 Callorhinchus milii (Elephant shark), 46 Calories, in seafood; see also Seafood and seafood products direct measurement, 274 food composition tables and databases, 276 indirect measurement, 275–276 Canadian Grain Commission, 258 Capillaria philippinensis, 595 Capillary electrophoresis (CE), 60–61, 845 Capillary zone electrophoresis (CZE), 291, 298–299 Carbohydrate content determination, in seafood, 273; see also Seafood and seafood products
891
Carbon rod atomizer (CRA), advantage, 755 Carnobacterium spp., 550 Carotenoids, in seafood and seafood products, 317–319, 328–332 Catalases enzyme, role, 313 Cation exchange chromatography (CEC), 291–292 CE coupled to electrospray ionization (ESI) MS (CE-ESI-MS), 299 Center for Disease Control (CDC), 558 Center for Food Safety and Applied Nutrition (CFSAN), 696 Cepola rubescens, 357 Certified reference materials (CRM), 819 Cestodes, in seafoods, 587–589 Cetyl–trimethyl ammonium bromide (CTAB) method, 693 Chamelea galena (Clam), 358 Channel catfish virus (CCV), 631 Charge coupled device (CCD), 758 Charge injection device (CID), 758 Charge transfer device (CTD), 758 Chemical and biochemical methods, in seafood freshness assessment ATP, 197–198 biogenic amines, 199 dimethylamine, 201 formaldehyde, 201 lipid hydrolysis, 203 lipid oxidation indicators, 201–203 pH, 199–200 trimethylamine, 200–201 TVB-N, 200 Chemical migration, definition, 852 Chemical residues in seafood, FCMs, 852–857 packaging materials, 860 active and intelligent packaging, 867–869 glass jars, 867 metal cans, 863–866 multilayer packaging materials, 867 paper and cartonboard, 862–863 plastics, 861–862 surface-active biocides, 869–870 residues test, 857–858 testing strategies, 858–860 Chemical sensor, technologies amperometric gas sensors, 158 color indicators, 159–160 conductance changes, sensor metal-oxide semiconductors, 157 polymer and molecular aggregates, 158 FET, 159 mass transducers, 158 Chemiluminescence (CL), 92 Chinolones, in aquaculture, 722–723 Chinook salmon embryo (CHSE-214), 614 Chip-based capillary electrophoresis system, usage, 682 Chlamydomonas reinhardtii, 47
892
◾
Index
Chloramphenicol, in aquaculture, 725–726 Chloroform–methanol method, in lipid analysis, 72; see also Marine lipid analysis Chondrostoma regium, 768 ChromaControl C, 398 Chroma Meter CR-300, usage, 397 Chromatography technique, in nucleosides and nucleotides analysis, 61–63 Chromium, in seafood, 354–358, 767 Chrysophrys auratus, 358 CIE 1931 2°, usage, 396 Ciguatera fish poisoning, 650–651 Ciguatoxins (CTXs) poisoning, 650; see also Marine toxins clinical symptoms, 651 detection methods, 651–653 Citrobacter freundii, 839 Clarias gariepinus (Catfish), 369 Clinical sensitivity, see Diagnostic sensitivity, definition Clonorchis sinensis, 582 Clostridium botulinum, 526 Clupea harengus (Herring), 111, 354, 360, 501 Cobalt in seafood, determination, 767 Codex Alimentarius General Standard, 774 Cod, volatile compounds, 101–103 Cold vapor atomic fluorescence spectrometry system (CV-AFS), 756 Collagen solubility, determination, 14 Cololabis saira (Pacific saury), 747 Color evaluation, novel method, 399–400; see also Fish and fishery products Colorimeters device, features, 397 Colorimetry, fish spoilage detection, 546–547 Color measurement, in fish and fishery products; see also Fish and fishery products aquaculture, 400–402 fish mince, surimi and surimi-based products, 403 of frozen products, 410–411 of heat-treated products, 412–414 in high pressure treated fish, 415–417 processing effect, 403–407 of refrigerated products, 408–410 Column chromatography (CC), in lipid classes analysis, 79 Commercial PCR kits, for fish species differentiation, 682–683 Commission Internationale de l’Eclairage (CIE), 396 Community Reference Laboratory (CRL), 875 Complementary DNA (cDNA), 45 Complex permittivity, definition, 176–177; see also Microwave theory, in seafood quality analysis Compression analysis, in seafood analysis, 429–430 Computer screen photoassisted technique (CSPT), 159–160, 196 Concerted actions (CA), 7 Conger myiaster (Common Japanese conger), 508
Conjugated diene hydroperoxides and primary oxidation products, 89 Connective tissue proteins, 13 Construct-specific PCR methods, for GMO analysis, 875 Contracaecum osculatum, 590 Controlled atmosphere packaging (CAP), 868 Cooked Cod Fillets, freshness evaluation, 194; see also Seafood and seafood products Coomassie Brilliant Blue G (CBBG), 15 Copper level in fish, determination, 768 Cosmos instrument, in fish quality assessment, 195 Covalent adduct chemical ionization (CACI), 387 Crangon crangon (Brown shrimp), 354 Crangon vulgaris, 358 Crassostrea ariakensis, 581 Crassostrea gigas (Pacific oysters), 358, 586 Crassostrea rhizophorae (Oyster), 360 Crawfish, metal concentration, 764 Creep test, usage, 431–432 Crustacean and molluscan shellfish, minerals and trace elements, 368 Cryptosporidium parvum, 581 ctxA gene, 568 Cyanidioschyzon merolae (Red alga), 47 3-[Cyclohexylamino]-1-propanesulfonic acid (CAPS), 61 Cynoscion regalis (Weakfish), 508 Cyprinus carpio, 768 Cytochrome oxidase I (COI), 680 Cytopathic effect (CPE), 607
D Danio rerio (Zebrafish), 23, 46 Daphnia magna, 659 Daphnia pulex (Zooplankton), 46 2DE applications, in seafood analysis allergen identification, 34–35 development, 31–32 quality involution aquaculture and antemortem effects, 33 protein autolysis and oxidation, 32–33 species authentication, 34 Degradome, 24–25 Dehydroascorbic acid (DHAA), 314–315, 343 2DE, in proteome analysis 2DE methods analysis, 28 equilibration, 27 first-dimension electrophoresis, 25–27 sample extraction and cleanup, 25 second-dimension electrophoresis, 27 staining, 28 protein identification, 28–31 sample matrix
Index ◾ degradome, 24–25 muscle proteomes, 24 whole larval proteomes, 22–24 Denaturing gradient gel electrophoresis (DGGE), 681, 700 Depuration method, for seafoods, 596 Descriptive sensory analysis, of seafood, 500 Diagnostic sensitivity, definition, 605 Diaminoxidase (DAO), 845 Diarrhetic shellfish poisoning (DSP), 5, 658–660 Diazomethane, usage, 382 Dielectric spectroscopy, in seafood quality analysis, 174–175; see also Seafood and seafood products Dietary folate equivalents (DFE), 340 Dietary intake, of dioxin, 798 Diethylaminoethyl (DEAE), 79 Difference gel electrophoresis (DIGE), 28 Differential pulse scanning anodic voltammetry (DPSAV), 352 Differential scanning calorimetry (DSC), 8 Diff use reflectance infrared Fourier transform (DRIFT) spectroscopy, 196–197 Dihydrothymidine (DiHT), 787 Dimethylamine and fish spoilage indicator, 201; see also Seafood and seafood products Dimethylamine (DMA), 105 4-Dimethyl-aminoazobenzene-4′-sulfonyl chloride (Dabsyl-Cl), 294 1-Dimethylamino-naphthalene-5-sulfonyl chloride (Dansyl-Cl), 294–295 4,4-Dimethyloxazoline (DMOX), 387 Dinophysistoxin (DTX), 658 Diode array detectors (DAD), 823 Dioxin analysis, from seafoods, 798–799; see also Seafood and seafood products automation of extraction and cleanup, 804–805 bioanalytical screening methods, 808–809 cleanup methods, 802–803 extraction techniques accelerated solvent extraction, 801–802 microwave oven, 802 SFE, 801 soxhlet extraction, 800 SPE and MSPD, 800–801 fractionation and group separation, 803–804 instrumental determination GC congener separation, 805–807 GC detectors, 807–808 sample pretreatment and recovery studies, 799–800 Diphyllobothriosis, in humans, 588 Diphyllobothrium latum, 588 Diphyllobothrium pacificum, 588–589 Dipstick immunoassay, for adulteration detection, 677–678 Direct epifluorescent fi lter count (DEFT), 788
893
Direct solid sampling Zeeman GFAAS methods, usage, 767 Dithiothreitol (DTT), 16 Dityrosine, usage, 17 Divinylbenzene (DVB), 448 DNA barcoding, usage, 45 DNA extraction methods, usage, 217–218 DNA technologies; see also Adulteration of seafood, detection; Food irradiation; Genetically modified organisms (GMOs) in food irradiation detection, 785–787 in GMO analysis, 874–877, 879–881 for seafood adulteration detection, 679–683 species identification techniques application, 691 DNA extraction and sample handling, 692–693 DNA sequencing methods, 693–697 non-DNA sequencing methods, 697–703 Docosahexaenoic acid (DHA), 70, 87, 92, 190, 378 2-Dodecylcyclobutanone (DCB), 782 Dogfish certified reference material (DORM), 764 Domoic acid, see Amnesic shellfish poisoning (ASP) Dried fish, development, 105; see also Volatile aroma compounds, in fish Dry extract spectroscopy by infrared reflection (DESIR), 742 Dual-energy x-ray absorptiometry (DXA), 132 Dumas method in fish proteins determination, 270 in protein content determination, 13 (see also Seafood and seafood products)
E Eagle’s minimum essential medium (EMEM), 613 Earle’s salt solution (ESS), 607, 613 Echelle Monochromator, usage, 758 Echinochasmus hortense, 586 Echinostoma japonicus, 586 Edwardsiella ictaluri, 51 Effective loss factor, definition, 177; see also Seafood and seafood products Eicosapentaenoic acid (EPA), 70, 190, 378 health effects, 87 Electrical resistance of fish, measurement, 737–738; see also Fresh and frozen-thawed fish, differentiation methods Electron-capture negative ionization (ECNI), 807 Electronic nose, 160; see also Seafood and seafood products application, 160–164 in fish aroma analysis, 452 (see also Fish and seafood aroma) in fish freshness determination, 196 Electron microscopy (EM), 140, 613 Electron paramagnetic resonance (EPR) spectroscopy, 781
894 ◾
Index
Electron spin resonance (ESR) spectroscopy, 92, 780–782; see also Food irradiation Electrophoresis-based methods, in proteins analysis, 16 Electrothermal atomizer (ETA), 754 Electrothermal vaporization (ETV), 760 Elemental composition in crustacean and molluscan shellfish, 368 in seafood, 367–368 aluminum, 353 arsenic, 353–354 chromium, 354–358 iodine, 358–359 manganese, 360 molybdenum, 361 selenium, 361–364 thallium, 364 vanadium, 364 zinc, 365–367 Emiliania huxleyi (Haptophyte), 47 End point temperature (EPT), 173 Enterobacter aerogenes, 839 Enterobacter cloacae, 839 Entire Color Index (ECI), 400 Environmental Protection Agency (EPA), 799, 873 Enzymatic methods in nucleotides analysis, 64–65 (see also Seafood and seafood products) usage, 739–741 (see also Fresh and frozen-thawed fish, differentiation methods) Enzymatic spoilage, of fish, 544 Enzyme-linked immunosorbent assay (ELISA), 16, 581, 615, 620–621, 874 for brevetoxin detection, 663–664 for DA determination, 661 DSP toxins detection, 659 for GMO analysis, 877–879 for NSP determination, 663 for seafood adulteration detection, 677 STX detection, 657 for veterinary drugs analysis, 717 Enzyme sensors, definition, 64 Ephemeral spoilage organisms (ESO), 538–544 Epithelioma papillosum cyprini (EPC), 614 Epizootic hematopoietic necrosis virus (EHNV), 618 Equivalent chain lengths (ECLs), 75 Erythrocytes of fish, examination, 738–739; see also Fresh and frozen-thawed fish, differentiation methods Escherichia coli, 774 Essential amino acids analysis, in seafood, 290–291 capillary zone electrophoretic technique, 298–299 free essential amino acids analysis, 288–289 GLC methods, 298 HPLC method, 291–298 MS, 299–300 total essential amino acid analysis, 289–290
Essential fatty acids (EFAs), 378 Ethanol detection, in fish, 104; see also Volatile aroma compounds, in fish Ether lipids, analysis, 77; see also Marine nonsaponifiable matter, analysis Ethylacetate method, role, 268; see also Seafood and seafood products Ethylenediaminetetraacetic acid (EDTA), 343 EU regulation, seafood, 476–477; see also Quality Index Method (QIM), for fish storage European Collection of Cell Culture (ECACC), 613 European Commission, in dioxin exposure prevention, 798 European Food Safety Authority, 597 European regulations, in SF, 249–250; see also Seafood and seafood products European Union (EU) freshness grading, in seafood, 191 ExPASy Tools web site, 29 Expressed sequence tags (ESTs), 45, 51 Eye lens opacity of fish, examination, 736–737; see also Fresh and frozen-thawed fish, differentiation methods
F Fathead minnow (FHM), 614 Fat-soluble vitamins, in seafood vitamin A and carotenoids, 328–332 vitamin D, 332 vitamin E, 332–333 vitamin K, 333–334 Fatty acid composition; see also Marine fatty acid analysis qualitative analysis, 75–76 quantitative estimation, 76 Fatty acid derivatives, preparation, 380–382; see also Total fatty acids, analysis Fatty acid methyl esters (FAME), 73 GC analysis, 385–386 GLC analysis, 75–76 preparation, 73–75 (see also Marine fatty acid analysis) Fatty seafood tissues, dioxin analysis, 798–799 Field-effect transistors (FET), 159 Fish and fishery products color evaluation, novel method, 399–400 color measurement aquaculture, 400–402 fish mince, surimi and surimi-based products, 403 processing effect, 403–407 instrumental texture compression analysis, 429–430 Kramer test, 427 puncture test, 428–429 stress relaxation test, 431–432
Index ◾ tension analysis, 429 TPA, 430–431 viscoelastic methods, 431 W-B test, 427–428 methods for analysis, 259–265 photoelectric color-measuring instruments, 397–399 test procedure and sample preparation, 426–427 texture measurement, 432–433 Fish and seafood aroma aroma components quantification, 450–451 separation and identification, 449–450 aroma extraction methods adsorption, 446–447 distillation/extraction, 443–445 geosmin and 2-Methylisoborneol, analysis, 449 headspace analysis, 446 high vacuum distillation, 445 solvent extraction, 442 SPME, 447–449 steam distillation and vacuum steam distillation, 443 TMA, analysis, 449 electronic nose, 452 future developments, 453 sample preparation, 442 sensory significance, detection, 451–452 study desirable aroma, 440–441 pre and postslaughter treatment, 441 undesirable aroma, 441 Fish and shellfish infection nematode species, 590 tapeworm species, 587–588 trematode species, 583–585 PAHs analysis, 818–819 chromatographic analysis, 823–824 clean-up of extracts, 821–822 methods of extraction, 819–821 PCBs analysis, 818, 824–825 chromatographic analysis, 828–829 clean-up of extracts, 827–828 extraction methods, 825–827 viral diseases, diagnosis and screening, 604–605 analytical techniques, 608 clinical, histological, and microscopical techniques, 609–613 factors affecting accuracy, 606–608 isolation in cell culture, 613–616 molecular diagnosis, 622–634 nonlethal methods, 634–635 serological and immunological techniques, 616–622 validation, 605–606 Fish aroma, development, 98–99 Fish DNA Barcode of Life (FishBOL) database, application, 694–695
895
Fish feed, GMOs, 872–873 Fish freshness determination, 191 electronic noses application, 160–164 (see also Electronic nose) Fish lipids, composition, 267 Fish muscle ATP degradation, 58 microstructure, 140–145 (see also Seafood and seafood products) hake, 146–148 herring, 145–146 proteins, types, 13 proteomes, 24 FishNose, role, 196; see also Seafood and seafood products Fish odors, fresh, 99–100 Fish proteins, functional properties, 12 Fish quality labeling and monitoring (FQLM), 7 Fish Shearing Device (FSD), 428 Fish species; see also Seafood and seafood products anatomy, 500–501 differentiation, commercial PCR kits, 682–683 (see also Adulteration of seafood, detection) spoilage, 537–538 determination methods, 544–550 types, 538–544 variation between individuals, 501–502 Fish texture, factors affecting, 426 FishTrace database, application, 694 Fish, volatile aroma compounds aroma development, 98–99 fresh odors, 99–100 quality indicators, identification, 100–103 microbial spoilage odors, 103–106 oxidatively derived odors, 106–110 processing odors, 111–113 Flame atomic absorption spectrometry (FAAS), 766 Flame ionization detector (FID), 76, 249, 298, 379, 824 Flame photometric detector (FPD), 298 Flavin adenine dinucleotide (FAD), 335 Flavin mononucleotide (FMN), 335 Florisil chromatography, disadvantages, 784–785 Flow injection analysis (FIA), 65, 844 Flow injection analysis-hydride generation (FIA-HG), 760 9-Fluorenylmethyl chloroformate (FMOC), 271, 295 Fluorescein isothiocyanate (FITC), 609 Fluorescence detectors (FLD), 823 Folate, in seafoods, 338–340 Folch method, in lipid extraction, 379–380 Food and Agricultural Organization (FAO), 735 Food and Drug Administration (FDA), 696, 836, 873 Foodborne diseases prevention, high energy irradiation, 774–775; see also Food irradiation
896
◾
Index
Foodborne pathogens, in seafoods and seafood-related environments, 557–559 detection, 559–560 Listeria monocytogenes, 568–570 Salmonella, 570–571 Vibrio cholerae in, 567–568 Vibrio parahaemolyticus in, 560–565 Vibrio vulnificus in, 565–567 Food composition databases, role, 276; see also Seafood and seafood products Food contact materials (FCMs) and chemical migration, 852–857; see also Seafood and seafood products packaging materials, 860 active and intelligent packaging, 867–869 glass jars, 867 metal cans, 863–866 multilayer packaging materials, 867 paper and cartonboard, 862–863 plastics, 861–862 surface-active biocides, 869–870 residues test, 857–858 testing strategies, 858–860 Food irradiation applications, 774–775 detection methods, 780–781 DNA methods, 785–787 electron spin resonance spectroscopy, 781–782 luminescence, 787–788 microbiological methods, 788–789 radiolytic chemicals analysis, 782–785 development, 775–779 global acceptance and attitudes, 779 Food preservation, high energy irradiation, 774–775; see also Food irradiation Food quality sensor, usage, 170; see also Seafood and seafood products Food simulants, usage, 859–860 Food systems, antioxidants and antioxidant capacity determination, 313; see also Seafood and seafood products Forensically informative nucleotide sequencing (FINS), 696 Formaldehyde; see also Seafood and seafood products; Smoke flavoring (SF) and fish spoilage indicator, 201 and smoked fish texture, 246 Fourier transform infrared (FT-IR) spectroscopy, 90, 196 Free amino acids (FAA), 834, 838–839 Free essential amino acids analysis, in seafood, 288–289; see also Seafood and seafood products Free fatty acids (FFA), 73, 173 Free-induction decay (FID), 223 Freezing, effect on fish, 736 Fresh and frozen-thawed fish, differentiation methods electrical resistance, measurement, 737–738
enzymatic methods, 739–741 eye lens opacity, examination, 736–737 hematocrit value determination and erythrocytes examination, 738–739 spectroscopic analysis, 742–747 FreshSense, usage, 196; see also Seafood and seafood products
G Gadus morhua (Atlantic cod), 23, 112, 354, 431–432, 466–467, 505 Gambierdiscus toxicus, 651 Gambusia affinis (Mosquito fish), 663 Gamma-irradiated seafood, microbiology, 526 Gas chromatographic congener separation, in dioxin analysis, 805–807; see also Dioxin analysis, from seafoods Gas chromatographic detectors, in dioxin analysis, 807–808; see also Dioxin analysis, from seafoods Gas chromatographic (GC) analysis, of fish and marine oils, 382–383; see also Total fatty acids, analysis Gas chromatography–mass spectrometry (GC–MS), 858 lipid oxidation product, determination, 92 Gas liquid chromatography (GLC), 73 analysis, of FAME, 75–76 (see also Fatty acid methyl esters (FAME)) applications, 298 Gasterosteus aculeatus (Three-spined stickleback), 46 Gastroallergic anisakiosis, 592 Gel permeation chromatography (GPC), 821–822 Gene technology, application, 872 Genetically modified (GM) product, for food use, 872 Genetically modified organisms (GMOs); see also Seafood and seafood products analysis DNA and protein methods, comparison, 879–881 DNA-based detection, 874–877 protein-based detection, 877–879 definition, 872 development in detection, 881–882 in fish feed, 872–873 international regulations, 873–874 Genetic analysis, in farmed and wild seafood, 217–218 Genomic library, definition, 45 Genomics and genetics, in seafood, 44–45 aquaculture and genomics, 49–51 fisheries and biodiversity management, 47–49 genomic resources and genome projects, 45–47 Genomics Research on All Salmon Project consortium (cGRASP), 46 Geosmin and 2-Methylisoborneol, analysis, 449; see also Fish and seafood aroma Giardia duodenalis, 581
Index ◾ Glass jars, in seafood packaging, 867 Glutathione peroxidase (GPx), role, 314 Glutathione reductase (GR), role, 314 Glutathione S-transferases (GST), role, 314 Gnathostoma doloresi, 595 Gnathostoma hispidum, 595 Gnathostoma nipponicum, 595 Gnathostoma spinigerum, 595 Gnathostomiosis, species causing, 595 Gonyautoxins (GNTXs), 655 Good Manufacturing Practice (GMP), 472–473 Gram-negative bacteria (GNB), 788 Graphite furnace atomic absorption spectrometry (GFAAS), 353, 358, 756 Grass carp rhabdovirus (GCRV), 618 Gravimetric methods, usage, 275; see also Seafood and seafood products Guided microwave spectrometer, usage, 181–182 Gymnodinium catenatum, 655 Gymnophalloides seoi, 586
H Haddock fi llets, volatile compounds, 101–103 Hafnia alvei, 839 Hake, muscle microstructure, 146–148; see also Seafood and seafood products Hanks’ balanced salt solution (HBSS), 607 Hazard analysis and critical control point (HACCP), 8, 171, 203, 597, 845 Headspace analyzers, in fish and seafood aroma analysis, 446; see also Fish and seafood aroma Heated graphite atomizer (HGA), 754 Height equivalent to a theoretical plate (HETP), 824 Helicolenus sp., 357 Hematocrit value of fish, determination, 738–739; see also Fresh and frozen-thawed fish, differentiation methods Hepatotoxins, symptoms and detection methods, 664–666 Herring muscle microstructure, 145–146 (see also Seafood and seafood products) sensory evaluation, 193 Heterobranchus longifilis (African catfish), 32 Heterophyes heterophyes, 586 Heterphyes nocens, 586 Highly unsaturated fatty acids (HUFAs), 379 High-performance capillary electrophoresis (HPCE), 61 High-performance liquid chromatography (HPLC), 77, 443, 679, 823, 843 in fish amino acids assessment CEC, 291–292 RP-HPLC, 292–298 in lipid classes analysis, 79–80
897
High-pressure processing, of fish and fishery products, 403–407; see also Fish and fishery products High-pressure TLC (HPTLC), 79 High-resolution gas chromatographic methods, for dioxin analysis, 805; see also Dioxin analysis, from seafoods High-resolution magic angle spinning (HR-MAS), 223 High-resolution mass spectrometry (HRMS), 825 High-resolution NMR (HR-NMR), 8, 131 High-resolution nuclear magnetic resonance spectroscopy (HR-NMR), 222–223 Hippoglossus hippoglossus L. (Atlantic halibut), 506 Histamine decarboxylase enzymes, in bacteria, 839 Histamine, in fish and fish products, 834–835 1H NMR spectra, usage, 92–93 Homarus vulgaris, 358 Hormones usage, in aquaculture, 730 Horseradish peroxidase (HRP), 609 Human consumption and seafood supply, 4; see also Seafood and seafood products Human infections, seafoods causing acanthocephalans, 595–596 anisakid nematodes, 590–595 cestodes, 587–589 diagnosis, 582 protozoa, 581–582 trematodes, 582–587 HunterLab MiniScan XE Plus 45/0 LAV spectrophotometer, 397–398 Huso huso, 364 Hydra magnipapillata, 47 Hydride generation atomic absorption spectrometry (HGAAS), 354, 364 Hydride generation atomic fluorescence spectrometry (HG-AFS), 354 Hydroperoxides, reaction, 91; see also Lipid oxidation, analysis Hydroxylysine, determination, 15 Hypoxanthine (Hx), 58 Hysterothylacium aduncum, 590
I Ichthyosarcotoxins; see also Marine toxins ciguatoxin, 650–653 tetrodotoxin, 653–655 Imaging spectroscopy, in seafood composition; see also Seafood composition basic constituents, analysis, 128–130 theory, measurement principles and data analysis, 128 Immobilized pH gradients (IPGs), 25 Immunoaffinity Chromatography (IAC), 804 Immunoassay techniques, for TTX determination, 654
898 ◾
Index
Immunodot, viral isolates identification, 619 Immunoenzymatic assays (IEA), 616 Immunofluorescence antibody test (IFAT), 609 Immunofluorescent antibody analysis (IFA), 581 Immunohistochemistry (IHC) usage, 609, 612–613 of viral infected cells, 619–620 Immunological diagnostic tools, advantages and disadvantages, 616–618 Impedance spectroscopy, in seafood quality analysis, 174; see also Seafood and seafood products Inductively coupled plasma (ICP), 352, 752 Inductively coupled plasma mass spectrometry (ICP-MS), 227, 752, 759–760 Industrial, scientific, and medical applications (ISM), 179 Infectious hematopoietic necrosis virus (IHNV), 606 Infectious pancreatic necrosis virus (IPNV), 606 Inosine (Ino), 58 Inosine monophosphate (IMP), 58, 197–198 Insoluble proteins, see Connective tissue proteins Instron Universal Testing Machine, 430 Instrumental analytical approaches, for veterinary drugs analysis, 717–721 Instrumental methods and seafood analyses, 8; see also Seafood and seafood products Instrumental texture methods, for seafood analysis; see also Fish and fishery products compression analysis, 429–430 Kramer test, 427 puncture test, 428–429 stress relaxation test, 431–432 tension analysis, 429 TPA, 430–431 viscoelastic methods, 431 W-B test, 427–428 Intellectron Fischtester VI, usage, 195 Internal amplification control (IAC), 564 International Atomic Energy Agency (IAEA), 777 International Consultative Group on Food Irradiation (ICGFI), 777 International Dairy Federation (IDF), 89 International regulations, for GMOs, 873–874 International Standard ISO 8914:1990, 560 Iodine, in seafood, 358–359 Ion chromatography (IC), 60 Ion-pair RP-HPLC, role, 63 Irradiated foods detection, EN Protocols, 780 Irradiation effect, in BA formation, 842; see also Biogenic amines (BA), in seafood IR spectroscopy, in seafood quality analysis, 173–174; see also Seafood and seafood products Isoelectric focusing (IEF), 25–26, 676 Isotope dilution mass spectrometry (IDMS), 799 Isotope ratio mass spectrometry (IRMS), 224
J Japan Agriculture Standard (JAS), 735 Jassa slatteryi, 47 Joint Expert Committee on Food Irradiation (JECFI), 775, 777
K Karenia brevis, 651 KDC Technology Corporation, role, 181 Kjeldahl method in fish proteins determination, 270 in protein content determination, 12 (see also Seafood and seafood products) Kjel-Foss® instrument, usage, 12 Klebsiella pneumoniae, 839 Kloekera brevis, 338 Koi herpesvirus (KHV), 631 Kramer test, in seafood analysis, 427 K value, definition, 59
L Lactobacillus delbrueckii subsp. lactis, 340 Lactobacillus leichmannii, 340 Lactobacillus plantarum, 337 Lactobacillus rhamnosus, 336, 339 Lactobacillus spp., 550 Lambert-Beer law, in protein concentration calculation, 14 Larmor frequency, definition, 781 L-ascorbate 2-sulfate (AAS), 314 Leucoraja erinacea (Little skate), 46 Light microscope (LM), application, 140 Likens–Nickerson apparatus, in aroma analysis, 443–444 Limulus Ameobocyte Lysate test (LAL), 788 Linear discriminant analysis (LDA), 743 Linear sorption energy relationship (LSER), 161 Line-specific PCR detection methods, for GMO analysis, 875–876 Lipid analysis, of marine products, 71 Lipid classes analysis SFC, 82 TLC, 79 Lipid classes, stereospecific analysis, 78–79; see also Marine lipid classes, analysis Lipid content analysis, in farmed and wild seafood, 221– 223; see also Seafood and seafood products Lipid extraction, methods, 379–380; see also Total fatty acids, analysis Lipid manipulation and storage, 73; see also Marine lipid analysis Lipid oxidation analysis
Index ◾ instrumental methods, 92–93 primary oxidation products, 88–89 secondary oxidation products, 89–92 sensory analysis of rancidity, 93 stability methods, 92 importance, 87 indicators, in fish quality measurement, 201–203 (see also Seafood and seafood products) types, 88 Lipid peroxidation, effects, 311; see also Oxidation, in seafood and seafood products Lipid quantification, 73; see also Marine lipid analysis Lipid removal, in dioxin analysis, 803 Lipid saponification, 76; see also Marine nonsaponifiable matter, analysis Lipids, in seafood; see also Seafood and seafood products isolation, 72 (see also Marine lipid analysis) methods comparison, 269 nondestructive methods, 268–269 nutritional aspects, 267 roles, 70 total lipids, determination, 267–268 Lipoxygenase (LOX), 98–99 Liquid–liquid partitioning, for PCBs extraction, 826 Liquid smoke atomization technique, usage, 239 chemical composition, 239–240 fractions, sensory taste intensities, 246 (see also Smoke flavoring (SF)) usage, 236 (see also Seafood and seafood products) Listeria monocytogenes, 549, 558, 568–570; see also Seafoods and seafood-related environments, foodborne pathogens Listeria monocytogenes blood agar (LMBA), 569 Listeria repair broth (LRB), 569 Litopenaeus Yannamei, 315 Loligo sp. (Squid), 353 Loop-mediated isothermal amplification (LAMP), 622, 626 Lophius americanus (Monkfish ), 507 Lopholatilus chamaeleonticeps (Tilefish), 507 Low-density lipoprotein (LDL), 319 Low-field (LF) NMR, 8 Low-resolution NMR (LR-NMR), 131 Lowry method in fish proteins determination, 272 in soluble protein analysis, 14–15 Luminescence, in food irradiation detection, 787–788; see also Food irradiation Lymphocystis disease virus (LDV), 631
M Magnetic resonance imaging (MRI), 8, 742 Malanogrammus aeglefinus (Haddock), 507 Malonaldehyde (MDA), 90
899
Manganese, in seafood, 360 Marine fatty acid analysis fatty acid methyl esters preparation, 73–75 GLC analysis, 75–76 Marine lipid analysis lipid manipulation and storage, 73 lipid quantification, 73 lipids isolation, 72 nonlipid contaminants, removal, 72 Marine lipid, characteristics, 70–71 Marine lipid classes, analysis, 77–78 CC, 79 HPLC, 79–80 MS, 81–82 NMR spectrometry, 80–81 SFC, 82 silver ion chromatography, 80 spectrophotometric assessments, 78 stereospecific analysis, 78–79 TLC, 79 Marine lipid oxidation, 311; see also Oxidation, in seafood and seafood products Marine nonsaponifiable matter, analysis analysis of ether lipids, 77 lipid saponification, 76 sterols analysis, 76–77 Marine organisms, AA, 315 Marine products, lipid analysis, 71 Marine species, fatty acids, 74 Marine toxins; see also Seafood and seafood products ichthyosarcotoxins ciguatoxin, 650–653 tetrodotoxin, 653–655 from phytoplanktons, 664–666 shellfish toxins amnesic poisoning, 660–662 diarrhetic poisoning, 658–660 neurotoxic poisoning, 662–664 paralytic poisoning, 655–657 Marker-assisted selection (MAS), 49–50 Mass spectrometry (MS) in amino acid analysis, 299–300 of fatty acid derivatives, 384–387 (see also Total fatty acids, analysis) in lipid classes analysis, 81–82 usage, 17 Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), 23 Matrix solid phase dispersion extraction (MSPD), 718, 798, 800–801 Maximum residue level (MRL), 719 Meat and bone meal (MBM), 678 Membrane immunobead assay (MIA), in CTX detection, 652 Menadione, 333 Menaquinones, production, 333
900
◾
Index
Merlangus merlangus (Whiting), 738 Merluccius bilinearis (Whiting), 507 Merluccius capensis (Hake), 34, 183, 431 Merluccius merluccius (European hake), 34, 358 Metagonimus yokogawai, 586 Metal cans, in seafood packaging, 863–866 Metal concentration determination, in M. cephalus, 768 Metal-oxide semiconductors, usage, 157; see also Chemical sensor, technologies Metals determination in seafood, spectrochemical methods; see also Seafood and seafood products AAS, 752–756 AES, 756–759 analytical atomic spectroscopic technique, 760–761 ICP-MS, 759–760 sample preparation, 761 selected applications, 762–769 Metanephrops andamanicus (Pacific scampi), 677 (1-Methyleneimino)-1-ethenylium (MIE), 387 Metorchis conjunctus, 586 Micellar electrokinetic capillary chromatography (MECC), 299, 664 Microarray technology, in GMO detection, 881–882 Microbial spoilage odors, in fish, 103–106; see also Volatile aroma compounds, in fish Microbial spoilage, of fish, 539 Microbiological analyses, of fish spoilage, 548 Microbiological methods in food irradiation detection, 788–789 (see also Food irradiation) in seafood freshness evaluation, 203–204 Microorganisms, in seafood, 543 Microscopical techniques, for seafood adulteration detection, 678–679 Microwave-assisted solvent extraction (MASE), 798, 802 Microwave digestion and atomic absorption spectroscopy (MD-AAS), 767–768 Microwave digestion method, for metals determination, 764 Microwave methods, advantages, 175–176; see also Seafood and seafood products Microwave spectroscopy–dielectric spectroscopy, 174–175 Microwave spectroscopy, in seafood quality analysis, 174–175; see also Seafood and seafood products Microwave technology applications, in seafood quality analysis, 179–180 freshness and salting process, 182–183 moisture content, determination, 180–182 Microwave theory, in seafood quality analysis, 176–179; see also Seafood and seafood products Mid-Atlantic Ridge (MAR), 767 Mid-infrared (MIR) spectroscopy, usage, 173 Minimal risk level (MRL), 798
Minimum inhibitory concentration (MIC), 869 Minimum required performance limit (MRPL), 726 MiniScan XE Plus, usage, 399 Misgurnus anguillicaudatus, 586 Modified atmosphere packaged (MAP), 196, 507, 842 Mollusks, metal concentration determination, 765–766 Molybdenum, in seafood, 361 Monoamine oxidase inhibitors (MAOI), 836 Monoclonal antibodies (MAbs), 609 Morganella (Proteus) morganii, 839 Morone saxatilis (Striped bass), 508 Most probable number (MPN), 560 Mouse bioassay DSP toxins, analysis, 658–659 TTX toxicity determination, 654 Mugil cephalus, 360, 586, 768 Muller–Kauff mann tetrathionate novobiocin (MKTTn), 570 Mullus barbatus, 353 metal concentration determination, 768 Mullus surmuletus, 357–358 Multilayer packaging materials, in seafood packaging, 854, 867 Multimetal determination method, for trace metals, 764 Multiplex PCR methods for GMO analysis, 876 in seafood adulteration detection, 680 (see also Adulteration of seafood, detection) Multiplicative scattering correction (MSC), 743 Multireaction mode (MRM), 721 Multiresidue methods, for veterinary drugs, 720–721 Multisensor techniques for monitoring the quality of fish (MUSTEC), 7 Multispectral imaging, see Imaging spectroscopy, in seafood composition Multivariate trace elemental analysis, usage, 226; see also Seafood and seafood products Mycteroperca microlepis (Grouper), 507 Myocommata, in fish, 426 Myofibrillar proteins, 13 Myripristis berndti (Soldierfish), 766 Mytella guiyanensis (Mussel), 360 Mytilus edulis (Mussel), 34, 273, 353–354 Mytilus galloprovincialis (Mussel), 34, 82, 358, 766 Mytilus trossulus, 34
N National Atmospheric and Oceanic Administration (NOAA), 694–695 National Centre for Biotechnology Information (NCBI), 29 Natural antioxidants, in seafood and seafood products, 321 Natural color system (NCS), 504
Index ◾ Near-infrared (NIR) spectroscopy, 8, 124–128, 678, 742; see also Seafood composition advantages, 13 composition, determination, 122 in fish components determination, 258 in fish freshness determination, 196–197 theory, measurement principles and data analysis, 122–124 Near-infrared transmittance (NIT), for fish analysis, 266 Nematostella vectensis, 47 Nephrops norvegicus (Breaded scampi), 677 Nested PCR (Nt-PCR), 623 Neurotoxic shellfish poisoning (NSP), 5, 662–664 Neutralization, viral isolates identification, 619 Niacin equivalents (NE), 337 Niacin, in seafoods, 336–337 Nicotinamide adenine dinucleotide (NADH), 336, 742 Nicotinamide adenine dinucleotide phosphate (NADP), 336 NIR spectrometer coupled to a microscope (NIRM), 678 NIR spectrum, definition, 258 Nitrofurans, in aquaculture, 724–725 Nitroimidazoles, in aquaculture, 726–727 NMR-mobile universal surface explores (NMRMOUSE), 131 2,6-Nonadienal compound, characteristics, 100 Non-DNA sequencing methods, for species identification, 697–703 Nonintentionally added substances (NIAS), 860 Nonlethal methods, for viral diseases diagnosis, 634–635 Nonlipid contaminants, removal, 72; see also Marine lipid analysis Nonoral methods, usage, 426; see also Fish and fishery products Nonprotein nitrogenous components (NPN), 98 n-3 PUFAs, source, 378 Nuclear magnetic resonance (NMR) spectroscopy, 61, 653, 742; see also Seafood composition in fish fat and water determination, 266 (see also Seafood and seafood products) in lipid classes analysis, 80–81 in seafood composition, 131–132 basic composition, determination, 130 theory and measurement principles, 130–131 Nucleic acid hybridization (NAH), 615, 622–623, 631–632 Nucleic acid sequence based amplification (NASBA), 622, 626 Nucleoside phosphorylase (NP), 58 Nucleosides, chemical structure, 59; see also Seafood and seafood products Nucleotides chemical structure, 59 (see also Seafood and seafood products) and nucleosides
901
CE in determination, 61 chromatographic analysis, 61–63 enzymatic analysis, 64–65 extraction, 60–61 31P-NMR in analysis, 61 Nutrition Committee of the American Heart Association, 190
O Objective methods, in seafood analyses, 7–8; see also Seafood and seafood products Octopus vulgaris, 358, 360, 364 Oil stability index (OSI), 92 Okadaic acid, see Diarrhetic shellfish poisoning (DSP) Oligonucleotide ligation assay (OLA), 218 o-, m-tyrosine, analysis, 785 Oncorhynchus keta, 588 Oncorhynchus mykiss (Rainbow trout), 24, 430, 505, 588, 681, 699 Online control technologies, for seafood quality analysis, 171–172; see also Seafood and seafood products IR spectroscopy, 173–174 microwave methods, advantages, 175–176 microwave spectroscopy-dielectric spectroscopy, 174–175 RF spectroscopy-impedance spectroscopy, 174 ultrasound-acoustic spectroscopy, 172–173 visible spectroscopy, 173 Online sensors, usage, 170; see also Seafood and seafood products o-Phenylenediamine (OPDA), 315 O-phthalaldehyde (OPA), 271, 295, 548, 844 Opisthorchis felineus, 586 Opisthorchis viverrini, 586 Oreochromis niloticus (Tilapia), 46 Organochlorinated pesticides (OCPs), 802 Oryzias latipes (Medaka fish), 46, 50 Ostrea edulis, 358 Ostreococcus tauri (Picoeukaryote), 47 Overpressure TLC (OPTLC), 79 Oxidation, in seafood and seafood products lipid peroxidation, 311 marine lipid oxidation, 311 oxidative stress, 310–311 Oxidograph technique, in oil stability measurement, 92
P Packaging formats, for seafood and seafood products, 853, 860; see also Seafood and seafood products active and intelligent packaging, 867–869 glass jars, 867 metal cans, 863–866 multilayer packaging materials, 867
902
◾
Index
paper and cartonboard, 862–863 plastics, 861–862 surface-active biocides, 869–870 Pagelus erythrinus, 358 Pagrus major (Red sea bream snapper), 508, 742 Pagrus pagrus, 400 Paleomon serratus, 360 Pantothenic acid, in seafoods, 341 Paper and cartonboard, in seafood packaging, 862–863 Paragonimidosis, symptoms and species causing, 582 Paragonimus westermani, 582 Paralytic shellfish poisoning (PSP), 5, 655–657 Parapenaeus longirostris, 360 Parasite infections in humans, from seafoods, 579–580; see also Seafood and seafood products acanthocephalans, 595–596 anisakid nematodes, 590–595 cestodes, 587–589 protozoa, 581–582 trematodes, 582–587 Partial least square (PLS), 124, 258, 678 Partial least-square regression (PLS-R), 196 Patinopectin yessoensis (Scallop), 747 PCR-based method, in Vibrio sp. detection, 561–562 PCR-restriction fragment length polymorphism (PCRRFLP), 680–681; see also Adulteration of seafood, detection PCR-sequencing, application, 680; see also Adulteration of seafood, detection PCR-single-stranded conformation polymorphism (PCR-SSCP), 680 Pecten maximus (Scallop), 273 Penaeus aztecus (Gulf brown shrimp), 509 Penaeus duorarum (Gulf pink shrimp), 509 Penaeus indicus (Tropical shrimp), 677 Penaeus japonicus (Kuruma prawn), 747 Penaeus monodon (Black Tiger Prawn), 715 Penaeus monodon (Tiger prawn), 34 Penaeus semisulcatus, 360 Penaeus setiferus (Gulf white shrimp), 509 Penaeus vannamei (Pacific White shrimp), 508, 715 Penicillin, in aquaculture, 724 Penicillium chrysogenum, 724 Penicillium notatum, 724 Peptide, characterization, 16–17 Peptide mass fingerprinting, protein identification, 28–31 Peptone Iron Agar (PIA), 522 Perna perna (Mussel), 360 Peroxides, features, 89 Peroxide value (PV), 88–89, 202–203 Peroxiredoxins (Prx), role, 314 Persistent organic pollutants (POPs), 6, 798, 818 Phaeodactylum tricornutum (Diatoms), 47 Phenylisothiocyanate (PITC), 292–294 Phenylthiocarbamyl (PTC), 292–293 Phospholipids, antioxidant activity, 320
Phospholipids (PL), 70 31Phosphorous nuclear magnetic resonance spectroscopy (31P-NMR), 61 Photobacterium damselae, 839 Photobacterium phosphoreum, 104, 839 Photoelectric color-measuring instruments, usage, 397–399 Physical methods, in seafood freshness assessment cosmos, 195 electronic nose, 196 Intellectron Fischtester VI, 195 near-infrared reflectance spectroscopy, 196–197 RT-Freshtester, 195 texture analysis, 194 torrymeter, 194–195 Phytoplanktons, toxins, 664–666; see also Marine toxins Pike fry rhabdovirus (PFRV), 618 Pinctada radiata (Pearl oyster), 360 Pinnatoxins, 666; see also Marine toxins Plaque neutralization test, for antibody detection, 621 Plastics, in seafood packaging, 861–862 Plecoglossus altivelis, 586 Plesiomonas shigelloides, 839 Pleuronectes platessa (Plaice), 505, 738 Polar organic solvents, usage, 72 Pollachius virens (Saithe), 505 Polychlorinated biphenyls (PCBs), in seafoods, 818, 824–825 chromatographic analysis, 828–829 clean-up of extracts, 827–828 extraction methods, 825–827 Polychlorinated dibenzofurans (PCDFs), 798 Polychlorinated dibenzo-p-dioxins (PCDDs), 798 Polycyclic aromatic hydrocarbons (PAHs), 234 European regulations, 249–250 extraction and analysis methods, 249 properties and toxicology, 248–249 in seafoods, 818–819 chromatographic analysis, 823–824 clean-up of extracts, 821–822 methods of extraction, 819–821 Polydimethylsiloxane (PDMS), 448 Polymerase chain reaction (PCR), 8, 581, 607, 676, 874 Polymorphic DNA markers, usage, 49 Polyunsaturated fatty acids (PUFA), 70, 87, 98, 190, 378 Pomadasys argenteus, 364 Pomotomus saltatrix (Bluefish), 508 Porous graphitic carbon (PGC), 804 Portunus pelagicus (Blue swimmer crab), 360, 678 Pressurized liquid extraction (PLE), 820–821, 826–827 Primary oxidation products, methods, 88–89; see also Lipid oxidation, analysis Primary smoke condensates (PSC), 236 Primary tar fraction (PTF), 236 Primer-extension assay (PER), 680 Principle component analysis (PCA), 107–108, 196, 742, 766
Index ◾ Processed animal proteins (PAPs), 679 Processed fish microstructure; see also Seafood and seafood products salted cod, 149–150 smoked salmon, 148–149 surimi, 150–151 Processed seafood adulteration, 691 Propanol detection, in fish, 104; see also Volatile aroma compounds, in fish Protective atmospheres, application, 842; see also Biogenic amines (BA), in seafood Protein analysis, in farmed and wild seafood, 218–221 (see also Seafood and seafood products) autolysis and oxidation, 32–33 (see also Seafood analyses, 2DE applications) based detection for GMOs analysis, 877–880 (see also Genetically modified organisms (GMOs)) in seafood sp. identification, 691–692 oxidation, 17 in seafood (see also Seafood and seafood products) analysis of soluble, 14–15 determination, 270 immunoassays, 16 modifications, 17–18 nitrogen, determination, 270 nondestructive analysis, 273 nutritional aspects, 269 solubility classes, 13–14 soluble protein, determination, 270–272 total content, 12–13 Proteolysis, role, 98 Proteome analysis, 22 2DE methods analysis, 28 equilibration, 27 first-dimension electrophoresis, 25–27 sample extraction and cleanup, 25 second-dimension electrophoresis, 27 staining, 28 protein identification, 28–31 sample matrix degradome, 24–25 muscle proteomes, 24 whole larval proteomes, 22–24 Proteomics, definition, 22 Proteus mirabilis, 839 Proteus vulgaris, 839 Proton-transfer reaction MS (PTR-MS), 453 Protozoa, in seafoods, 581–582 Pseudomonas fluorescens, 518, 839 Pseudomonas fragi, 105, 521 Pseudomonas nigrifaciens, 521 Pseudomonas perolens, 521 Pseudomonas putida, 518, 839 Pseudonitzschia australis, 660
903
Pseudonitzschia pseudodelicataissima, 660 Pseudonitzschia pungens f. multiseries, 660 Pseudopleuronectes americanus (Blackback flounder), 507 Pseudoterranova decipiens, 590 Pterygotrigla hemistica (Spottyback searobin), 766 Pulsed electric fields (PEF), 149 Pulsed-field gel electrophoresis (PFGE), 559 Pulsed infrared stimulation (PSL), 787 Pulsed xenon arc (PXA), 397 Puncture test, in seafood analysis, 428–429 Pyrodinium bahamense, 655
Q Quality index method (QIM), 8–9, 426, 544 in fish freshness evaluation, 191–192 for fish storage, 463–468 development, 472–476 EU-scheme, 476–477 principle, 464 usage, 468–472 Quantitative descriptive analysis (QDA), for fish freshness assessment, 192–194 Quartz microbalances (QMB), 158, 162
R Radiation hybrid (RH), 45 Radioimmunoassay (RIA), in CTX detection, 652 Radioisotope dilution methods, limitation, 340; see also Water-soluble vitamins, in seafood Radiolytic chemicals, analysis, 782–785; see also Food irradiation Radiolytic hydrogen and carbon monoxide, detection, 785 Random amplified polymorphic DNA (RAPD), 44, 559, 680, 699 Raoultella planticola, 839 Ribonuclease protection assay (RPA), 622 Reactive nitrogen species (RNS), 310 Reactive oxygen species (ROS), 310 Real-time PCR, usage, 681–682; see also Adulteration of seafood, detection Regulatory Fish Encyclopedia (RFE), 692, 696 Relative standard deviations (RSD), 764 Repeatability and reproducibility (R&R), 605 Restriction endonuclease digestion profi les (REDP), 570 Restriction fragment length polymorphism (RFLP), 44, 622 Retention index (RI), 113 Retinol activity equivalents (RAE), 331–332 Retinol equivalents (RE), definition, 331 Reversed-phase high-performance liquid chromatography (RP-HPLC), 61–62 in fish amino acids assessment, 292–298
904
◾
Index
RF spectroscopy, in seafood quality analysis, 174; see also Seafood and seafood products Riboflavin, in seafoods, 335–336 RT-Freshtester, role, 182, 195
S Saccharomyces cerevisiae (Yeast), 25 Saccharomyces uvarum, 338 Salmonella spp., 774 Salmonella spp, in seafoods, 559, 570–571; see also Seafoods and seafood-related environments, foodborne pathogens Salmon, sensory profi ling, 485–486 Salmo salar (Atlantic salmon), 111, 216, 501, 681, 699 Salt-soluble proteins, see Myofibrillar proteins Salvelinus alpinus (Arctic charr), 25 Saponification, in PAHs isolation, 819–820 Sarcoplasmic proteins, 13 Sardina pilchardus, 319, 357–358 Sarotherodon mossambicus (Red tilapia), 652 Saurida undosquamis, 353 Saxitoxin (STX), see Paralytic shellfish poisoning (PSP) Scanning electron microscopes (SEM), usage, 140, 146, 149 Scomber scombrus (Atlantic mackerel), 357, 508, 738 Scombroid poisoning, definition, 834–835 Seafood analyses, 2DE applications allergen identification, 34–35 development, 31–32 quality involution aquaculture and antemortem effects, 33 protein autolysis and oxidation, 32–33 species authentication, 34 Seafood and seafood products, 367–368; see also Fresh and frozen-thawed fish, differentiation methods; Veterinary drugs, in aquaculture added antioxidants natural antioxidants, 321 synthetic antioxidants, 320–321 adulteration by foreign proteins chromatographic techniques, 679 DNA technologies, 679–683 electrophoresis, 676–677 immunological techniques, 677–678 microscopic methods, 678–679 visible and near-infrared spectrometry, 678 adulteration, species identification DNA-based techniques, 692–703 future perspectives, 703–704 protein based methods, 691–692 species replacement, 688–691 analytical methods, 7–8 antioxidants, 311–313 antioxidant enzymes, 313–314 ascorbic acid, 314–315 carotenoids, 317–319
endogenous, 320 ubiquinone, 319–320 vitamin E, 315–317 ATP-related compounds, analysis, 59–60 nucleotides and nucleosides determination, 61–65 nucleotides and nucleosides, extraction, 60–61 benefits and risks, 6 biogenic amines determination, 843–846 formation, 838–842 legal limits, 837–838 as quality index, 836–837 toxicity, 834–836 borne parasites, infections, 580 acanthocephalans, 595–596 anisakid nematodes, 590–595 cestodes, 587–589 protozoa, 581–582 trematodes, 582–587 calories direct measurement, 274 food composition tables and databases, 276 indirect measurement, 275–276 carbohydrate content, determination, 273 chemical and biochemical methods, in freshness evaluation ATP, 197–198 biogenic amines, 199 dimethylamine, 201 formaldehyde, 201 lipid hydrolysis, 203 lipid oxidation indicators, 201–203 pH, 199–200 trimethylamine, 200–201 TVB-N, 200 chemical migration from contact materials, 852–857 packaging materials, 860–870 residues test, 857–858 testing strategies, 858–860 chemical sensor, technologies amperometric gas sensors, 158 color indicators, 159–160 conductance changes, sensor, 157–158 FET, 159 mass transducers, 158 determination of antioxidants, 313 developments, 598 dioxin analysis, 798–799 automation of extraction and cleanup, 804–805 bioanalytical screening methods, 808–809 extraction techniques, 800–802 fractionation and group separation, 803–804 instrumental determination, 805–808 sample pretreatment and recovery studies, 799–800
Index ◾ electronic noses application, 160–164 elemental composition aluminum, 353 arsenic, 353–354 chromium, 354–358 iodine, 358–359 manganese, 360 molybdenum, 361 selenium, 361–364 thallium, 364 vanadium, 364 zinc, 365–367 essential amino acids, analysis, 290–291 capillary zone electrophoretic technique, 298–299 free essential amino acids analysis, 288–289 GLC methods, 298 HPLC method, 291–298 MS, 299–300 total essential amino acid analysis, 289–290 farmed and wild genetic analysis, 217–218 lipid analysis, 221–223 morphological differentiation, 216–217 proteins analysis, 218–221 stable isotopes, 224–226 trace element fingerprint, 226–227 fat-soluble vitamins vitamin A and carotenoids, 328–332 vitamin D, 332 vitamin E, 332–333 vitamin K, 333–334 fish muscle microstructure, 140–145 hake, 146–148 herring, 145–146 food quality sensor, usage, 170 freshness assessment, physical methods cosmos, 195 electronic nose, 196 Intellectron Fischtester VI, 195 near-infrared reflectance spectroscopy, 196–197 RT-Freshtester, 195 texture analysis, 194 torrymeter, 194–195 freshness evaluation, microbiological methods, 203–204 genomics and genetics, 44–45 aquaculture and genomics, 49–51 fisheries and biodiversity management, 47–49 genomic resources and genome projects, 45–47 handling, 502 human senses chemical sense, 484 gustatory sense, 483–484 olfactory sense, 483 sense of hearing, 484 sense of touch, 483 sense of vision, 482–483
905
instrumental texture compression analysis, 429–430 Kramer test, 427 puncture test, 428–429 stress relaxation test, 431–432 tension analysis, 429 TPA, 430–431 viscoelastic methods, 431 Warner–Bratzler test, 427–428 limitations, 8–9 lipids methods comparison, 269 nondestructive methods, 268–269 nutritional aspects, 267 total lipids, determination, 267–268 metals determination, spectrochemical methods AAS, 752–756 AES, 756–759 analytical atomic spectroscopic technique, 760–761 ICP-MS, 759–760 sample preparation, 761 selected applications, 762–769 microscopy techniques, 139–140 microwave technology, applications, 179–180 freshness and salting process, 182–183 moisture content, determination, 180–182 microwave theory, 176–179 nucleosides and nucleotides, chemical structure, 59 objective and subjective sensory analysis, 482 online control technologies, 171–172 IR spectroscopy, 173–174 microwave methods, advantages, 175–176 microwave spectroscopy-dielectric spectroscopy, 174–175 RF spectroscopy-impedance spectroscopy, 174 ultrasound-acoustic spectroscopy, 172–173 visible spectroscopy, 173 oxidation lipid peroxidation, 311 marine lipid oxidation, 311 oxidative stress, 310–311 PAHs analysis, 818–819 chromatographic analysis, 823–824 clean-up of extracts, 821–822 methods of extraction, 819–821 PCBs analysis, 818, 824–825 chromatographic analysis, 828–829 clean-up of extracts, 827–828 extraction methods, 825–827 poisoning, marine toxins ichthyosarcotoxins, 650–655 from phytoplanktons, 664–666 shellfish toxins, 655–664 processed fish microstructure
906
◾ Index
salted cod, 149–150 smoked salmon, 148–149 surimi, 150–151 proteins analysis of soluble, 14–15 determination, 270 immunoassays, 16 modifications, 17–18 nitrogen, determination, 270 nondestructive analysis, 273 nutritional aspects, 269 solubility classes, 13–14 soluble protein, determination, 270–272 total content, 12–13 proximate composition, nondestructive analysis, 258–266 quality assessment, 517–518 quality control importance, 170–171 safety for allergic and immunosuppressed persons, 597–598 for consumers and restaurateurs, 597 production and handling, 596 thermal processing, 596–597 sampling, 6–7 sensory analysis, 482, 502 assessors and sessions, 503–505 examples, 507–510 preparation of heat-treated samples, 505–506 sensory attributes, 506 sensory descriptors appearance, 487–488 odor, 488 taste, 488–492 texture, 492–494 usage, 484–486 sensory methods, in freshness evaluation, 190–191 EU freshness grading, 191 QDA, 192–194 QIM, 191–192 Torry Scheme, 192 smoke flavoring (SF), 234–236 European regulations on PAH, 249–250 extraction and analysis methods of PAH, 249 liquid smokes, 236 organoleptic roles of volatile compounds, 241–247 in preservation, 247 smoke by-products, 237–238 smoke oils, 237 smoke powders, 237 usage, 238–239 in volatile compounds generation, 240–241 spoilage Aeromonas genus, 525 Alteromonas genus, 521 bacterial identity confirmation tests, 528–532
Brochothrix genus, 524 chemical causes, 517 gamma-irradiated seafood, microbiology, 526 genera Flavobacterium and Cytophaga, 524 genera Moraxella and Acinetobacter, 523 Lactobacillus genus, 525 MA stored seafood, microbiology, 525–526 Photobacterium genus, 524–525 Pseudomonas genus, 518–521 Shewanella genus, 521–523 varius bacterial counts, determination, 527–528 Vibrio genus, 525 squid microstructure, 151 storage conditions, 4 test procedure and sample preparation, 426–427 texture measurement, 432–433 total fatty acids, analysis column, 383–384 GC analysis, 382–383 lipid extraction, 379–380 MS of fatty acid derivatives, 384–387 preparation of fatty acid derivatives, 380–382 trends and outlook, 9 water content, determination, 274 water-soluble vitamins biotin, 341–342 folate, 338–340 niacin, 336–337 pantothenic acid, 341 riboflavin, 335–336 thiamin, 334–335 vitamin A, 328–332 vitamin B2, 335–336 vitamin B3, 336–337 vitamin B5, 341 vitamin B6, 337–338 vitamin B12, 340–341 vitamin C, 342–343 vitamin D, 332 vitamin E, 315–317, 332–333 vitamin K, 333–334 world catch and harvest, 3–4 Seafood composition imaging spectroscopy basic constituents, analysis, 128–130 theory, measurement principles and data analysis, 128 NIR, 124–128 composition, determination, 122 theory, measurement principles and data analysis, 122–124 NMR spectroscopy, 131–132 basic composition, determination, 130 theory and measurement principles, 130–131
Index ◾ X-ray imaging analysis, 132–133 theory and measurement principles, 132 Seafood lipids, in human diet, 70 Seafoods and seafood-related environments, foodborne pathogens, 557–559 Listeria monocytogenes, 568–570 Salmonella, 570–571 Vibrio cholerae in, 567–568 Vibrio parahaemolyticus in, 560–565 Vibrio vulnificus in, 565–567 Seafood species and protein sequences, 30 Selective ion monitoring (SIM), 807, 824, 828 Selenium, in seafood, 361–364 Semipermeable membranes (SPMs), 803 Sensor parameters, 154–157 in quality assessment, 170 Sensory analysis, of seafood, 482, 500, 507–510; see also Seafood and seafood products assessors and sessions, 503–505 for fish spoilage, 545 human senses chemical sense, 484 gustatory sense, 483–484 olfactory sense, 483 sense of hearing, 484 sense of touch, 483 sense of vision, 482–483 objective and subjective sensory analysis, 482 preparation of heat-treated samples, 505–506 sensory attributes, 506 sensory descriptors appearance, 487–488 odor, 488 taste, 488–492 texture, 492–494 usage, 484–486 Sensory methods in seafood analyses, 7–8 (see also Seafood and seafood products) in seafood freshness evaluation, 190–191 EU freshness grading, 191 QDA, 192–194 QIM, 191–192 Torry Scheme, 192 Sensory Texture Profi le, 426 Sepia officinalis, 358 Sequence-characterized amplified regions (SCARs), 681, 699 Sequence-tagged sites (STS), 45 SEQUID project, 7, 183 Serratia fonticola, 839 Serratia liquefaciens, 839 Shellfish
907
anatomy, 500 toxins (see also Marine toxins) amnesic poisoning, 660–662 diarrhetic poisoning, 658–660 neurotoxic poisoning, 662–664 paralytic poisoning, 655–657 Shewanella putrecfaciens, 105, 522 Short-wavelength near-infrared spectroscopic method (SW-NIR), 549 Silurus glanis, 768 Silver ion chromatography, in lipid classes analysis, 80 Silver staining methods, usage, 15; see also Seafood and seafood products Simultaneous distillation/extraction (SDE), 443–445 Single nucleotide polymorphism (SNP), 44, 51, 217, 698 Single-strand conformation polymorphisms (SSCP), usage, 700 Size exclusion chromatography (SEC), 798 Small and medium sized (SMEs), 8 Smoked fish odors, 111–112; see also Volatile aroma compounds, in fish Smoked salmon, volatile compounds, 101–103 Smoke flavoring (SF) process, in seafood, 234–236 liquid smokes, 236 organoleptic roles of volatile compounds in color, 246–247 in flavor, 245–246 in odor, 241–245 in texture, 246 in preservation, 247 smoke by-products, 237–238 smoke oils, 237 smoke powders, 237 usage, 238–239 in volatile compounds generation, 240–241 Smoke oils, usage, 237; see also Seafood and seafood products Smoke powders, usage, 237; see also Smoke flavoring (SF) process, in seafood Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 25 Sodium dodecyl sulfate (SDS) electrophoresis, 16, 676 Soft independent modeling of class analogy (SIMCA), 124, 743–744 Solea aegyptiaca (Egyptian sole), 873 Solea solea (Common sole), 32, 354 Solid phase extraction (SPE), 718, 800–801 PAHs determination, 822 Solid-phase microextraction (SPME), 90, 447–449, 545–546, 785 Soluble proteins, analysis, 14–15; see also Seafood and seafood products Solvent-assisted flavor evaporation (SAFE), 445 Sonication method for PAHs extraction, 820 for PCBs extraction, 826
908
◾ Index
Soxhlet extraction method of dioxins, 800 (see also Dioxin analysis, from seafoods) in fish lipids determination, 268 for PAHs, 820 of PCBs, 825–826 usage, 73 (see also Marine lipid analysis) Spanish Agency of Food Safety and Nutrition, 597 Sparus aurata, 353 Species replacement and adulterations, 688–691; see also Adulteration of seafood, detection Species-specific PCR, in seafood adulteration detection, 680; see also Adulteration of seafood, detection Specific spoilage organisms (SSO), 8, 98, 203–204 Spectral–QC, usage, 398 Spectrochemical methods, for seafood metals determination analytical atomic spectroscopic technique, 760–761 atomic absorption spectrometry, 752–756 atomic emission spectrometry, 756–759 ICP-MS, 759–760 sample preparation, 761 selected applications, 762–769 Spectrophotometer, function, 398 Spectroscopic analysis, usage, 742–747; see also Fresh and frozen-thawed fish, differentiation methods Sphyrna lewini, 360 Spisula subtruncata (Shell), 358 Spring viremia of carp virus (SVCV), 618 Squid, microstructure, 151 S. salar (Whole Farmed Salmon), QIM scheme, 465–466 Stable isotopes, in farmed and wild seafood, 224–226; see also Seafood and seafood products Stable micro systems (SMS), 427 Standard Error of Prediction (SEP), 475 Sterols, analysis, 76–77; see also Marine nonsaponifiable matter, analysis Stictodora lari, 586 Stress relaxation test, in seafood analysis, 431–432 Strongylocentrotus purpuratus (Purple sea urchin), 46 Sulfide-producing bacteria (SPB), 549 Sulfitocobalamin, 340 Sulfonamides, in aquaculture, 721–722 Supercoiled mitochondrial DNA, usage, 787 Supercritical fluid chromatography (SFC), 77, 82 Supercritical fluid extraction (SFE), 442, 783, 798, 801, 821, 827 Superoxide dismutases (SOD), role, 313 Surface-active biocides, in seafood packaging, 869–870 Surface plasmon resonance (SPR) system, 882 Surimi and surimi products, color measurements, 404–407 Synthetic antioxidants, in seafood and seafood products, 320–321
T Takifugu rubripes, 46 tdh gene, 563–564 Technicon AutoAnalyzer, usage, 12 Tenax TA, usage, 446–447; see also Fish and seafood aroma Tension analysis, in seafood analysis, 429 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 798 Tetracycline, in aquaculture, 722 2-Tetradecylcyclobutanone (TCB), 782–783 Tetrahydrofolic acid (THF), 339 Tetramethylsilane (TMS), 223 Tetraodon nigroviridis, 46 Tetrodotoxin (TTX) poisoning; see also Marine toxins clinical symptoms, 653–654 detection methods, 654–655 Texture analyses, of seafood, 194; see also Seafood and seafood products Texture profi le analysis (TPA), 427 in seafood analysis, 430–431 Thalassiosira pseudonana (Diatoms), 47 Thallium, in seafood, 364 Thermal infrared (TIR), 173 Thermal processing, of fish and fishery products, 403; see also Fish and fishery products Thermal processing, of seafood, 596–597 Thermography technology, application, 174 Thermoluminescence (TL) technique, usage, 787 Thiamin, in seafoods, 334–335 Thin-layer chromatography (TLC), 61, 77, 79, 655 Thiobarbituric acid-reactive substances (TBARS), 89–90, 109 Thiobarbituric acid (TBA), 203 Thiosulfate-citrate-bile salts-sucrose agar (TCBS), 560 Thunnus alalunga (Albacore tuna), 82 Time domain spectroscopy (TDR), 8 Time-of-flight (TOF), usage, 450; see also Fish and seafood aroma tlh gene, 563–564 a-Tocopherol, usage, 316 Todarodes sagittatus (Squid), 358 Tolerable daily intake (TDI), 798 Torrymeter in fish freshness determination, 194–195 usage, 737–738 Torry Scheme, for fish freshness assessment, 192 Total body electrical conductivity (TOBEC), 266 Total essential amino acid analysis, in seafood, 289–290; see also Seafood and seafood products Total fatty acids, analysis; see also Seafood and seafood products column, 383–384 GC analysis, 382–383 lipid extraction, 379–380 MS of fatty acid derivatives, 384–387 preparation of fatty acid derivatives, 380–382
Index ◾ Total lipids, determination, 267–268 Total quality management (TQM), 171 Total viable count (TVC), 8, 203 Total volatile bases (TVB), 105, 545 Total volatile basic nitrogen (TVB-N), 7, 200 Totox value, 90; see also Lipid oxidation, analysis Toxins risk, in mussels and fish, 6 Trace element fingerprint, in farmed and wild seafood, 226–227; see also Seafood and seafood products Trachurus trachurus (Whole scad), 195, 357 Tranquilizers, in aquaculture, 728 Transcriptomics, usage, 51; see also Seafood and seafood products Transmission electron microscopes (TEM), usage, 125, 140, 146; see also Seafood composition Trematodes, in seafoods, 582–587 trh gene, 564 Triacylglycerols (TG), 70 Trichloroacetic acid (TCA), 545 Trienzyme extraction method, importance, 339 Triglyceride (TG), 221 Trimethylamine and fish spoilage indicator, 200–201; see also Seafood and seafood products Trimethylamine oxide (TMAO), 200–201, 270, 441, 517, 541 Trimethylamine (TMA), 441, 449, 517 Trimethylsilyl (TMS), 77 Trinitrobenzene-sulfonic acid (TNBS), 17 Triphenylmethane dyes, in aquaculture, 727 Triphenyltetrazolium chloride soya tryptone agar (TSAT), 560 Trypticase Soy Agar supplemented with yeast extract (TSAYE), 569 Tryptose soy broth (TSB), 560 Tubular TLC (TTLC), 79 Turbo livona pica, 651 Two-dimensional gel electrophoresis (2-DE), 22, 677
U Ubiquinone, in seafood and seafood products, 319–320 Ultrasound-acoustic spectroscopy, for seafood quality analysis, 172–173; see also Seafood and seafood products United Nations Environment Programme (UNEP), 818 Universal pre-enrichment broth (UPB), 569 Urophycis tenuis (White hake), 507 U.S. Environmental Protection Agency (US-EPA), 249 U.S. Food and Drug Administration, 597 UV detection, in folic acid detection, 340
V Vacuum packaging, of seafoods, 868 Vanadium, in seafood, 364
909
Veterinary drugs, in aquaculture, 714–716 analytical techniques for analysis, 717–721 anthelmintic drugs, 729–730 antimycotica, 727–728 bactericidal drugs, 721–727 future perspectives, 730–731 hormones, 730 origin of residues, 716–717 tranquilizers and antiparasitica, 728–729 Vibrio alginolyticus, 839 Vibrio cholerae, in seafoods, 558, 567–568; see also Seafoods and seafood-related environments, foodborne pathogens Vibrio parahaemolyticus, in seafoods, 558, 560–565; see also Seafoods and seafood-related environments, foodborne pathogens Vibrio vulnificus, in seafoods, 558, 565–567; see also Seafoods and seafood-related environments, foodborne pathogens Viral cell line, selection, 614 Viral diseases, in fish and shellfish aquaculture; see also Seafood and seafood products diagnosis and screening, 604–605 analytical techniques, 608 clinical, histological, and microscopical techniques, 609–613 factors affecting accuracy, 606–608 isolation in cell culture, 613–616 molecular diagnosis, 622–634 nonlethal methods, 634–635 serological and immunological techniques, 616–622 validation, 605–606 Viral hemorrhagic septicemia virus (VHSV), 606, 714 Viral isolation, in cell culture, 614–615 Viral nucleic acid, extraction, 627 Virus detection, IFAT, 620 Virus extraction, methods, 607 Viscoelastic methods, in seafood analysis, 431 Visible spectroscopy, in seafood quality analysis, 173; see also Seafood and seafood products Visible spectroscopy/near infrared spectroscopy (VS/NIRS), 173 Volatile aroma compounds, in fish aroma development, 98–99 fresh odors, 99–100 quality indicators, identification, 100–103 microbial spoilage odors, 103–106 oxidatively derived odors, 106–110 processing odors, 111–113 Volatile basic nitrogen (VBN), 545 Volatile compounds generation, SF process, 240–241; see also Smoke flavoring (SF) Volatile hydrocarbons, analysis, 784–785 Volvox carteri, 47
910
◾
Index
W
X
Wall-coated open tubular (WCOT), 75, 379, 383 Warner-Bratzler (W-B) test, in seafood analysis, 427–428 Washed cod muscle system, usage, 108–110; see also Volatile aroma compounds, in fish Water content determination, in seafood, 274; see also Seafood and seafood products Water-soluble vitamins, in seafood biotin, 341–342 folate, 338–340 niacin, 336–337 pantothenic acid, 341 riboflavin, 335–336 thiamin, 334–335 vitamin B6, 337–338 vitamin B12, 340–341 vitamin C, 342–343 Western blot technique, application, 877 White spot syndrome virus (WSSV), 609 World catch and harvest, of seafood, 3–4
Xanthine oxidase (XO), 58 Xanthine (Xa), 58 Xiphias gladius (Sword-fish), 508 X-ray imaging, of seafood composition; see also Seafood composition analysis, 132–133 theory and measurement principles, 132 Xylose lysine desoxycholate (XLD), 570
Y Yellow head disease virus (YHDV), 618 Yesilirmak River fish, metal concentration determination, 767 Yessotoxins (YTXs), 658
Z Zeeman atomic absorption spectrometry, usage, 360 Zinc, in seafood, 365–367 Zosterisessor ophiocephalus, 368
