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Chemical and Functional Properties of Food Components Third Edition
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Chemical and Functional Properties of Food Components Series SERIES EDITOR
Zdzisław E. Sikorski
Chemical and Functional Properties of Food Components, Third Edition Edited by Zdzisław E. Sikorski
Carcinogenic and Anticarcinogenic Food Components Edited by Wanda Baer-Dubowska, Agnieszka Bartoszek and Danuta Malejka-Giganti
Methods of Analysis of Food Components and Additives Edited by Semih Ötleş
Toxins in Food Edited by Waldemar M. Dąbrowski and Zdzisław E. Sikorski
Chemical and Functional Properties of Food Saccharides Edited by Piotr Tomasik
Chemical and Functional Properties of Food Lipids Edited by Zdzisław E. Sikorski and Anna Kolakowska
Chemical and Functional Properties of Food Proteins Edited by Zdzisław E. Sikorski
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Chemical and Functional Properties of Food Components Third Edition EDITED BY
Zdzislaw E. Sikorski Gda´nsk University of Technology Gdansk, ´ Poland
Boca Raton London New York
CRC Press is an imprint of the Taylor & Francis Group, an informa business
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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & 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-10: 0-8493-9675-1 (Hardcover) International Standard Book Number-13: 978-0-8493-9675-5 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. 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 Chemical and functional properties of food components / edited by Zdzislaw E. Sikorski. -- 3rd ed. p. cm. Includes bibliographical references and index. ISBN 0-8493-9675-1 (alk. paper) 1. Food--Analysis. 2. Food--Composition. I. Title. TX545.C44 2007 664’.07--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com
2006047537
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Preface Water, saccharides, proteins, lipids, and mineral compounds constitute the main building materials of the tissues and are responsible for the nutritional value and most sensory properties of foodstuffs. A large number of other constituents present in lower quantities, especially colorants, flavor compounds, vitamins, prebiotics, probiotics, and additives, also contribute to food quality. These compounds undergo various biochemical and chemical changes during postharvest storage and processing of raw materials. The extent of changes depends on the chemical properties of these food components, on the conditions of storage, and on the parameters of freezing, salting, drying, smoking, marinating, frying, cooking, and other methods of preservation or processing. The material presented in this book emphasizes the role of the chemical properties of different food constituents, and shows how the reactions that take place in the conditions of storage and processing affect the quality of foodstuffs. In Chapters 1 and 2, the content of various food components is described, as well as their role in the structure of raw materials and the formation of different attributes of quality. The components contained in foods in the largest amounts are presented in detail in Chapters 3 through 7. Chapter 8 deals with the flow properties of food materials and presents numerous examples of the rheological behavior of various products. Chapters 9 and 11 describe factors affecting the color and flavor of foods, respectively, and Chapter 13, the most important food additives. Interactions among different constituents are key with respect to many features of the quality of industrially processed and home-prepared foods. These are treated in Chapter 12. Chapters 10 and 14 through 20 of the book deal with different aspects of the biological value and safety of foodstuffs, including allergenic activity, the role of prebiotics and probiotics, as well as the effect of food on the moods and health of people. The characteristics that make this volume very different from the second edition are the new contributions on lipids and rheology, the integrating chapter on interactions, and new chapters dealing with the safety and biological aspects of foods. The volume contains a well-documented presentation of the current state of knowledge concerning food in the form of concise monographs. The text is based on the personal research and teaching experience of the authors, as well as on critical evaluation of the current literature. According to the editor’s suggestions, most lists of cited references contain only the indispensable sources. The book is addressed to food science graduate students, professionals in the food industry, nutritionists, personnel responsible for food safety and quality control, and to all persons interested in the roles and attributes of different food constituents. In preparing the book I have had the privilege of working with a large group of colleagues from several universities and research institutions in Asia, Australia, Europe, and North America, who have agreed to share their knowledge and experi-
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ence with the reader. Their acceptance of my editorial suggestions and the timely preparation of the high-quality manuscripts are sincerely appreciated. I dedicate this volume to the scores of researchers, especially Ph.D. students, whose investigations in universities all over the world contribute to a better understanding of the nature and interactions of food components, and thus lead to improving the quality of foods. A small fraction of the results of these investigations has been used in preparing this new edition of the book. Zdzisław E. Sikorski Gdańsk University of Technology
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About the Editor Zdzisław E. Sikorski earned his B.S., M.S, Ph.D., and D.Sc. in food technology and chemistry from the Gdańsk University of Technology (GUT), and his Dr honoris causa from the Agricultural University in Szczecin. He gained practical experience in breweries, in fish, meat, and vegetable processing plants in Poland and Germany, and on a deep-sea fishing trawler. He was an organizer, professor, and head of the Department of Food Chemistry and Technology, and served two terms as dean of the Faculty of Chemistry at GUT, 7 years as chairman of the Committee of Food Technology and Chemistry of the Polish Academy of Sciences, chaired the scientific board of the Sea Fisheries Institute in Gdynia for 9 years, and was an elected member of the Main Council of Science and Tertiary Education in Poland for 11 years. Dr. Sikorski was one of the founders and is now an honorary member of the Polish Society of Food Technologists. He also was a researcher and professor at Ohio State University in Columbus, Ohio; the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Hobart, Australia; the Department of Scientific and Industrial Research (DSIR) in Auckland, New Zealand; and the National Taiwan Ocean University, Keelung. His published works include about 210 papers, 15 books, 9 chapters on marine food science and food chemistry in edited volumes, and he holds seven patents in the area of fish and krill processing. Several of his books have appeared in multiple editions. He is a member of the editorial board of the Journal of Food Biochemistry and of two Polish food science journals. His research deals mainly with food preservation, the functional properties of food proteins, and the interactions of food components. In 2003 he was elected a Fellow of the International Academy of Food Science and Technology.
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Contributors Agnieszka Bartoszek Gdańsk University of Technology Gdańsk, Poland
Wiesława Łysiak-Szydłowska Medical Academy of Gdańsk Gdańsk, Poland
Maria Bielecka Polish Academy of Sciences Olsztyn, Poland
Michał Nabrzyski Medical Academy of Gdańsk Gdańsk, Poland
Maria H. Borawska Medical Academy of Białystok Białystok, Poland
Krystyna Palka Agricultural University of Cracow Cracow, Poland
Emilia Barbara Cybulska The Elbląg University of Humanities and Economy Elbląg, Poland
Barbara Piotrowska Merck Warsaw, Poland
Peter Edward Doe University of Tasmania Hobart, Tasmania
Anna Pruska-Kędzior Agricultural University of Poznań Poznań, Poland
Norman F. Haard University of California Davis, California
Bob Rastall University of Reading Reading, United Kingdom
Julie Miller Jones College of St. Catherine St. Paul, Minnesota
Adriaan Ruiter Professor Emeritus Utrecht University Utrecht, The Netherlands
Zenon Kędzior Agricultural University of Poznań Poznań, Poland Jen-Min Kuo Department of Seafood Science National Kaohsiung Marine University Kaohsiung, Taiwan
Grażyna Sikorska-Wiśniewska Medical Academy of Gdańsk Gdańsk, Poland Zdzisław E. Sikorski Gdańsk University of Technology Gdańsk, Poland
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Piotr Siondalski Medical Academy of Gdańsk Gdańsk, Poland
Alphons G.J. Voragen Wageningen University Wageningen, The Netherlands
Andrzej Stołyhwo Warsaw Agricultural University Warsaw, Poland
Jadwiga Wilska-Jeszka Technical University of Łódż Łódż, Poland
Bonnie Sun-Pan Department of Food Science National Taiwan Ocean University Keelung, Taiwan
Barbara Wróblewska Polish Academy of Sciences Olsztyn, Poland
Małgorzata Szumera Medical Academy of Gdańsk Gdańsk, Poland Piotr Tomasik Agricultural University of Cracow Cracow, Poland
Chung-May Wu Hungkuang Institute of Technology Chai-Nan University of Pharmacy and Science National Taiwan Ocean University Keelung, Taiwan
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Contents Chapter 1
Food Components and Quality ............................................................ 1
Zdzisław E. Sikorski and Barbara Piotrowska Chapter 2
Chemical Composition and Structure of Foods ................................ 15
Krystyna Palka Chapter 3
Water and Food Quality..................................................................... 29
Emilia Barbara Cybulska and Peter Edward Doe Chapter 4
Mineral Components.......................................................................... 61
Michał Nabrzyski Chapter 5
Saccharides......................................................................................... 93
Piotr Tomasik Chapter 6
The Role of Proteins in Food .......................................................... 129
Zdzisław E. Sikorski Chapter 7
Lipids and Food Quality .................................................................. 177
Andrzej Stołyhwo Chapter 8
Rheological Properties of Food Systems ........................................ 209
Anna Pruska-Kędzior and Zenon Kędzior Chapter 9
Food Colorants................................................................................. 245
Jadwiga Wilska-Jeszka Chapter 10 Food Allergens ................................................................................. 275 Barbara Wróblewska
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Chapter 11 Flavor Compounds in Foods............................................................ 295 Bonnie Sun-Pan, Jen-Min Kuo, and Chung-May Wu Chapter 12 Interactions of Food Components ................................................... 329 Zdzisław E. Sikorski and Norman F. Haard Chapter 13 Main Food Additives........................................................................ 357 Adriaan Ruiter and Alphons G.J. Voragen Chapter 14 Food Safety ...................................................................................... 375 Julie Miller Jones Chapter 15 Prebiotics.......................................................................................... 391 Bob Rastall Chapter 16 Probiotics in Food............................................................................ 413 Maria Bielecka Chapter 17 Mood Food....................................................................................... 427 Maria H. Borawska Chapter 18 Food Components in the Protection of the Cardiovascular System..................................................................... 439 Piotr Siondalski and Wiesława Łysiak-Szydłowska Chapter 19 Mutagenic, Carcinogenic, and Chemopreventive Compounds in Foods............................................................................................ 451 Agnieszka Bartoszek Chapter 20 The Role of Food Components in Children’s Nutrition ................. 487 Grażyna Sikorska-Wiśniewska and Małgorzata Szumera Index...................................................................................................................... 517
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1
Food Components and Quality Zdzisław E. Sikorski and Barbara Piotrowska
CONTENTS 1.1
Food Components ............................................................................................ 1 1.1.1 Components in Food Raw Materials and Products............................. 1 1.1.2 Factors Affecting Food Composition................................................... 3 1.1.3 The Role of Food Components ........................................................... 4 1.2 Functional Properties of Food Components.................................................... 5 1.3 The Role of Postharvest Changes, Handling, and Processing in the Quality of Foods.................................................................................... 6 1.3.1 Introduction .......................................................................................... 6 1.3.2 Attributes of Quality ............................................................................ 6 1.3.3 Safety and Nutritional Value................................................................ 7 1.3.4 Sensory Quality.................................................................................... 8 1.4 Chemical Analysis in Ensuring Food Quality................................................. 9 1.4.1 Introduction .......................................................................................... 9 1.4.2 Requirements of the Producer ............................................................. 9 1.4.3 Requirements of the Consumer ......................................................... 12 1.4.4 Limits of Determination .................................................................... 12 References................................................................................................................ 13
1.1 FOOD COMPONENTS 1.1.1 COMPONENTS
IN
FOOD RAW MATERIALS
AND
PRODUCTS
Foods are derived from plants, carcasses of animals, and single-cell organisms. Their main components include water, saccharides, proteins, lipids, and minerals (Table 1.1), as well as a host of other compounds present in minor quantities, albeit of significant impact on the quality of many products. The nonprotein nitrogenous compounds, vitamins, colorants, flavor compounds, and functional additives belong here. The content of water in various foods ranges from a few percent in dried commodities (e.g., milk powder), about 15% in grains, 16 to 18% in butter, 20% in honey, 35% in bread, 65% in manioc (cassava), and 75% in meat, to about 90% in many fruits and 1
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Chemical and Functional Properties of Food Components
TABLE 1.1 Typical Products as Rich Sources of the Main Food Components Water
Saccharides
Proteins
Lipids
Minerals
Vitamins
Juices Fruits Milk Vegetables Jellies Lean fish Lean meat
Saccharose Honey Cereals Chocolate Potato Cassava Fruits
Soybean Beans Meat Fish Wheat Cheese Eggs
Oils Lard Butter Chocolate Nuts Egg yolk Pork
Vegetables Fruits Meat Fish products Dairy products Cereals Nuts
Vegetables Fruits Fish liver Meat Cereals Milk Yeast
vegetables. Saccharides are present in food raw materials in quantities ranging from about 1% in meats and fish, 4.5% in milk, 18% in potatoes, and 15 to 21% in sugar beets, to about 70% in cereal grains. The protein content in foods is present mainly as crude protein (i.e., as N × 6.25). The nitrogen-to-protein conversion factor (N:P) of 6.25 has been recommended for most plant and animal food products under the assumption that the N content in their proteins is 16%, and they do not contain nonprotein N. The N content in the proteins in various foods, however, is different because it depends on the amino acid composition. Furthermore, the total N consists of protein N and of N contained in numerous nonprotein compounds, such as free peptides and amino acids, nucleic acids and their degradation products, amines, betains, urea, vitamins, and alkaloids. In some foods the nonprotein N may constitute up to 30% of total N. In many of these compounds the C:N ratio is similar to the average in amino acids. However, the N content in urea, at 47%, is exceptionally high. The average conversion factor for the estimation of true protein, based on the ratios of total amino acid residues to amino acid N determined for 23 various food products, is 5.68 and for different classes of foods is between 5.14 and 6.61 (Table 1.2). The N:P factor of 4.39, based on analysis of 20 different vegetables, has been proposed by Fujihara et al. (2001) for estimating the true protein content in vegetables. A common N:P factor of 5.70 for use with respect to blended foods or diets has been recommended by Sosulski and Imafidon (1990).
TABLE 1.2 The Nitrogen to Protein Conversion Factors in Foods Product
Factor
Product
Factor
Dairy products Egg Meat and fish Cereals and legumes
6.02–6.15 5.73 5.72–5.82 5.40–5.93
Potato Leafy vegetables Fruits Microbial biomass
5.18 5.14–5.30 5.18 5.78–6.61
Source: Data from Sosulski, F.W. and Imafidon, G.I. 1990. J. Agric. Food Chem. 38, 1351, 1990).
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Crude protein makes up from about 1% of the weight of fruits and 2% of potatoes, 3.2% of bovine milk, 12% of eggs, 12 to 22% of wheat grain, about 20% of meat, to 25 to 40% of various beans. During their development, cereal grain and legume seeds deposit large quantities of storage proteins in granules also known as protein bodies. In soybeans these proteins constitute 60 to 70% of the total protein content, and the granules are 80% proteins. The lipid content in foods is given in nutrition information labeling predominantly as total fat, which is often called crude fat. This is a mixture of various classes of lipids, mainly different triacylglycerols. The lipids of numerous food fishes, such as orange roughy, mullet, codfish, and shark, as well as some crustaceans and mollusks also include wax esters. Some shark oils are very rich in hydrocarbons, particularly in squalene. Furthermore, the lipid fraction of food raw materials harbors different sterols, vitamins, and pigments that are crucial for metabolism. Thus the composition of the extracted crude fat depends on the kind of food and the polarity of the solvent used for extraction. Lipids constitute from less than 1% of the weight of fruits, vegetables, and lean fish muscle, 3.5% of milk, 6% of beef meat, and 32% of egg yolk, to 85% of butter.
1.1.2 FACTORS AFFECTING FOOD COMPOSITION The content of different components in food raw materials depends on the species and variety of the animal or plant crop; on the conditions of cultivation and time of harvesting of the plants; on the feeding, conditions of life, and age of the farm animals or the fishing season for fish and marine invertebrates; and on postharvest changes that take place in the crop during storage. The food industry, by establishing quality requirements for raw materials, can encourage producers to control, within limits, the contents of the main components in their crops; for example, starch in potatoes, fat in various meat cuts, pigments in fruits and vegetables and in the flesh of fish from aquaculture, or protein in wheat and barley, as well as the fatty acid composition of lipids in oilseeds and meats. The contents of desirable minor components can also be effectively controlled; for example, the amount of natural antioxidants to retard the oxidation of pigments and lipids in beef. Contamination of the raw material with organic and inorganic pollutants can be controlled by observing recommended agricultural procedures in using fertilizers, herbicides, and insecticides, and by seasonally restricting certain fishing areas to avoid marine toxins. The size of predatory fish like swordfish, tuna, or shark, which are fished commercially, can be limited to reduce the risk of excessive mercury and arsenic in the flesh. The composition of processed foods depends on the recipe applied and on changes taking place due to processing and storage. These changes are mainly brought about by endogenous and microbial enzymes, active forms of oxygen, heating, chemical treatment, and processing at low or high pH (Haard 2001). Examples of such changes are listed below: • •
Leaching of soluble, desirable and undesirable components, such as vitamins, minerals, and toxins during washing, blanching, and cooking Dripping after thawing or due to cooking
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Chemical and Functional Properties of Food Components
• • •
•
• •
Loss of moisture and volatiles due to evaporation and sublimation Absorption of desirable or harmful compounds during salting, pickling, seasoning, frying, or smoking Formation of desirable or harmful compounds due to enzyme activity, such as the development of typical flavor in cheese or decarboxylation of amino acids in fish marinades Generation of desirable or objectionable products due to interactions of reactive groups induced by heating or chemical treatment, such as flavors or carcinogenic compounds in roasted meats, or trans-fatty acids in hydrogenated fats Formation of different products of oxidation of food components, mainly of lipids, pigments, and vitamins Loss of nutrients and deterioration of dried fish due to attacks by flies, mites, and beetles
1.1.3 THE ROLE
OF
FOOD COMPONENTS
The indigenous water that is immobilized in the plant and animal tissues by the structural elements and various solutes contributes to buttressing the conformation of the polymers, serves as a solvent for different constituents, and interacts in metabolic processes. Polysaccharides, proteins, and lipids serve as the building material of different structures of the plant and animal tissues used for food. The structures made of these materials are responsible for the form and tensile strength of the tissues, and create the necessary conditions for metabolic processes to occur. Compartmentalization resulting from these structures plays a crucial biological role in the organisms. Some of the main components, as well as other constituents, are bound to different cell structures or are distributed in soluble form in the tissue fluids. Many saccharides, proteins, and lipids are stored for reserve purposes. Polysaccharides are present in plants as starch in the form of granules and in muscles as glycogen. Other saccharides are dissolved in tissue fluids or perform different biological functions, such as in free nucleotides or as components of nucleic acids, or in being bound to proteins and lipids. Proteins also play crucial metabolic roles in plants and animals as enzymes and enzyme inhibitors, participate in the transport and binding of oxygen and metal ions, and perform immunological functions. The distribution of lipids in food raw materials depends on their role in the living animal and plant organisms. In an animal body, lipids occur primarily as an energyrich store of neutral fat in the subcutaneous adipose tissue; as kidney, leaf, and crotch fat; as the intramuscular fat known as marbling; and as intermuscular or seam fat. In fatty animals, the largest portion of lipids is stored as depot fat in the form of triacylglycerols. In lean fish species, most of the fat occurs in the liver. The lipids contained in the food raw materials in low quantities serve mainly as components of protein-phospholipid membranes and have metabolic functions. The main food components supply the human body with the necessary building material and source of energy, as well as elements and compounds indispensable for metabolism. Some plant polysaccharides are only partly utilized for energy.
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5
However, as dietary fiber they affect, in different ways, various processes in the gastrointestinal tract. Many of the minor components originally present in the raw materials are nutritionally essential, such as vitamins. Some of them, although not indispensable, can be utilized by the body, including most free amino acids, or impart desirable sensory properties to food products. Numerous groups, including tocopherols, ubiquinone, carotenoids, ascorbic acid, thiols, amines, and several other nonprotein nitrogenous compounds serve as endogenous muscle antioxidants, playing an essential role in postmortem changes in meat (Decker et al. 2000). Other minor components are useless or even harmful if present in excessive amounts. Most food raw materials are infected with different microorganisms, putrefactive and often pathogenic, and some contain parasites and the products of microbial metabolism. A variety of compounds are added intentionally during processing, to be used as preservatives, antioxidants, colorants, flavorings, sweeteners, and emulsifying agents, or to fulfill other technological purposes.
1.2 FUNCTIONAL PROPERTIES OF FOOD COMPONENTS The term functional properties has a broad range of meanings. The term technological properties implies that the given component present in optimum concentration, subjected to processing at optimum parameters, contributes to the expected desirable sensory characteristics of the product, usually by interacting with other food constituents. Hydrophobicity, hydrogen bonds, ionic forces, and covalent bonding are involved in these interactions. Thus, the functional properties of food components are affected by the number of accessible reactive groups and by the exposure of hydrophobic areas in the given material. Therefore, in a system of given water activity and pH, and in the given range of temperature, the functional properties can be to a large extent predicted from the structure of the respective saccharides, proteins, and lipids. They can also be improved by appropriate, intentional enzymatic or chemical modifications of the molecules, mainly those that affect the size, charge density, or the hydrophilic/hydrophobic character of the compounds, or by changes in the environment of both the solvent and other solutes. The functional properties of food components make it possible to manufacture products of desirable quality. Thus, pectins contribute to the characteristic texture of ripe apples and make perfect jellies. Other polysaccharides are efficient thickening and gelling agents at different ranges of acidity and concentration of various ions. Alginates in the presence of Ca2+ form protective, unfrozen gels on the surface of frozen products. Some starches are resistant to retrogradation, thereby retarding staling of bread. Fructose retards moisture loss from biscuits. Mono- and diacylglycerols, phospholipids, and proteins are used for emulsifying lipids and stabilizing food emulsions and foams. Antifreeze proteins inhibit ice formation in various products, and gluten plays a major role in producing the characteristic texture of wheat bread. Technologically required functional effects can also be achieved by intentionally employing various food additives, such as food colors, sweeteners, and a host of
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Chemical and Functional Properties of Food Components
other compounds. These additives are per se not regarded as foodstuffs, but are used to modify the rheological properties or acidity, increase the color stability or shelf life, and act as humectants or flavor enhancers. During the two most recent decades, the term functional has been predominantly given to a large group of products and components, also termed designer foods, pharmafoods, nutraceuticals, or foods for specific health use, which are regarded as health enhancing or potentiating the performance of the human organism. These foods, mainly drinks, meals, confectionery, ice cream, and salad dressings contain various ingredients, including oligosaccharides, sugar alcohols, or choline, which are claimed to have special physiological functions like neutralizing harmful compounds in the body and promoting recovery and general good health (Goldberg 1994). Foods containing prebiotics (various oligosaccharides) and probiotics (mainly dairy products) are treated in detail in Chapters 15 and 16 of this volume, respectively.
1.3 THE ROLE OF POSTHARVEST CHANGES, HANDLING, AND PROCESSING IN THE QUALITY OF FOODS 1.3.1 INTRODUCTION The chemical nature of food components is of crucial importance for all aspects of food quality. It determines the nutritional value of the product, its sensory attractiveness, development of desirable or deteriorative changes due to interactions with other constituents and to processing, and susceptibility or resistance to spoilage during storage. Food components, which contain reactive groups, many of them essential for the quality of the products, are generally labile and easily undergo different enzymatic and chemical changes, especially when treated at elevated temperature or in conditions promoting the generation of active species of oxygen.
1.3.2 ATTRIBUTES
OF
QUALITY
The quality of a food product—the characteristic properties that determine its degree of excellence—is a sum of the attributes contributing to the consumer’s satisfaction with the product. The composition and the chemical nature of the food components affect all aspects of food quality. The total quality reflects at least the following attributes: •
•
•
Compatibility with the local or international food law regulations and standards regarding mainly the proportions of main components, presence of compounds serving as identity indicators, contents of contaminants and additives, hygienic requirements, packaging, and labeling Nutritional aspects, such as the contents and availability of nutritionally desirable constituents, mainly proteins, essential amino acids, essential fatty acids, saccharides, vitamins, fiber, and mineral components Safety aspects affected by the concentration of compounds that may constitute health hazards for consumers and affect the digestibility and
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Food Components and Quality
•
• •
•
7
nutritional availability of the food, such as heavy metals, toxins of various origins, some enzymes and enzyme inhibitors, factors decreasing the availability of some metal components, pathogenic microorganisms, and parasites Sensory attributes, such as the color, size, form, flavor, taste, and rheological properties, obviously affected by the chemical composition of the product, and by changes resulting from processing and culinary preparation Shelf life under specific storage conditions Convenience aspects related to the size and ease of opening and reclosing the container, the suitability of the product for immediate use or for different types of thermal treatment, ease of portioning or spreading, and transport and storage requirements Ecological aspects regarding suitability for recycling of the packaging material and pollution hazards
For many foods, one of the most important quality criteria is freshness. This is especially so in the case of numerous species of vegetables, fruits, and seafood. Fish of valuable species in a state of prime freshness, suitable to be eaten raw, may sell at a market price ten times higher than the same fish after several days storage in ice, even though the stored fish is still very fit for human consumption.
1.3.3 SAFETY
AND
NUTRITIONAL VALUE
Food is regarded as safe if it does not contain harmful organisms or compounds in concentrations above the accepted limits (see Chapter 14). The nutritional value of foods depends primarily on the levels of nutrients and nutritionally objectionable components in the products. Processing may increase the safety and biological value of food by inducing chemical changes increasing the digestibility of the components or by inactivating undesirable compounds, such as toxins or enzymes catalyzing the generation of toxic agents from harmless precursors. Freezing and short-term frozen storage of fish inactivates the parasite Anisakis, which could escape detection during visual inspection of herring fillets used as raw material for cold marinades produced under mild conditions. Thermal treatment brings about inactivation of myrosinase, the enzyme involved in hydrolysis of glucosinolanes. This arrests the reactions that lead to the formation of goitrogenic products in oilseeds of Cruciferae. Heat pasteurization and sterilization reduce to an acceptable level the number of vegetative forms and spores, respectively, of pathogenic microorganisms. Several other examples of such improvements in the safety and biological quality of foods are covered in the following chapters of this book. There are also, however, nutritionally undesirable side effects of processing, such as destruction of essential food components as a result of heating, chemical treatment, and oxidation. As is generally known, the partial thermal decomposition of vitamins, especially thiamine, loss of available lysine- and sulfur-containing amino acids, or generation of harmful compounds such as carcinogenic heterocyclic aromatic amines, lysinoalanine and lanthionine, or position isomers of fatty acids, not present
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Chemical and Functional Properties of Food Components
originally in foods. In recent years new evidence of side effects has been accumulated with respect to chemical processing of oils and fats. Commercial hydrogenation of oils brings about not only the intended saturation of selected double bonds in the fatty acids and thereby the required change in the rheological properties of the oil, but also results in the generation of a large number of trans-trans and cis-trans isomers that are absent in the unprocessed oils.
1.3.4 SENSORY QUALITY Many of the desirable sensory attributes of foods stem from the properties of the raw material. The natural color of meat, fish muscles and skin, vegetables, and fruits depends on the presence of a host of different pigments, which are water or lipid soluble. Chlorophylls impart the green color to vegetables, but also to olive oil. Some natural oils are yellow or red due to different carotenoids. Carotenes are also present in the flesh oil of redfish (Sebastes marinus), while different carotenoproteins are responsible for the vivid colors of fish skin. Many hydroxycarotenoids (xanthophylls) occur in plants in the form of esters of long-chain fatty acids. The red, violet, or blue color of fruits and flowers is caused by anthocyanins. Betalains impart color to red beets. The flavor, taste, and texture of fresh fruits and vegetables, as well as the taste of nuts and milk, depend on the presence of natural compounds. These properties are in many cases carried through to the final products. In other commodities, the characteristic sensory attributes are generated as a result of processing. The texture of bread develops due to interactions of proteins, lipids, and saccharides among themselves and with various gases, while that of cooked meats appears as the results of thermal protein denaturation. The bouquet of wine is due to the presence of volatile components in the grape as well as the result of fermentation of saccharides and a number of other biochemical and chemical reactions. The delicious color, flavor, texture, and taste of smoked salmon are generated in enzymatic changes in the tissues and the effect of salt and smoke. The flavor of various processed meats develops due to thermal degradation of predominantly nitrogenous compounds, the generation of volatile products of the Maillard reaction, interactions of lipid oxidation products, and the effect of added spices. Optimum foam performance of beer depends on the interactions of peptides, lipids, the surface-active components of hops, and gases. The flavor, texture, and taste of cheese result from fermentation and ripening, while the appealing color and flavor of different fried products are due to reactions of saccharides and amino acids. The sensory attributes of foods are related to the contents of many chemically labile components. These components, however, just as most nutritionally essential compounds, are prone to deteriorative changes in conditions of severe heat treatment, oxidizing conditions, or application of considerably high doses of chemical agents, such as acetic acid or salt, which are often required to ensure safety and sufficiently long shelf life of the products. Thus, loss in sensory quality takes place, for example, in oversterilized meat products due to the degradation of sulfur-containing amino acids and development of an off-flavor; toughening of the texture of overpasteurized ham or shellfish due to excessive shrinkage of the tissues and drip; deterioration of the texture and arresting of ripening in herring preserved at too high a concentration of salt.
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Optimum parameters of storage and processing ensure the retention of the desirable properties of the raw material and lead to the development of intended attributes of the product. In the selection of these parameters, the chemistry of food components and of the effect of processing must be studied. The eager food technology student can find all the necessary information in at least two excellent textbooks on food chemistry by Belitz et al. (2001) and Fennema (1996), in numerous books on food lipids, proteins, and saccharides, as well as in current international journals.
1.4 CHEMICAL ANALYSIS IN ENSURING FOOD QUALITY 1.4.1 INTRODUCTION The aspects of food quality described in Section 1.3.2 can be assured only by applying appropriate control in the manufacturing process and storage, based on sensory, physical, chemical, biochemical, and microbial techniques. For the purposes of analysis, appropriate techniques and hardware are used, from the most simple procedures and gadgets, to the very sophisticated analytical instruments known in analytical chemistry. A rational system of control is necessary for the producers of the raw material, the food processor, the retailer, and even for consumer organizations. The results of chemical and microbiological analyses are indispensable for selecting the most suitable parameters of processing and for their implementation, for designing and operating the hazard analysis and critical control points system of quality assurance in processing plants, and for securing safety of the food products available in the market.
1.4.2 REQUIREMENTS
OF THE
PRODUCER
Thanks to the possibility of rapid and reliable determination of food composition and contaminants by applying contemporary techniques, the raw materials can be optimally used for manufacturing various products; loss in quality and health hazards can be avoided. In the relationship between the primary producer and the food processor, the requirements regarding the contents and characteristics of the most important components, as well as freshness grades of the raw materials are agreed upon, often in the form of contracts. Depending on the commodity, it may be saccharose in sugar beets; fat in milk or in mackerel as raw material for hot smoking; the color of vegetables and egg yolks, depending on the concentration of carotenoid pigments; the proportion of lean tissue and marbling in pig or beef carcasses; connective tissue in meats used for least-cost formulations of sausages; the contents and characteristics of gluten in wheat grains; starch and protein in barley used for malting; extract in tomatoes; oil in oil-bearing raw materials; free fatty acids and peroxide value in fat-containing commodities; trimethylamine, hypoxanthine, or other freshness indicators in marine fish; or the elasticity of kamaboko, the Japanese-type fish cake. These components and characteristics are usually determined using standard, simple
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chemical or physicochemical analyses or enzymatic sensors. For example, the texture of kamaboko is commonly determined by folding a 5-mm-thick slice of the product and observing the formed edge. The highest quality kamaboko can be folded twice without any cracking; the lowest-quality product falls apart after the first folding. Although this test is very simple, it may decide the price of a large consignment of surimi or kamaboko (Suzuki 1981). Nowadays many companies supply the hardware, reagents, and analytical procedures for numerous applications in the food plant and for water field analysis (Table 1.3). For example, to assist in the routine analyses in dairy production, many tests based on photometric or reflectometric techniques are offered, such as reflectometric detection of alkaline phosphatase for controlling milk pasteurization, photometric control of lactose fermentation and determination of urea, or photometric assay of ammonia in milk. Thanks to enormous progress in analytical methodology and instrumentation, the food chemist can use automated equipment for assaying water, proteins, lipids, saccharides, fiber, and mineral components. Online analyses provide for continuous control of processing parameters.
TABLE 1.3 Examples of Some Tests Offered for Rapid Food Analysis Measured parameter
Example of food
Technique
Ascorbic acid
Apple purée, apple sauce, banana purée, candies, fruit and vegetable juices Beer
Reflectometric determination after reaction with molybdophosphoric acid to phosphomolybdenum blue Photometric determination with cresolphthalexone Reflectometric determination after reaction with phthalein purple Photometric determination with glyoxal-bis(2hydroxyanil) or reflectometric determination after reaction with phthalein purple Photometric determination with cresolphthalexone Photometric determination as Fe(III)thiocyanate in hot water extract with the Fe(III)-Hg(II)-thiocyanate method Photometric determination with diphenylcarbazide after decomposition with H2SO4 and perhydrol Photometric determination with cuprizon subsequent to decomposition with H2SO4 and perhydrol Reflectometric determination after reaction with 4-amino-3-hydrazino-5-mercapto1,2,4-triazole Reflectometric determination after reaction with glucose oxidase and peroxidase
Calcium
Cheese Milk
Wine Chloride
Meat and sausage products, pickled cabbage
Chromium
Dairy products
Copper
Various foods
Formaldehyde
Fish products
Glucose
Jam, juices
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The characteristic freshness attributes of different foods are usually evaluated by sensory methods and by determination of specific indices, predominantly by biochemical sensors. A typical example may be the examination of fish freshness by a taste panel, and by chemical tests or biochemical sensors suitable for assaying the volatile odorous compounds and products of nucleotide catabolism (Figures 1.1 and 1.2). The results of these kinds of analyses serve as the basis for technological decisions regarding the suitability of the raw materials for further storage or the given treatment, as well as for adjusting the processing parameters; they also often decide the price of the commodity. Adenosine triphosphate (ATP) ↓ Adenosine diphosphate (ADP) ↓ Adenosine monophosphate (AMP) ↓ ↓ ↓ ↓ Adenosine(Ado) inosine monophosphate (IMP) ↓ ↓ Inosine (Ino) ↓ Hypoxanthine (Hx) ↓ Xanthine ↓ Uric acid K = 100(Ino + Hx)/ATP + ADP + AMP + IMP + Ino + Hx
FIGURE 1.1 Degradation of ATP in fish muscle and the K-value as a freshness indicator.
5
100
3
1
40
20
2
0
2
4
6
8
10
12
14
Sensory score
60
4 Vo latile base N, TM A N
K Value, %
80
16
Time of storage in ice [days]
FIGURE 1.2 Typical sensory and chemical tests of fish freshness: (1) flavor of cooked fish after cooking, (2) K-value, (3) volatile base nitrogen, (4) trimethylamine nitrogen, (5) flavor of raw fish.
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The producer needs chemical analysis to ascertain that the raw material used in his plant does not contain any harmful components or contaminants in quantities higher than those accepted by national or international regulations; for example, nitrates (V) and (III) in vegetables, pesticide residues in various crops, heavy metals in many plant and animal tissues including Hg in large predatory fish, histamine in fish meat, or mycotoxins in peanuts.
1.4.3 REQUIREMENTS
OF THE
CONSUMER
The results of routine analyses performed by the producer and by food inspection laboratories must ascertain that most consumer expectations regarding nutritious and wholesome food of high sensory quality be fulfilled. The consumer generally requires that foods offered on the market contain the components typical for the type of product and that their proportions are those represented on the label. This concerns, for example, the contents of protein and fat in meat products, milk fat in butter, vitamin C in fruit juices, the unique fatty-acid composition of the product sold as extra virgin olive oil or as n-3 polyenoic fatty acids–rich preparation, absence of pork in products declared as being made of other meats, or meat of fish species other than that specified in comminuted commodities. Food adulteration is an age-old vice and chemical analysis helps to combat it. The nutrition-cautious person looks on the label for information regarding essential amino acids, polyunsaturated fatty acids, vitamins, mineral components, fiber, and recently functional additives or genetically modified products. Many consumers carefully study the labels on packaged foods because their health or even life may depend on the information regarding the presence of different ingredients rich in allergens in the produce, such as gluten or peanuts. However, small amounts of such compounds may originate from residues in processing machinery or stem from additives used by the processor. The safety of food products is safeguarded by determining, for example, heavy metals and their speciation (see Chapter 4), polycyclic aromatic hydrocarbons (PAHs) in oils, heavily smoked fish and meat products, acrylamide in French fries, mycotoxins in a variety of commodities, and various additives. For determination of the very large number of hazardous components, additives, and impurities, many specialized chromatographic, spectroscopic, and physical techniques, as well as enzyme, microbial, and immunological sensors are used (Tunick 2005).
1.4.4 LIMITS
OF
DETERMINATION
By applying efficient procedures of enrichment and separation of analytes combined with the use of highly selective and sensitive detectors, it is now possible to determine different additives and contaminants, as well as the products of various chemical and biochemical reactions in foods in extremely low concentrations. This is often necessary because the national and international bodies responsible for the safety of foods require that the producers conform to regulations allowing very low amounts of various natural toxic compounds and contaminants in their produce. The contents of nitrates in potatoes and other vegetables for children less than three years of age in Poland should not exceed 250 mg NO3/kg. The tolerance for various pesticide
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residues ranges in different foods from about 0.01 to 20 mg/kg (Lehotay and Mastovska 2005). According to German regulations introduced in 1973, the content of benzo[a]pyrene (BaP), the recognized representative of the carcinogenic PAHs, should be no higher than 1 µg/kg in smoked meat products; for meats treated with smoke preparations, the upper limit of 0.03 µg/kg has been set by the European Union. In Europe the countries producing olive residual oil have established a maximum level of 2 µg/kg for each of the eight highly carcinogenic PAHs, but not above 5 µg/kg for the total amount of all eight compounds. In smoked meat and fishery products, in baby foods, and in food oils, according to the new regulation of EC, No. 208/2005, the maximum permissible level of BaP is 5 µg/kg, 1 µg/kg, and 2 µg/kg wet weight, respectively. In selecting the most appropriate analytical procedure for detection or determination of a compound in a food sample, the properties of the matrix must be considered. This is especially important in the step of separation of the analyte from the food material, be it by digestion, membrane techniques, solvent extraction, supercritical fluid extraction, sorption, headspace technique, or steam distillation. An interesting treatment of suitability of various procedures for extraction of lipids from food samples is presented in Chapter 7. In gas chromatography/mass spectrometry (GC/MS) determination of acrylamide, the methanol extraction of the analyte from the food sample during several days in a Soxhlet apparatus yields about 7 times higher results than homogenizing with the solvent followed by centrifuging. The detection limit of acrylamide in foods is actually about 10 µg/kg wet weight (Food and Agriculture Organization/World Health Organization [FAO/WHO] 2002). By using procedures comprising extraction of hydrocarbons from the food matrix, cleanup, separation by gas chromatography (GC) or high-performance liquid chromatography (HPLC), followed by detection and quantification by mass spectrometry or in fluorescence detectors, it is possible to determine the individual carcinogenic PAHs at concentrations on the order of 0.1 or even 0.01 µg/kg wet weight (Stołyhwo and Sikorski 2005). The accuracy of the results depends significantly on the quality of standards used for calibration. Certified reference materials are now available containing up to 15 PAHs in food samples. For quantitative analysis internal GC/MS calibration with stable isotopes added prior to extraction and an MS detector in selected ion mode may be used (van Rooijen 2005). In studies and routine monitoring regarding nutritional requirements and food safety aspects, many toxic elements are determined in trace concentrations of 0.01 to 10 mg/kg or even in ultratrace amounts of less than 10 µg/kg by using mainly spectrometric techniques (Capar and Szefer 2005). The lowest dose-inducing symptoms of allergy in highly sensitive persons is about 0.1 mg of peanut or egg protein. This means that the applied chemical examination must guarantee the detection of a few micrograms of peanut material in 1 gram of food (Williams et al. 2005).
REFERENCES Belitz, H.D., Grosch, W., and Schieberle, P. 2001. Lehrbuch der Lebensmittelchemie. 4th ed., Springer Verlag, Berlin.
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Capar, S.G. and Szefer, P. 2005. Determination and speciation of trace elements in foods, in Methods of Analysis of Food Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton, FL, chap. 6. Decker, E.A., Livisay, S.A., and Zhou S. 2000. Mechanism of endogenous skeletal muscle antioxidants: chemical and physical aspects, in Antioxidants in Muscle Foods. Nutritional Strategies to Improve Quality, Decker, E., Faustman, C., and Lopez-Bote, C.J., Eds., Wiley-Interscience, New York, chap. 25. FAO/WHO (Food and Agricultural Organization/World Health Organization). 2002. Health Implications of Acrylamide in Food, Report of a Joint FAO/WHO Consultation, Geneva, 25–27 June. Fennema, O.R., Ed. 1996. Food Chemistry, 3rd ed., Marcel Dekker, New York. Fujihara, S., Kasuga, A., and Aoyagi, Y. 2001. Nitrogen-to-protein conversion factors for common vegetables in Japan, J. Food Sci. 66, 412. Goldberg, I. 1994. Functional Foods: Designer Foods, Pharmafoods, Nutraceuticals, Chapman and Hall, New York. Haard, N.F. 2001. Enzymic modification in food systems, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Lancaster, PA, chap. 7. Lehotay, S.J. and Mastovska, K. 2005. Determination of pesticide residues, in Methods of Analysis of Food Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton, FL, chap. 12. Sosulski, F.W. and Imafidon, G.I. 1990. Amino acid composition and nitrogen-to-protein conversion factors for animal and plant foods, J. Agric. Food Chem. 38, 1351. Stołyhwo, A. and Sikorski, Z.E. 2005. Polycyclic aromatic hydrocarbons in smoked fish—a critical review, Food Chem., 91, 3.3. Suzuki, T., 1981. Fish and Krill Proteins: Processing Technology, Applied Science Publishers Ltd., London. Tunick, M.H. 2005. Selection of techniques used in food analysis, in Methods of Analysis of Food Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton, FL, chap. 1. van Rooijen, J.J.M. 2005. The control of polycyclic aromatic hydrocarbons in food ingredients, International Review of Food Science and Technology, Winter 2005/2006, 116. Williams, K.M. et al. 2005. Determination of food allergens and genetically modified components, in Methods of Analysis of Food Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton, FL, chap. 11.
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Chemical Composition and Structure of Foods Krystyna Palka
CONTENTS 2.1 2.2
Introduction .................................................................................................... 15 Protein Food Products.................................................................................... 16 2.2.1 Meat.................................................................................................... 16 2.2.2 Milk and Milk Products..................................................................... 19 2.2.3 Eggs.................................................................................................... 20 2.3 Saccharide Food Products.............................................................................. 21 2.3.1 Cereal and Cereal Products ............................................................... 21 2.3.2 Potatoes .............................................................................................. 23 2.3.3 Honey ................................................................................................. 25 2.3.4 Nuts .................................................................................................... 25 2.3.5 Seeds of Pulses .................................................................................. 25 2.4 Edible Fats...................................................................................................... 25 2.5 Fruits and Vegetables ..................................................................................... 26 References................................................................................................................ 28
2.1 INTRODUCTION Foods are edible fragments of plant or animal organisms in a natural or processed state, which after being eaten and digested in the human organism, may be a source of different nutrients. Taking as a base the dominant nutritional component, food products may be divided into four groups: 1. 2. 3. 4.
Protein food products Saccharide food products Edible fats Fruits and vegetables
The particular groups of chemical constituents participate in building the structure of food products as components of specialized tissues. For this reason this chapter presents, in addition to chemical composition, the morphology of the selected products from each group. 15
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2.2 PROTEIN FOOD PRODUCTS 2.2.1 MEAT Meat is the edible part of animal, chicken, or fish carcasses. Its chemical composition is as follows: 60 to 85% water, 8 to 23% protein, 2 to 15% lipids, 0.5 to 1.5% saccharides, and about 1% inorganic substances (Table 2.1). These quantities change significantly depending on type, age, sex, level of fattening, and part of animal carcass. The largest fluctuations are observed in the contents of water and lipids. Water is a solvent of organic and inorganic substances and an environment of biochemical reactions. It also participates in the maintenance of meat protein conformation.
TABLE 2.1 Chemical Composition of Foods Rich in Proteins
Muscle food
Beef, lean Pork, lean Veal Lamb Chicken Light meat Dark meat Herring Oyster
Milk and milk products
Cow milk Sheep milk Sour cream 25% Yogurt, low fat Quarg Ripened cheese Milk powder
Eggs and egg product
Product
Whole egg without shell White Yolk Whole egg powder
Water %
Crude protein N×6.25 %
Lipids %
Saccharides %
Mineral components %
71.5 72.0 75.0 71.5
21.0 20.0 20.0 19.5
6.5 7.0 3.5 7.0
1.0 1.0 1.0 1.5
1.0 1.0 1.0 1.0
75.0 76.0 60.0 85.0
23.0 20.0 18.0 7.5
2.0 4.5 15.5 1.5
1.0 1.0 0.5–1.5 0.5–1.5
88.0 82.0 68.0 85.0 64.0–75.0 35.0–50.0 3.0
3.0 6.0 3.0 5.0 9.0–14.0 20.0–35.0 26.0
3.5 6.5 25.0 1.0 12.0–18.0 20.0–30.0 26.0
4.5 4.5 4.0 7.5 2.5 2.0 38.0
1.0 1.0 0.5 0.7 1.5 5.0 6.0
73.5 88.0 48.5 3.5
13.0 11.0 16.0 47.5
12.0 traces 32.0 43.0
1.0 0.5 1.0 to 0.5
1.0 0.5 1.0 4.0
Source: Adapted from Hedrick, H.B. et al., Principles in Meat Science, Kendal-Hunt Publ. Comp., Dubuque, 1994; Kirk, R.S. and Sawyer, R., Pearson’s Composition and Analysis of Foods, Longman Science, London, 1991; Renner, E., Cheese: Chemistry, Physics and Microbiology, Vol. 1, Fox, P.F., Ed., Chapman & Hall, London, 1993; Sikorski, Z.E., Seafood Raw Materials, WNT, Warsaw, 1992; Tamime, A.Y. and Robinson, R.K., Yoghurt. Science and Technology, CRC Press. Boca Raton, FL, 1999.
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Meat proteins include sarcoplasmic, myofibrillar, and connective tissue proteins. Among the sarcoplasmic proteins are heme pigments and enzymes, which influence the color, smell, and structure of meat. Myofibrillar proteins and collagen are able to retain and hold water in meat structure and to emulsify fat. Therefore, they influence the rheological properties of meat products. Mineral elements are in enzymatic complexes and other structures that play an important biochemical role. They can affect the technological properties of meat, for example, water-holding capacity, as well as the sensory characteristics. Meat is also a good source of the B group of vitamins. The main structural unit of striated muscle tissue is a multinucleus cell called muscle fiber. Its length varies from several millimeters to hundreds of millimeters, and the diameter is between 10 and 100 µm (Figure 2.1a). The thickness of muscle fibers affects the meat’s tenderness. The muscle fiber contains typical somatic cell compounds, sarcoplasmic reticulum, and myofibrils. The sarcoplasmic reticulum has the capacity of reversible binding of calcium ions. Myofibrils are the main structural element of muscle fiber, making up 80% of its volume. They have a diameter of 1 to 2 µm and are situated parallel to the long axis of the fiber (Figure 2.1b). The spaces between myofibrils are filled up with a semiliquid sarcoplasm, which forms the environment of enzymatic reactions and takes part in conducting nervous impulses into the muscle. Each myofibril consists of two different protein structures: myosin thick filaments (15 nm × 1.5 µm) and thin (7 nm × 1 µm) filaments composed of actin, tropomyosin, and troponin. Inside the muscle fiber there is also a cytoskeleton—the protein structures assuring the integrity of muscle cells. Cytoskeletal proteins such as titin and nebulin are located in myofibrils and anchored in the Z-band. Desmin is made up of costamers, which connect the myofibrils; vinculin connects myofibrils and sarcolemma (Figure 2.2). Postmortem degradation of cytoskeletal proteins plays a role in the improvement of meat functional properties, especially its tenderness and water-holding capacity. The muscle fiber is covered by a thin membrane called the sarcolemma and a layer of connective tissue called the endomysium. Bundles of muscle fibers are
10 µm
5 µm
FIGURE 2.1 Scanning electron microscope (SEM) micrographs of bovine semitendinosus muscle: transverse section (a) and longitudinal section (b). F, muscle fiber; MF, myofibril. (From Palka, K., unpublished. With permission.)
18
FIGURE 2.2 Schematic structure of muscle cytoskeleton: titin (1), nebulin (2), vinculin (3), skelemin (4), and desmin (5). C, costameres; CM, cell membrane; F, muscle fiber; M, M-band; Z, Z-band.
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FIGURE 2.3 Schematic structure of skeletal muscle: tendon (1), epimysium (2), perimysium (3), endomysium (4), sarcolemma (5), myofibril (6), muscle fiber (7), and bundle of muscle fibers (8).
surrounded by the perimysium, and the whole muscle by the epimysium. At the ends of the muscle, the epimysium forms tendons, which connect the muscle to the bone (Figure 2.3). Both the quantity and kind of connective tissue affect the technological and nutritional properties of meat.
2.2.2 MILK
AND
MILK PRODUCTS
Milk is a liquid secretion of the mammary glands of female mammals, consisting of 80 to 90% water and 10 to 20% dry mass. It is an oil-in-water (O/W) emulsion composed of fat and fat-soluble vitamins; the aqueous phase contains proteins, mineral salts, lactose, and water-soluble vitamins. The chemical composition of milk (Table 2.1) depends on species and breed, lactation period, as well as nutritional and health conditions of the animal. Milk proteins are made up of caseins and whey proteins. Milk proteins, caseins, and several enzymes, mainly hydrolases and oxidoreductases, are very important in the manufacturing of cheeses and yogurts (Figure 2.4). After drying they are used in the food industry as milk powder, caseinates, and casein hydrolyzates. Nonprotein nitrogenous compounds constitute about 0.2% of milk. Milk fat is made up of about 98% triacylglycerols and 1% phospholipids. It also contains smaller amounts of di- and monoacylglycerols, sterols, higher fatty acids, carotenoids, and vitamins. In cow milk fat, over 500 various fatty-acid residues have been identified. The polyenoic fraction constitutes about 3% of the total fatty acids and is composed mainly of linoleic acid and α-linolenic acids. Milk fat is easily digestible because of a relatively low melting temperature and great dispersion (droplets of 5 to 10 µm in diameter). Because of the latter, it is susceptible to hydrolysis and oxidation. The main saccharide of milk is lactose. During heat treatment of milk, lactose is involved in Maillard reactions. Lactose is used for the production of baby formulas, low-calorie foods, bread, drugs, and microbiological media. The milk minerals are composed mainly of calcium and phosphorus in the form of calcium phosphate. Phosphorus is also present in milk in the form of phosphoproteins. These components have important nutritional and technological significance. The total content of Ca and P in milk is about 0.12 and 0.10%, respectively. About 6 to 9% of milk volume is made up of gases, mainly CO2, N2, and O2. Oxygen present in milk may cause oxidation of unsaturated fatty acids. For this reason air is removed from milk during processing.
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Chemical and Functional Properties of Food Components
FIGURE 2.4 SEM micrograph of protein matrix in yogurt. (From Domagała, J., unpublished. With permission.)
Milk contains vitamins essential for the growth and development of young organisms, especially vitamins from the B group and vitamin A. The quantity of vitamin A depends on the season.
2.2.3 EGGS The hen egg consists of a shell, the egg white, and yolk (Figure 2.5). The 0.2- to 0.4-mm thick shell constitutes up to 10 to 12% of the egg mass and consists of about 3.5% organic and 95% mineral components. The shell has a multilayer structure. Two of the layers are made of keratin and collagen fibers. The next two layers are calcinated and on the surface are covered by a thin membrane (cuticula), which contains two-thirds of the shell pigments. The shell protects the egg against microbiological contamination and makes the exchange of gases possible.
FIGURE 2.5 Schematic structure of a hen egg: shell (1), membranes (2), air chamber (3), rare white (4), dense white (5), yolk (6), and chalazae (7).
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The egg white (about 60% of the egg’s mass) is composed mainly of water and a mixture of proteins, and also has a multilayer structure. Starting from the shell there are four fractions of white: external thin, external thick, internal thin, and internal thick, which make up 23, 57, 17, and 2.5%, respectively, of the egg white mass. Mucin structures called chalazae keep the yolk in a central position in the egg. During long storage, the chalazae lose their elasticity, and the egg white loses part of its water due to evaporation. The egg yolk (about 30% of the egg’s mass) has a spherical shape, a diameter of about 3 to 3.5 cm, and a color ranging from dark to light orange, depending on the quantity of lipids and carotenoid pigments in the fodder. It is surrounded by a thin and elastic vittelin membrane built of keratin and mucin fibers. The egg yolk, being an O/W emulsion stabilized by lecithin, has a very high viscosity. The viscosity of the yolk decreases during storage, as a result of water permeation from the white through the vittelin membrane. Egg yolks are utilized as a stabilizer in the manufacturing of mayonnaise. The chemical composition of the egg (Table 2.1) is rather stable. As the only source of food for the embryo, it contains all substances essential for life. There is about 6.6 g of very well-balanced proteins in one egg. About two-thirds of the yolk mass are lipids, mainly unsaturated. Cholesterol makes up about of 2.5% of the dry mass of the yolk. The egg is also a source of vitamins A, B, D, E, and K, and the best dietary source of choline. The minerals S, K, Na, P, Ca, Mg, and Fe are in free form or bound to proteins and lipids. Eggs and egg products, thanks to their texture-improving properties, emulsifying effect, and foaming ability, are multifunctional additives used in food technology in liquid or dried form.
2.3 SACCHARIDE FOOD PRODUCTS 2.3.1 CEREAL
AND
CEREAL PRODUCTS
Cereals are fruits of cultivated grasses that may be used as raw materials for production of food and feed. The major cereals are wheat, rye, barley, oats, millet, rice, sorghum, and maize. The share of cereal products in the human diet is estimated as 50 to 60%. The shape of grains varies from elongated (rye) to spherical (millet), but the anatomical structures of cereal grains are rather similar. The essential anatomical elements of cereal grains are seed coat (bran), endosperm, and germ. Commercially the most important cereal is wheat. A wheat grain is about 1-cm long and has a diameter of 0.5 cm. It is egg-shaped with a deep crease running along one side and a number of small hairs, called the beard, at one end (Figure 2.6). The grain is surrounded by a five-layer coat called bran, which makes up 15% of the mass of the whole grain. It is rich in B vitamins and contains about 50% of the total mass of minerals in the grain. The bran consists of cellulose and is indigestible for humans. It is separated during flour production and used as animal fodder. The germ, about 3% of the mass of the grain, is situated at the base of the grain. It contains the embryo, which is rich in lipids, proteins, B vitamins, vitamin E, and
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Chemical and Functional Properties of Food Components
FIGURE 2.6 Schematic structure of wheat grain: longitudinal section (a) and transverse section (b). Beard (1), bran (2), endosperm (3), crease (4), scutellum (5), germ (6).
minerals, mainly iron. A membranous tissue called the scutellum separates the germ from the endosperm. It is a rich source of thiamine—about 60% of all its content in the grain. The starchy endosperm makes up 80 to 90% of the wheat grain and is a reserve of food for the germ. The starch granules are embedded in a protein matrix, while the periphery of the endosperm is composed of a single aleurone layer. The aleurone layer is rich in proteins and contains high amounts of minerals, vitamins, and enzymes, but it is usually removed during milling. Considering the size, most of the starch granules in the endosperm cells of wheat may be located in two ranges: large (15 to 40 µm in diameter) and small (1 to 10 µm in diameter), whereas those in the subaleurone endosperm cells are 6 to 15 µm in diameter. The chemical composition of cereal (Table 2.2) is dependent on the species, means of cultivation, and the time and conditions of growth, harvest, and storage. Starch constitutes about 80% of the grain dry mass. In bread making the most important properties of starch are its water-holding capacity, gelatinization, and susceptibility to hydrolysis. The protein content of cereal grains is in the range of 7 to 18%. From the technological point of view, proteins, mainly gluten proteins, as well as enzymes (amylases, proteases, and lipases) are important during dough making. Cereal grains also contain 2 to 4% lipids, mainly triacylglycerols of unsaturated fatty acids and phospholipids. The mineral elements, mainly P and K, and to a smaller extent Mg and Ca, make up about 2% of the grain mass. Vitamins of the B group and vitamin E are also present in grains. The milling technique can be modified to increase or decrease the yield of flour from a given amount of grain. The percentage of flour produced is termed the
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TABLE 2.2 Chemical Compositions of Cereals and Cereal Products
Bread
Flour
Grains
Product Wheat Rye Maize Rice paddy Millet Wheat flour 97% Wheat flour 50% Rye flour 97% Rye flour 60% Wheat bread Rye bread Rusks
Water %
Crude protein N×6.25 %
Saccharides %
Lipids %
Mineral components %
15.0 15.0 15.0 15.0 15.0 13.5 13.5 13.5 13.5 37.5 46.0 7.0
11.0 9.0 10.0 7.5 10.5 10.0 8.5 7.5 5.5 8.0 6.5 8.5
68.5 70.5 67.0 75.5 65.0 70.5 75.0 73.0 78.5 57.5 45.0 75.0
2.0 1.5 4.5 0.5 4.0 3.0 1.5 2.0 1.5 1.5 1.0 5.5
1.5 1.5 1.5 1.0 3.0 1.5 0.5 1.5 0.5 2.0 2.0 1.5
Source: Adapted from Fox, B.A. and Cameron, A.G., Food Science: A Chemical Approach, Hodder and Stoughton, London, 1986; Kent, N.L., Technology of Cereals with Special Reference to Wheat, Pergamon Press, Oxford, 1975.
extraction rate of flour. Whole flour, containing the bran, germ, scutellum, and endosperm of the grain, has an extraction rate of 100%. The extraction rate of 70% means that the flour is almost entirely composed of crushed endosperm. As the percentage of flour increases, the amount of dietary fiber in flour increases, too. This is an important nutritional aspect of cereal products.
2.3.2 POTATOES The potato is a swollen underground stem or tuber that contains a store of food for the plants. In the tuber, a bud end and a stem end can be distinguished. The hollows, called eyes, are spirally arranged around the tuber surface. The tuber section is divided into pith, parenchyma, vascular system, cortex, and periderm (Figure 2.7).
FIGURE 2.7 Schematic structure of potato, longitudinal section: eye (1), periderm (skin) (2), parenchyma (3), vascular ring (4), and pith (5).
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TABLE 2.3 Chemical Composition of Potato and Honey Potato
[%]
Honey
[%]
Water Dry matter: Starch Saccharides Proteins Cellulose Lipids Mineral components
76.0 24.0 17.5 1.5 2.0 1.0 0.5 1.0
Water Saccharides: Fructose Glucose Maltose Trisaccharides Saccharose Proteins, vitamins, and mineral components
17.0 82.5 38.5 31.0 7.0 4.0 1.5 0.5
Source: Adapted from Lisińska, G. and Leszczyski, W., Potato Science and Technology, Elsevier Applied Science, London, 1989; Ramsay, I., Honey as a food ingredient, Food Ingredients and Process. Int., 10, 16, 1992.
Each potato tuber is a single living organism, and its water is indispensable in all the vital processes. Water transports any substances moving in the interior of the tuber. It also protects the tubers against overheating (by transpiration). Water constitutes about 75% of the potato (Table 2.3). The second major constituent of potato is starch (about 20%). With regard to starch content, there are potato cultivates of a low (to 14%), medium (15 to 19%), and high (above 20%) starch content. Potato is also a valuable source of ascorbic acid—up to 55 mg/100 g. Its ash consists of about 60% K and 15% P2O5. The chemical composition of potato tubers changes during storage due to evaporation and catabolic processes. In many parts of the world, potatoes are the main saccharide source in human food and animal fodder and are also widely used as raw material for starch manufacture and in the fermentation industry. The shape and size of starch granules are specific for different starchy raw materials (Figure 2.8). a
b
20 µm
c
20 µm
20 µm
FIGURE 2.8 SEM micrographs of starch granules in different starchy raw materials: potato (a), wheat (b), and maize (c). (From Juszczak, L., unpublished. With permission.)
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2.3.3 HONEY Honey is produced by honeybees from the flower nectar of plants. Fresh honey is a clear, very aromatic, dark amber-colored liquid. It is very sticky and hygroscopic, with a density of about 1.40 g/cm3. Honey is an oversaturated solution of glucose and fructose, and easily crystallizes. After crystallization its color is brighter. It is a very stable product. At a temperature of 8 to 10°C and humidity of 65 to 75% it may be stored for many years. Honey is a high-calorie food easily assimilated by the human organism. Honey is used in the manufacturing of alcoholic beverages (i.e., in wine production). In medicine it is prescribed for heart, liver, stomach, skin, and eye illnesses. In the food industry honey is used as a very effective sweetener (25% sweeter than sucrose), a very well-binding, concentrating, and covering additive, and a taste intensifier. The chemical composition of honey (Table 2.3) is dominated by glucose and fructose. Honey also contains many other valuable components, such as enzymes, organic acids, mineral elements, nonprotein nitrogenous compounds, vitamins, aroma substances, and pigments.
2.3.4 NUTS Nuts are composed of a wooden-like shell and a seed, covered by a yellow or brown skin. Each part makes up about 50% of the nut mass. Inside of the seed is a germ. The seeds of nuts consist of about 60% lipids rich in unsaturated fatty acids, 16 to 20% easily digested proteins, 7% saccharides, vitamin B1 (10 mg/100g) and C (30–50 mg/100 g), and P, Mg, K, and Na. Because of their high quantity of easily assimilated nutrients, nuts may be used in the diets of convalescents and children.
2.3.5 SEEDS
OF
PULSES
To this group belong peas, beans, lentils, soybeans, and peanuts. All of them have fruits in the form of pods. Their shape and size depend on the cultivar. Inside the pod there are seeds used as raw material in the food industry. The dry mass of pulse seeds consists of saccharides (14 to 63%), proteins (28 to 44%), and lipids (1 to 50%). The other constituents are mineral elements (mainly K and P), and vitamins from the B group. Soybean is the most valuable pod plant, due to its high quantity and good quality of protein. Soy products in the form of meat extenders and analogues are used all over the world. Soybean is also a raw material in the oil industry.
2.4 EDIBLE FATS Food products such as butter, lard, margarine, or plant oils are regarded as visible fats. They make up about 45% of the total fat consumed by humans, while the invisible fats, which are natural components of foods such as meat, fish, eggs, and bakery products, make up about 55%.
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Chemical and Functional Properties of Food Components
Visible fats are composed mainly of triacylglycerols. They also contain fatsoluble vitamins A, D, and E, and additives added during processing, for example, antioxidants, colorants, or preservatives. The consistency of fats depends on the content of unsaturated fatty-acid residues. The oils of plant and fish origin are rich in long-chain polyenolic fatty acids. Butter consists of 16 to 18% water, 80 to 82.5% lipids, 0.5 % proteins, and 0.5% saccharides.
2.5 FRUITS AND VEGETABLES Fruits and vegetables are rich sources of vitamins and minerals, as well as terpenes, flavonoids, tannins, chinons and phytoncides. They make food more attractive because of smell and color. Fruits and vegetables are living organisms and their chemical compositions are very changeable. The predominant constituent of fruits and vegetables is water, which may represent up to about 96% of the total weight of the crop. The water in fruits and vegetables may be in free or bound form. A relatively high amount of free water improves the taste of fruits and vegetables consumed in their raw state, as well as the accessibility of soluble components. Most of the solid matter of fruit and vegetables is made of saccharides and smaller amounts of protein and fat. The total saccharide content in the fresh weight of fruits and vegetables ranges from about 2% in some pumpkin fruits to above 30% in starchy vegetables. Generally, vegetables contain less than 9% saccharides. The polysaccharides (cellulose and hemicelluloses) are largely confined to the cell walls. The di- and monosaccharides (sucrose, glucose, and fructose) are accumulated mainly in the cell sap. The proportions of the different saccharide constituents can fluctuate due to metabolic activity of the plant, especially during fruit ripening. The majority of proteins occurring in fruits and vegetables play enzymatic roles that are very important in the physiology and postmortem behavior of the crop. The protein content in vegetables is lower than 3%, except in sweet maize (above 4%). In fruits it ranges from below 1% to above 1.5%. Proteins are found mainly in the cytoplasmic layers. The lipids of fruits and vegetables are, like the proteins, largely confined to the cytoplasmic layers, in which they are especially associated with the surface membranes. Their content in fruits and vegetables is always lower than 1%. Lipid and lipidlike fractions are particularly prominent in the protective tissues at the surfaces of plant parts—the epidermal and corky layers. Plant tissues also contain organic acids formed during metabolic processes. For this reason fruits and vegetables are normally acidic in reaction. The quantity of organic acids is different, from very low, about 2 milliequivalents of acid/100 g in sweet maize and pod seeds to very high, up to 40 milliequivalents/100 g in spinach. For the majority of fruits and vegetables the dominant acids are citric acid and malic acid, each of which can, in particular examples, constitute over 2% of the fresh weight of the material. Lemons contain more than 3% citric acid. Tartaric acid accumulates in grape and oxalic acid in spinach. The fruits in general show a decrease in overall acidity during the ripening process.
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TABLE 2.4 Mineral Components of Fruits and Vegetables (mg/100 g of raw mass) Component K Na Ca Mg P Cl S Fe
mg
Rich source
350 65 150 50 120 90 80 2
Parsley (above 1000 mg) Celery Spinach (up to 600 mg) Sweet maize Seeds and young growing parts Celery Plants with higher quantity of proteins Parsley (up to 8 mg)
Source: Adapted from Duckworth, R.B., Fruit and Vegetables, Pergamon Press, London, 1966.
The total amount of mineral components in fruits and vegetables is in the range of 0.1% (in sweet potatoes) up to about 4.4% (in kohlrabi). The most abundant mineral constituent in fruits and vegetables is potassium (Table 2.4). Generally, vegetables are a better source of minerals than fruits. The mineral elements influence not only the growth and crop of fruits and vegetables, but also their texture (Ca), color (Fe), and metabolic processes (microelements). The diversity of form shown by fruit and vegetable structures is extremely wide. Among the vegetables there are representatives of all the recognizable morphological divisions of the plant body—whole shoots, roots, stems, leaves, and fruits. Fruits may also be classified into a number of structural types. The individual seed-bearing structures of the flower (called carpels) constitute the gynoecium. The seed-containing cavity of a carpel is called the ovary, and its wall develops into the pericarp of the fruit. The edible fleshy part of a fruit most commonly develops from the ovary wall, but it may also be derived from the enlarged tip of stem from which floral organs arise, and sometimes leaflike structures protecting the flowers may also become fleshy, for example, in pineapple. Most of the metabolic activity of plants is carried out in the tissue called parenchyma, which generally makes up the bulk of the volume of all soft edible plant structures. The epidermis, which is sometimes replaced by a layer of corky tissue, is structurally modified to protect the surface of the organ. The highly specialized tissues collenchyma and sclerenchyma provide mechanical support for the plant. Water, minerals, and products of metabolism are transported through the plant via the vascular tissues, xylem and phloem, which are the most characteristic anatomical features of plants in the cross-section. The structure of fruits is dominated by soft parenchymatous tissue, while conducting and supporting structures are rather poorly developed. An exception is the pineapple, in which conducting tissues are very prominently represented. The subtle structure and proportions of individual tissues influence the texture, properties, and suitability for processing of fruits and vegetables.
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REFERENCES Duckworth, R.B., Fruit and Vegetables. Pergamon Press, London, 1966. Fox, B.A. and Cameron, A. G., Food Science: A Chemical Approach. Hodder and Stoughton, London, 1986. Hedrick, H.B. et al., Principles of Meat Science. Kendal/Hunt Publish Comp., Dubuque, IA, 1994. Kent, N.L., Technology of Cereals with Special Reference to Wheat. Pergamon Press, Oxford, 1975. Kirk, R.S. and Sawyer, R., Pearson’s Composition and Analysis of Foods. Longman Science, London, 1991. Kristensen, L. and Purslow, P.P., The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins, Meat Science, 58, 17–23, 2001. Lisińska, G. and Leszczyński, W., Potato Science and Technology. Elsevier Applied Science, London, 1989. Ramsay, I., Honey as a food ingredient, Food Ingredient and Processing International, 10, 16, 1992. Renner, E., Nutritional aspects of cheese, in Cheese: Chemistry, Physics and Microbiology, Vol. 1, Fox, P.F. (Ed.). Chapman & Hall, London, 1993, p. 557. Sikorski, Z.E., Seafood Raw Materials. WNT, Warsaw, 1992 (in Polish). Tamime, A.Y. and Robinson, R.K., Yoghurt. Science and Technology. CRC Press, Boca Raton, FL, 1999.
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Water and Food Quality Emilia Barbara Cybulska and Peter Edward Doe
CONTENTS 3.1 3.2
Introduction .................................................................................................... 30 Structure and Properties of Water.................................................................. 31 3.2.1 The Water Molecule........................................................................... 31 3.2.2 Hydrogen Bonds ................................................................................ 31 3.2.3 Properties of Bulk Water ................................................................... 33 3.2.4 Thermal Properties of Water.............................................................. 36 3.2.5 Water as a Solvent ............................................................................. 37 3.2.6 Water in Biological Materials............................................................ 41 3.2.6.1 Properties ............................................................................ 41 3.2.6.2 Water Transport .................................................................. 43 3.3 Water in Food................................................................................................. 44 3.3.1 Introduction ........................................................................................ 44 3.3.2 Sorption Isotherms and Water Activity ............................................. 46 3.3.2.1 Principle .............................................................................. 46 3.3.2.2 Measurement of Water Activity ......................................... 48 3.3.2.3 Water Activity and Shelf Life of Foods............................. 48 3.3.3 Bottled Water ..................................................................................... 50 3.3.3.1 Classification....................................................................... 50 3.3.3.2 Natural Mineral Water ........................................................ 50 3.3.4 Bottled Water Other than Natural Mineral Water ............................. 52 3.3.4.1 Definition ............................................................................ 52 3.3.4.2 Water Defined by Origin .................................................... 52 3.3.4.3 Hygiene, Labeling, and Health Benefits ............................ 53 3.3.5 Water Supply, Quality, and Disposal................................................. 53 3.3.5.1 Water Supply ...................................................................... 53 3.3.5.2 Water Quality: Standards and Treatment ........................... 54 3.3.6 Water Pollution .................................................................................. 56 3.3.7 Wastewater Treatment and Disposal.................................................. 57 References................................................................................................................ 58
29
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3.1 INTRODUCTION Water is the most popular and most important chemical compound on our planet. It is a major chemical constituent of the Earth’s surface and it is the only substance that is abundant in solid, liquid, and vapor form. Because it is ubiquitous, it seems to be a mild and inert substance. In fact, it is a very reactive compound characterized by unique physical and chemical properties that make it very different from other popular liquids. The peculiar water properties determine the nature of physical and biological world. Water is the major component of all living organisms. It constitutes 60% or more of the weight of most living things, and it pervades all portions of every cell. It existed on our planet long before the appearance of any form of life. The evolution of life was doubtlessly shaped by physical and chemical properties of the aqueous environment. All aspects of living cells’ structure and function seem to be adapted to water’s unique properties. Water is the universal solvent and dispersing agent, as well as a very reactive chemical compound. Biologically active structures of macromolecules are spontaneously formed only in aqueous media. Intracellular water is not only a medium in which structural arrangement and all metabolic processes occur, but an active partner of molecular interactions, participating directly in many biochemical reactions as a substrate or a product. Its high heat capacity allows water to act as a heat buffer in all organisms. Regulation of water contents is important in the maintenance of homeostasis in all living systems. Only 0.003% of all freshwater reserve participates in its continuous circulation between the atmosphere and the hydrosphere. The remaining part is confined in the Antarctic ice. The geography of water availability has determined, to a large degree, the vegetation, food supply, and habitation in the various areas of the world. For example, Bangladesh has one of the world’s highest population densities, made possible through the regular flooding of the Ganges River and the rich silts it deposits in its wake. In Bangladesh, the staple food—rice—grows abundantly and is readily distributed. In other societies, the food must be transported long distances or kept over winter. Human well-being is closely linked to the availability of water and food. An expected increase in the world population by the year 2050 (65% or 3.7 billion) will create enormous pressure on freshwater resources and food production. Agriculture is by far the largest consumer of water and the key issue is to look for ways to improve water use efficiency. The solution lies in producing more food from existing water and land resources (Wallace and Gregory, 2002). Stability, wholesomeness, and shelf life are significant features of foods that are, to a large degree, influenced by the water content. Dried foods were originally developed to overcome the constraints of time and distance before consumption. Canned and frozen foods were developed next. The physical properties, quantity, and quality of water within food have a strong impact on food effectiveness, quality attributes, shelf life, textural properties, and processing.
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3.2 STRUCTURE AND PROPERTIES OF WATER 3.2.1 THE WATER MOLECULE Water is a familiar material, but it has been described as the most anomalous of chemical compounds. Although its chemical composition, HOH or H2O, is universally known, the simplicity of its formula belies the complexity of its behavior. Its physical and chemical properties are very different from compounds of similar complexity, such as HF and H2S. To understand the reasons for water's unusual properties, it is necessary to examine its molecular structure in some detail. Although a water molecule is electrically neutral as a whole, it has a dipolar character. The high polarity of water is caused by the direction of the H-O-H bond angle, which is 104.5°, and by an asymmetrical distribution of electrons within the molecule. In a single water molecule, each hydrogen atom shares an electron pair with the oxygen atom in a stable covalent bond. However, the sharing of electrons between H and O is unequal because the more electronegative oxygen atom tends to draw electrons away from the hydrogen nuclei. The electrons are more often in the vicinity of the oxygen atom than in the vicinity of the hydrogen atom. The result of this unequal electron sharing is the existence of two electric dipoles in the molecule, one along each of the H-O bonds. The oxygen atom bears a partial negative charge δ–, and each hydrogen a partial positive charge δ+. Because the molecule is not linear, H-O-H has a dipole moment (Figure 3.1). Because of this, water molecules can interact through electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of another.
3.2.2 HYDROGEN BONDS Such interactions, which arise because the electrons on one molecule can be partially shared with the hydrogen on another, are known as hydrogen bonds. The H2O molecule, which contains two hydrogen atoms and one oxygen atom in a nonlinear arrangement, is ideally suited to engage in hydrogen bonding. It can act both as a donor and as an acceptor of hydrogen. The nearly tetrahedral arrangement of the δ+ Η δ–
δ+
H
O
–
δ–
104.5°
Dipole moment H
O
δ+
δ– +
FIGURE 3.1 Water molecule as an electric dipole.
δ–
H δ+
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orbital about the oxygen atom allows each water molecule to form hydrogen bonds with four of its neighbors (Figure 3.2). An individual, isolated hydrogen bond is very labile. It is longer and weaker than a covalent O-H bond (Figure 3.3). The hydrogen bond’s energy, that is, the energy required to break the bond, is about 20 kJ/mol. These bonds are intermediate between those of weak van der Waals interactions (about 1.2 kJ/mol) and those of covalent bonds (460 kJ/mol). Hydrogen bonds are highly directional; they are stronger when the hydrogen atom and the two atoms that share it are in a straight line (Figure 3.4). Hydrogen bonds are not unique to water. They are formed between water and different chemical structures, as well as between other molecules (intermolecular) or even within a molecule (intramolecular). They are formed wherever an electronegative atom (oxygen or nitrogen) comes in close proximity to a hydrogen atom covalently bonded to another electronegative atom. Some representative hydrogen bonds of biological importance are shown in Figure 3.5. H 1 H O H 2O H H H O 3 4 O H H
H 5O H
FIGURE 3.2 Tetrahedral hydrogen bonding of five water molecules.
FIGURE 3.3 Two water molecules connected by hydrogen bonds.
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FIGURE 3.4 Directionality of the hydrogen bonds.
FIGURE 3.5 Some hydrogen bonds of biological importance.
Intra- and intermolecular hydrogen bonding occurs extensively in biological macromolecules. A large number of the hydrogen bonds and their directionality confer very precise three-dimensional structures upon proteins and nucleic acids.
3.2.3 PROPERTIES
OF
BULK WATER
The key to understanding water structure in solid and liquid form lies in the concept and nature of the hydrogen bonds. In the crystal of ordinary hexagonal ice (Figure 3.6),
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Chemical and Functional Properties of Food Components
FIGURE 3.6 Structure of ice.
each molecule forms four hydrogen bonds with its nearest neighbors. Each HOH acts as a hydrogen donor to two of the four water molecules, and as a hydrogen acceptor from the remaining two. These four hydrogen bonds are spatially arranged according to tetrahedral symmetry (Bjerrum, 1952) The crystal lattice of ice occupies more space than the same number of H2O molecules in liquid water. The density of solid water is thus less than that of liquid water, whereas simple logic would have the more tightly bound solid structure more dense than its liquid. One explanation for ice being lighter than water at 0°C proposes a reforming of intermolecular bonds as ice melts, so that on average, a water molecule is bound to more than four of its neighbors, thus increasing its density. But as the temperature of liquid water increases, the intermolecular distances also increase, giving a lower density. These two opposite effects explain the fact that liquid water has a maximum density at a temperature of 4°C. At any given instant in liquid water at room temperature, each water molecule forms hydrogen bonds with an average of 3.4 other water molecules (Lehninger et al., 1993). The average translational and rotational kinetic energies of a water molecule are approximately 7 kJ/mol, the same order as that required to break hydrogen bonds; therefore, hydrogen bonds are in a continuous state of flux, breaking and reforming with high frequency on a picosecond time scale. A similar dynamic process occurs in aqueous media with substances that are capable of forming hydrogen bonds. At 100°C liquid water still contains a significant number of hydrogen bonds, and even in water vapor there is strong attraction between water molecules. The very large number of hydrogen bonds between molecules confers great internal cohesion on liquid water. This feature provides a logical explanation for many of its unusual properties. For example, its large values for heat capacity, melting point, boiling point, surface tension, and heat of various phase transitions are all related to the extra energy needed to break intermolecular hydrogen bonds.
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That liquid water has structure is an old and well-accepted idea; however, there is no consensus among physical chemists as to the molecular architecture of the hydrogen bond’s network in the liquid state. It seems that the majority of hydrogen bonds survive the melting process, but obviously rearrangement of molecules occurs. The replacement of crystal rigidity by fluidity gives molecules more freedom to diffuse about and to change their orientation. Any molecular theory for liquid water must take into account changes in the topology and geometry of the hydrogen bond network induced by the melting process. Many models have been proposed, but none has adequately explained all properties of liquid water. Historically, there are two competing theoretical approaches used to describe the molecular structure of liquid water: (1) the continuum (uniform) models, and (2) the mixture (cluster) models (Starzak and Mathlouthi, 2003; Dautchez et al., 2003). According to continuum models, liquid water is depicted as a continuous, threedimensional network in which water molecules are interconnected by somewhat distorted hydrogen bonds; hydrogen bonding is almost complete, the structural parameters (distances and angles) and bond energies have continuous distribution; all water molecules are qualitatively the same and a whole-water sample is considered as a single entity with temperature-dependent local structure. The continuum models are incompatible with particular water properties such as the compressibility minimum and the density maximum. Most mixture models describe liquid water as an equilibrium mixture of a few classes of different structural species more or less defined. A paper by Röntgen (1892) in which water was described as a saturated solution of ice in a liquid composed of simpler molecules, started the mixture model history. Over the next century a variety of structural arrangements, based on the equilibrium of small water aggregates were proposed and used to explain the properties of water and aqueous solutions. The most popular, the flickering clusters model (Figure 3.7), suggests that liquid water is highly organized on a local basis: the hydrogen bonds break and reform spontaneously, creating and destroying transient structural domains (Frank and Quist, 1961; Frank and Wen, 1957). However, because the half-life of any hydrogen bond is less than a nanosecond, the existence of these clusters has statistical validity only; even this has been questioned by some authors who consider water to be a continuous polymer. Experimental evidence obtained by x-rays and neutron diffractions strongly supports the persistence of a tetrahedral hydrogen bond order in the liquid water, but with substantial disorder present. Since the high-resolution Raman technique became available the spectra have been carefully analyzed in favor of the mixture models. The first computer simulation was performed by Rahman and Stillinger (1971) with a model of 216 water molecules. The view that emerges from these studies is the following: liquid water consists of a macroscopically connected, random network of hydrogen bonds. This network has a local preference for tetrahedral geometry, but it contains a large proportion of strained and broken bonds, which are continually undergoing topological reformation. The properties of water arise from the competition between relatively bulky ways of connecting molecules into local patterns
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Chemical and Functional Properties of Food Components
FIGURE 3.7 Flickering clusters of H2O molecules in bulk water.
characterized by strong bonds and nearly tetrahedral angles and more compact arrangements characterized by more strain and bond breakage (Stillinger, 1980). With the advent of supercomputers, a flood of quantitative studies on water structure based on quantum and statistical mechanics have been carried out. A number of models have been proposed in which more and more complicated structural units as liquid water components have been suggested (Starzak and Mathlouthi, 2003). According to a model proposed by Wiggins (1990, 2002), two types of structure can be distinguished: high-density water and low-density water. In the high-density water, the bent, relatively weak hydrogen bonds predominate over straight, stronger ones. Low-density water has many icelike straight hydrogen bonds. Although hydrogen bonding is still continuous through the liquid, the weakness of the bonds allows the structure to be disrupted by thermal energy extremely rapidly. High-density water is extremely reactive and more liquid, whereas low-density water is inert and more viscous. A continuous spectrum of water structures between these two extremes can be imagined. The strength of water–water hydrogen bonding, which is the source of water density and reactivity, has great functional significance; this explains water’s solvent properties and its role in many biological events. A common feature of all theories is that a definite structure of liquid water is due to the hydrogen bonding between molecules, and that the structure is in the dynamic state as the hydrogen bonds break and reform with high frequency.
3.2.4 THERMAL PROPERTIES
OF
WATER
The unusually high melting point of ice, as well as the heat of water vaporization and specific heat, is related to the ability of water molecules to form hydrogen bonds and the strength of these bonds.
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A large amount of energy, in the form of heat, is required to disrupt the hydrogenbonded lattice of ice. In the common form of ice, each water molecule participates in four hydrogen bonds. When ice melts, most of the hydrogen bonds are retained by liquid water, but the pattern of hydrogen bonding is irregular, due to the frequent fluctuation. The average energy required to break each hydrogen bond in ice has been estimated to be 23 kJ/mol, while the energy to break each hydrogen bond in water is less than 20 kJ/mol (Ruan and Chen, 1998). The heat of water vaporization is much higher than that of many other liquids. As is the case with melting ice, a large amount of thermal energy is required for breaking hydrogen bonds in liquid water, to permit water molecules to dissociate from one another and to enter the gas phase. Perspiration is an effective mechanism of decreasing body temperature because the evaporation of water absorbs so much heat. A relatively large amount of heat is required to raise the temperature of 1 g of water by 1°C because multiple hydrogen bonds must be broken in order to increase the kinetic energy of the water molecules. Due to the high quantity of water in the cells of all organisms, temperature fluctuation within cells is minimized. This feature is of critical biological importance because most biochemical reactions and macromolecular structures are sensitive to temperature. The unusual thermal properties of water make it a suitable environment for living organisms, as well as an excellent medium for the chemical processes of life.
3.2.5 WATER
AS A
SOLVENT
Many molecular parameters, such as ionization, molecular and electronic structure, size, and stereochemistry, will influence the basic interaction between a solute and a solvent. The addition of any substance to water results in altered properties of that substance and of the water itself. Solutes cause a change in water properties because the hydrate envelopes that are formed around dissolved molecules are more organized and therefore more stable than the flickering clusters of free water. The properties of solutions that depend on a solute and its concentration are different from those of pure water. The differences can be seen in such phenomena as the freezing point depression, boiling point elevation, and increased osmotic pressure of solutions. The polar nature of the water molecule and the ability to form hydrogen bonds determine its properties as a solvent. Water is a good solvent for charged or polar compounds and a relatively poor solvent for hydrocarbons. Hydrophilic compounds interact strongly with water by an ion–dipole or dipole–dipole mechanism, causing changes in water structure and mobility and in the structure and reactivity of the solutes. The interaction of water with various solutes is referred to as hydration. The extent and tenacity of hydration depends on a number of factors, including the nature of the solute, salt composition of the medium, pH, and temperature. Water dissolves dissociable solutes readily because the polar water molecules orient themselves around ions and partially neutralize ionic charges. As a result, the positive and negative ions can exist as separate entities in a dilute aqueous solution without forming ion pairs. Sodium chloride is an example where the electrostatic attraction of Na+ and Cl– is overcome by the attraction of Na+ with the negative
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FIGURE 3.8 Hydration shell around Na+ and Cl–.
charge on the oxygen and Cl– with the positive charge on the hydrogen ions (Figure 3.8). The number of weak charge–charge interactions between water and the Na+ and Cl– ions is sufficient to separate the two charged ions from the crystal lattice. To acquire their stabilizing hydration shell, ions must compete with water molecules, which need to make as many hydrogen bonds with one another as possible. The normal structure of pure water is disrupted in a solution of dissociable solutes. The ability of a given ion to alter the net structure of water depends on the strength of its electric field. Among ions of a given charge-type (e.g., Na+ and K+ or Mg2+ and Ca2+), the smaller ions are more strongly hydrated than the larger ions, in which the charge is dispersed over a greater surface area. Most cations, except the largest ones, have a primary hydration sphere containing four to six molecules of water. Other water molecules, more distant from the ion, are held in a looser secondary sphere. Electrochemical transfer experiments indicate a total of 16 molecules of water around Na+ and about 10 around K+. The bound water is less mobile and denser than HOH molecules in bulk water. At some distance, the bonding arrangements melt into a dynamic configuration of pure water. Water is especially effective in screening the electrostatic interaction between dissolved ions because, according to Coulomb’s law, the force (F) between two charges q+ and q– separated by a distance r is given as: F = q+ ⋅ q–/εr2
(3.1)
where ε is the dielectric constant of the medium. For a vacuum, ε = 1 Debye unit, whereas for bulk water, ε = 80; this implies that the energies associated with electrostatic interactions in aqueous media are approximately 100 times smaller than the energies of covalent association, but increase considerably in the interior of a protein molecule. In thermodynamic terms, the free energy change, ∆G, must have a negative value for a process to occur spontaneously. ∆G = ∆H – T∆S
(3.2)
where ∆G represents the driving force, ∆H (the enthalpy change) is the energy from making and breaking bonds, and ∆S (the entropy change) is the increase in randomness. Solubilization of a salt occurs with a favorable change in free energy. As salt such as NaCl dissolves, the Na+ and Cl– ions leaving the crystal lattice acquire greater
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freedom of motion. The entropy (∆S) of the system increases; where ∆H has a small positive value and T∆S is large and positive, ∆G is negative. Water in the multilayer environment of ions is believed to exist in a structurally disrupted state because of conflicting structural influences of the innermost vicinal water and the outermost bulk-phase water. In concentrated salt solutions, the bulkphase water would be eliminated, and the water structure common in the vicinity of ions would predominate. Small or multivalent ions, such as Li+, Na+, H3O+, Ca2+, Mg2+, F–, SO42–, and PO43–, which have strong electric fields, are classified as water structure formers because solutions containing these ions are less fluid than pure water. Ions that are large and monovalent, most of the negatively charged ions and large positive ions, such as K+, Rb+, Cs+, NH4+, Cl–, Br–, I–, NO3–, ClO4–, and CNS– disrupt the normal structure of water; they are structure breakers. Solutions containing these ions are more fluid than pure water (Fennema, 1996). Through their varying abilities to hydrate and to alter water structure and its dielectric constant, ions influence all kinds of water–solute interactions. The conformation of macromolecules and the stability of colloids are greatly affected by the kinds and concentrations of ions present in the medium. Water is a good solvent for most biomolecules, which are generally charged or polar compounds. Solubilization of compounds with functional groups such as ionized carboxylic acids (COO–), protonated amines (NH3+), phosphate esters, or anhydrides is also a result of hydration and charge screening. Uncharged but polar compounds possessing hydrogen bonding capabilities are also readily dissolved in water, due to the formation of hydrogen bonds with water molecules. Every group that is capable of forming a hydrogen bond to another organic group is also able to form hydrogen bonds of similar strength with water. Hydrogen bonding of water occurs with neutral compounds containing hydroxyl, amino, carbonyl, amide, or imine groups. Saccharides dissolve readily in water, due to the formation of many hydrogen bonds between the hydroxyl groups or carbonyl oxygen of the saccharide and water molecules. Water–solute hydrogen bonds are weaker than ion–water interactions. Hydrogen bonding between water and polar solutes also causes some ordering of water molecules, but the effect is less significant than with ionic or nonpolar solutes. The introduction into water of hydrophobic substances such as hydrocarbons, rare gases, and the apolar groups of fatty acids, amino acids, or proteins is thermodynamically unfavorable because of the decrease in entropy. The decrease in entropy arises from the increase in water–water hydrogen bonding adjacent to apolar entities. Water molecules in the immediate vicinity of a nonpolar solute are constrained in their possible orientations, resulting in a shell of highly ordered water molecules around each nonpolar solute molecule (Figure 3.9a). The number of water molecules in the highly ordered shell is proportional to the surface area of hydrophobic solute. In the case of dissolved hydrocarbons, the enthalpy of formation of the new hydrogen bonds often almost exactly balances the enthalpy of creation in water, a cavity of the right size to accommodate the hydrophobic molecule. However, the restriction of water mobility results in a very large decrease in entropy. To minimize contact with water, hydrophobic groups tend to aggregate; this process is known as hydrophobic interaction (Figure 3.9b). The existence of hydrophobic
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Hydrophylic head group”
(a)
(b)
FIGURE 3.9 Cagelike water structure around the hydrophobic alkyl chain (a) and hydrophobic interactions (b).
substances barely soluble in water but readily soluble in many nonpolar solvents, and their tendency to segregate in aqueous media, has been known for a long time. However, the origin of this hydrophobic effect is still somewhat controversial. The plausible explanation is that hydrophobic molecules disturb the hydrogen bonded state of water, without having any compensatory ordering effects. Apolar molecules are water structure formers; water molecules cannot use all four possible hydrogen bonds when in contact with hydrophobic, water-hating molecules. This restriction results in a loss of entropy, a gain in density, and increased organization of bulk water. Amphipathic molecules, compounds that contain both polar or charged groups and apolar regions, disperse in water if the attraction of the polar group for water can overcome possible hydrophobic interactions of the apolar portions of the molecules. Many biomolecules are amphipathics: proteins, phospholipids, sterols, certain vitamins, and pigments have polar and nonpolar regions. When amphipathic compounds are in contact with water, the two regions of the solute molecule experience conflicting tendencies: the polar or charged hydrophilic regions interact favorably with water and tend to dissolve, but the nonpolar hydrophobic regions tend to avoid contact with water. The nonpolar regions of the molecules cluster together to present the smallest hydrophobic area to the aqueous medium, and the polar regions are arranged to maximize their interactions with the aqueous solvent. In aqueous media, many amphipathic compounds are able to form stable structures, containing hundreds to thousands of molecules, called micelles. The forces that hold the nonpolar regions of the molecules together are due to hydrophobic interactions. The hydrophobic effect is a driving force in the formation of clathrate hydrates and the self-assembly of lipid bilayers. Hydrophobic interactions between lipids and proteins are the most important determinants of biological membrane structure. The
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three-dimensional folding pattern of proteins is also determined by hydrophobic interactions between nonpolar side chains of amino acid residues.
3.2.6 WATER
IN
BIOLOGICAL MATERIALS
3.2.6.1 Properties Water behaves differently in different environments. Properties of water in heterogeneous systems, such as living cells or food, remain a field of debate (Mathlouthi, 2001; Rückold et al., 2003). Water molecules may interact with macromolecular components and supramolecular structures of biological systems through hydrogen bonds and electrostatic interactions. Solvation of biomolecules such as lipids, proteins, nucleic acids, or saccharides resulting from these interactions, determines their molecular structure and function. Various physical techniques, such as nuclear magnetic resonance (NMR), x-ray diffraction, and chemical probes (exchange of H by D), indicate that there is a layer of water bound to protein molecules, phospholipid bilayers, and nucleic acids, as well as at the surface of the cell membranes and other organelles. Water associated at the interfaces and with macromolecular components may have quite different properties from those in the bulk phase. Water can be expected to form locally ordered structures at the surface of water-soluble, as well as waterinsoluble macromolecules and at the boundaries of the cellular organelles. Biomacromolecules generally have many ionized and polar groups on their surfaces and tend to align near polar water molecules. This ordering effect exerted by the macromolecular surface extends quite far into the surrounding medium. According to the association–induction theory proposed by Ling (1962), fixed charges on macromolecules and their associated counterions constrain water molecules to form a matrix of polarized multilayers having restricted motion, compared with pure water. The monolayer of water molecules absorbed on the polar sorption site of the molecule is almost immobilized and thus behaves, in many respects, like part of the solid or like water in ice. It has different properties from additional water layers defined as multilayers. The association–induction theory has been shared by many researchers for many years. Unfortunately, elucidation of the nature of individual layers of water molecules has been less successful, due to the complexity of the system and lack of appropriate techniques. Measurements of the diffusion coefficients of globular protein molecules in solution yield values for molecular size that are greater than the corresponding radii determined by x-ray crystallography. The apparent hydrodynamic radius can be calculated from the Stokes-Einstein relation: D = kBT/6πηaH
(3.3)
where D is the diffusion coefficient, kB is the Boltzmann constant, T is the temperature, η is the solution viscosity, and aH is the molecule radius (Nossal and Lecar, 1991). Similarly, studies utilizing NMR techniques show that there is a species of associated water that has a different character than water in the bulk phase. By these
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and other methods, it was found that for a wide range of protein molecules, approximately 0.25 to 0.45 g of H2O are associated with each gram of protein. The hydration forces can stabilize macromolecular association or prevent macromolecular interactions with a strength that depends on the surface characteristic of the molecules and the ionic composition of the medium. The interaction between a solute and a solid phase is also influenced by water. Hydration shells or icebergs associated with one or the other phase are destroyed or created in this interaction and often contribute to conformational changes in macromolecular structures—and ultimately to changes in biological and functional properties important in food processing. Biophysical processes involving membrane transport are also influenced by hydration. The size of the hydration shell surrounding small ions and the presence of water in the cavities of ionic channels or in the defects between membrane lipids strongly affect the rates at which the ions cross a cell membrane. The idea that intracellular water exhibits properties different from those of bulk water has been around for a long time. The uniqueness of the cytoplasmic water was deduced from: The observation that cells may be cooled far below the freezing point of a salt solution iso-osmotic with that of the cytoplasm. Properties of the cytoplasm, which in the same conditions should bind water like a gel. Osmotic experiments in which it has often been observed that part of cell water is not available as a solvent. This water has been described as osmotically inactive water, bound water, or compartmentalized water. According to a recent view, three different kinds of intracellular water can be distinguished: a percentage of the total cell water appears in the form of usual liquid water. A relevant part is made up of water molecules that are bound to different sites of macromolecules in the form of hydration water, while a sizeable amount, although not fixed to any definite molecular site, is strongly affected by macromolecular fields. This kind of water has been termed vicinal water. Most of the vicinal water surrounds the elements of the cell cytoskeleton. Vicinal water has been extensively investigated, and it has been found that some of its properties are different from those of normal water. It does not have a unique freezing temperature, but an interval ranging from –70 to –50°C; it is a very bad solvent for electrolytes, but nonelectrolytes have the same solubility properties as in usual water; its viscosity is enhanced, and its NMR response is anomalous (Giudice et al., 1986). The distribution of various types of water inside living cells is a question that cannot be answered yet, especially because in many cells marked changes have been noted in the state of intracellular water as a result of biological activity. The possibility that water in living cells may differ structurally from bulk water has prompted a search for parameters of cell water that deviate numerically from those of bulk water. The diffusion coefficient for water in the cytoplasm of various cells has been determined with satisfactory precision. It has been found that the movement of water molecules inside living cells is not much different and is reduced by a factor of
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between 2 and 6, compared with the self-diffusion coefficient for pure water. According to Mild and Løvtrup (1985), the most likely explanation of the observed values is that part of the cytoplasmic water, the vicinal water close to the various surface structures in the cytoplasm, is structurally changed to the extent that its rate of motion is significantly reduced compared with the bulk phase. In heterogeneous biological materials and foods, water exists in different states. It is thought that water molecules in different states function differently. Water associated with proteins and other macromolecules has traditionally been referred to as bound water. However, to designate such water as bound can be misleading because for the most part, the water molecules are probably only transiently associated, and at least a portion of the associated water has to be constantly rearranged due to the thermal perturbations of weak hydrogen bonds. Water molecules are constantly in motion, even in ice. In fact, the translational and rotational mobility of water directly determines its availability. Water mobility can be measured by a number of physical methods, including NMR, dielectric relaxation, electron spin resonance (ESR), and thermal analysis (Chinachoti, 1993). The mobility of water molecules in biological systems may play an important role in a biochemical reaction’s equilibrium and kinetics, formation and preservation of chemical gradients and osmotic pressure, and macromolecular conformation. In food systems, the mobility of water may influence the engineering processes, such as freezing, drying, and concentrating, chemical and microbial activities, and textural attributes (Ruan and Chen, 1998). Water determines quality, stability, shelf life and physical properties of food. It has influence on rheological, thermal, mass transfer, electrical, optical, and acoustic physical properties (Lewicki, 2004). 3.2.6.2 Water Transport Water transport is associated with various physiological processes in whole living organisms and single cells. When cells are exposed to hyper- or hypoosmotic solutions, they immediately lose or gain water, respectively. Even in an isotonic medium, a continuous exchange of water occurs between living cells and their surroundings. Most cells are so small and their membranes are so leaky that the exchange of water molecules measured with isotopic water reaches equilibrium in a few milliseconds. The degree of water permeability differs considerably between tissues and cell types. Mammalian red blood cells and renal proximal tubules are extremely permeable to water molecules. Transmembrane water movements are involved in diverse physiological secretion processes. How water passes through cells has begun to become clear only in the last few years. Water permeates living membranes through both the lipid bilayer and specific water transport proteins. In both cases water flow is passive and directed by osmosis. Water transport in living cells is therefore under the control of ATP (adenosine triphosphate) and ion pumps. The most general water transport mechanism is diffusion through lipid bilayers, with a permeability coefficient of 2 to 5 × 104 cm/sec. The diffusion through lipid bilayers depends on lipid structure and the presence of sterol (Subczyński et al.,
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1994). It is suggested that the lateral diffusion of the lipid molecules and the water diffusion through the membrane is a single process (Haines, 1994). A small amount of water is transported through certain membrane transport proteins, such as a glucose transporter or the anion channel of erythrocytes (aquapores). The major volume of water passes through water transport proteins. The first isolated water transporting protein was the channel-forming integral protein from red blood cells. The identification of this protein has led to the recognition of a family of related water-selective channels, the aquaporins, which are found in animals, plants, and microbial organisms. In addition to water, they permeate some other small molecules. The pore is formed by six membrane-spanning helices. In the membrane they aggregate into tetramers, but each monomer acts as a separate water channel. Water flow through the protein channel is controlled by the number of protein copies in the membrane. In red blood cells, there are 200,000 copies/cell that account for up to 90% of the water permeability of the membrane; in apical brush border cells of renal tubules, it constitutes 4% of the total protein (Engel et al., 1994). It is assumed that another important function of aquaporins is the detection of osmotic and turgor pressure gradients (Hill et al., 2004).
3.3 WATER IN FOOD 3.3.1 INTRODUCTION Water, with a density of 1000 kg ⋅ m–3, is denser than the oil components of foods; oils and fats typically have densities in the range 850 to 950 kg ⋅ m–3. Glycerols and sugar solutions are denser than water. Unlike solid phases of most other liquids, ice is less dense than liquid water, and ice has a lower thermal conductivity than water. These properties have an effect on the freezing of foods that are predominantly water based; the formation of an ice layer on the surface of the liquids and the outside of solids has the effect of slowing down the freezing rate. Because a molecule of water vapor is lighter (molecular weight = 18) than that of dry air (molecular weight about 29), moist air is lighter than dry air at the same temperature. This is somewhat unexpected in that the popular conception is that humid air (which contains more water) is heavier than dry air. At room temperature, water has the highest specific heat of any inorganic or organic compound with the sole exception of ammonia. It is interesting to speculate why the most commonly occurring substance on this planet should have one of the highest specific heats. One of the consequences of this peculiarity in the food industry is that heating and cooling operations for essentially water-based foods are more energy demanding. To heat a kilogram of water from 20 to 50°C requires about 125 kJ of energy, whereas heating the same mass of vegetable oil requires only 44 kJ. A sponge holds most of its water as liquid in the interstices of the sponge structure. Most of the water can be wrung out of the sponge, leaving a matrix of air
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TABLE 3.1 Classification of Water States in Foods
Class of water
Description
Constitutional Vicinal
An integral part of nonaqueous constituent Bound water that strongly acts with specific hydrophilic sites of nonaqueous constituents to form a monolayer coverage; Water–ion and water–dipole bonds. Bound water that forms several additional layers around hydrophilic groups; water–water and water–solute hydrogen bonds Flow is unimpeded; properties close to dilute salt solutions; water–water bonds predominate. Free water held within matrix or gel, which impedes flow
Multilayer
Free Entrapped
Proportion of typical 90% (wet basis) moisture content in food <0.03% 0.1 to 0.9%
1 to 5%
5% to about 96% 5% to about 96%
Source: After Fennema, O.R., Food Chemistry, 2nd ed., Marcel Dekker, Inc., New York, 1985.
and damp fibers. Within the sponge fibers the residual water is more strongly held— absorbed within the fiber of the sponge. If the sponge is left to dry in the sun, this adsorbed water will evaporate leaving only a small proportion of water bound chemically to the salts and to the cellulose of the sponge fibers. As with the familiar example of water in a sponge, water is held in food by various physical and chemical mechanisms (Table 3.1). It is a convenient oversimplification to distinguish between free and bound water. The definition of bound water in such a classification poses problems. Fennema (1996) reports seven different definitions of bound water. Some of these definitions are based on the freezability of the bound component and others rely on its availability as a solvent. He prefers a definition in which bound water is that which exists in the vicinity of solutes and other nonaqueous constituents and exhibits properties that are significantly altered from those of “bulk water” in the same system. Moisture content can be measured simply by weighing a sample, then oven drying it, usually at 105°C overnight—the difference in mass being the moisture in the original sample. However, much confusion is caused by reporting the moisture content simply as a percentage without specifying the basis of the calculation. It should be made clear whether the moisture content is calculated on a wet basis (moisture content divided by original mass) or on a dry basis (moisture content divided by the bone dry or oven dry mass). Even the term bone dry mass can cause confusion among non-English speakers; it was once misinterpreted as the mass of the dry bones! In foods containing significant quantities of fat or salt, for example, moisture content may be calculated as the mass of water in a sample divided by the dry solids that are not salt or fat, in which case the moisture content should be reported as salt free, fat free, dry basis.
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3.3.2 SORPTION ISOTHERMS
AND
WATER ACTIVITY
3.3.2.1 Principle Since 1929 it has been recognized that the chemical and microbial stability, and hence the shelf life of foods, is not directly related to its moisture content, but to a property called water activity (Tomkins, 1929). Essentially, water activity is the measure of the degree to which water is bound within the food, and hence is unavailable for further chemical or microbial activity. Water activity is defined as the ratio of the partial pressure of water vapor in or around the food to that of pure water at the same temperature. Relative humidity of moist air is defined in the same way except that by convention, relative humidity is reported as a percentage whereas water activity is expressed as a fraction. Thus if a sample of meat sausage is sealed within an airtight container, the humidity of the air in the head space will rise and eventually equilibrate to a relative humidity of, say, 83%, which means that the water activity (aw) of the meat sausage is 0.83. The relationship between water activity and moisture content for most foods at a particular temperature is sigmoid-shaped in a curve called the sorption isotherm (Figure 3.10). The phrase equilibrium moisture content curve is also used. Sorption isotherms at different temperatures can be calculated using the Clausius–Clapeyron equation from classical thermodynamics, namely, d (ln aw ) ∆H = d (1 / T ) R where: T is the absolute temperature ∆H is the heat of sorption R is the gas constant A complication arises from one of the methods of measuring sorption isotherms for a food. A food that has previously been dried and then is rehydrated will have a different sorption isotherm (adsorption isotherm) from that which is in the process of drying (desorption isotherm). This difference is due to a change in the waterbinding capacity in foods that have been previously dried. Many mathematical descriptors for sorption isotherms have been proposed. One of the more famous is that of Brunauer, Emmett, and Teller (1938) (the BET isotherm), which is based on the concept of a measurable amount of monomolecular layer (vicinal) water for a particular food. Wolf, Speiss, and Jung (1985) compiled 2201 references to sorption isotherm data for foods. An example of the type, detail, and accuracy of sorption isotherm data available in the literature is presented in Table 3.2.
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Moisture content (dry basis)
4.0
3.0
2.0
Desorption
Adsorption 1.0
0.2
0.4 0.6 Water activity
0.8
1.0
FIGURE 3.10 A typical sorption isotherm for a food.
TABLE 3.2 Sorption Isotherm Data for Cod and Corn Product
Specifications
Coda Cornb
Adsorption Desorption Desorption Desorption Desorption Desorption Desorption
Temperature (°C)
Xm
r
a′
30 4.5 15.5 30 38 50 60
7.68 8.30 7.68 7.30 6.35 6.89 5.11
1.2398 2.2345 2.4862 2.5663 2.3711 2.1203 2.2185
1.3490 1.9748 2.0949 1.7950 1.8618 1.5936 1.7430
a
Adsorption, after Jason, AC., A study of evaporation and diffusion processes in the drying of fish muscle, in Fundamental Aspects of the Dehydration of Foodstuffs, Soc. Chem. Ind., London, 1958, p. 103. b Desorption, after Chen, C.S. and Clayton, J.T., Trans. A.S.A.E., 14, 927, 1971. Source: From Iglesias, H.A, Chirife, J, and Lombardi, J.L., J. Food Technol., 10, 289, 1975.
Iglesias et al. (1975) propose the following three-parameter equation to fit sorption isotherm data for a range of foods: aw = exp(a′ θr)
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Chemical and Functional Properties of Food Components
where: a′ and r = the parameters as listed in Table 3.2 θ = X/Xm X = the equilibrium moisture content Xm, in units of g/100 g dry basis = the BET monomolecular moisture content for the particular food as listed in Table 3.2 However, there are nearly as many equations for sorption isotherms as there are researchers in this field. 3.3.2.2 Measurement of Water Activity Many methods of measuring water activity have been developed. These include direct vapor pressure measurement, equilibration with a stable hygroscopic substance that has a known sorption behavior, and various types of hygrometers (Doe, 1998). Water activity is most conveniently measured by the measurement of relative humidity in the head space over a food sample in a sealed container. Commercially available instruments for water activity determination use various methods for measuring the relative humidity: hair hygrometer (Lluft), electrical hygrometer (Nova Sina), and dewpoint temperature (Aqualab). The hygrometer-based instruments are prone to drift, and must be calibrated regularly against saturated solutions of various inorganic salts. Hygrometer-based instruments are also prone to hysteresis at high humidities. The Aqualab CX2 water activity meter (Decagon Devices Inc., Pullman, WA) detects water condensation on a chilled mirror (dewpoint temperature). The instrument is sensitive to less than 0.001 water activity units. Readings take 5 minutes or less and are accurate to ± 0.003 water activity units. Care must be taken with any measurement of water activity to ensure that the sample is representative of the food being tested. Dried fish, for example, will have moisture and salt content, and hence water activity, varying widely from thin, exposed flesh to the relatively moist interior. If the worst-case scenario for the growth of potentially toxic or spoilage organisms is of interest, the sample of flesh for water activity determination should be excised from the thickest, most moist region of the fish. 3.3.2.3 Water Activity and Shelf Life of Foods Many of the chemical and biological processes that cause deterioration of foods, and ultimately spoilage, are water dependent. Microbial growth is directly linked to water activity. No microbes can multiply at a water activity below 0.6. Dehydration is arguably the oldest form of food preservation; the sun drying of meat and fish has been traced to the beginning of recorded history. Drying relies on removing water, thus making it unavailable for microbial growth. Salting or curing has the same effect. A saturated solution of common salt has a water activity of close to 0.75. Thus by adding sufficient salt to foods, the water
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activity can be lowered to a level where most pathogenic bacteria are inactivated, but the moisture content remains high. Intermediate moisture content foods (IMF), such as pet food and continental sausages, rely on fats and water-binding humectants such as glycerol to lower water activity. Fat, being essentially hydrophobic, does not bind water, but acts as a filler for IMF to increase the volume of the product. The effect of several humectants is for each to sequester an amount of water independently of the other humectants that may be present in the food. Each thus lowers the water activity of the system according to the equation in Ross (1975): awn = aw0 ⋅ aw1 ⋅ aw2 ⋅ aw3 ⋅ etc where: awn = the water activity of the complex food system aw0, etc. = the water activities associated with each component of the system For example, the water activity of a food with a moisture content of 77% (wet basis) and a salt content of 3% (wet basis) can be calculated as follows: 100 g of the food comprises 77 g of water, 20 g of bone dry matter, and 3 g of salt. The contribution to the water activity due to the salt can be calculated (according to Raoult’s law of dilute solutions) and the molecular weights of water (18) and salt (58.5) as aw1 = (77 × 18) / (77 × 18 + 3 × 58.5) = 0.89 The water activity for the salt-free solid matter of the food is found from its sorption isotherm at that moisture content, aw0 = 0.90, say. Thus the water activity of the salted food is awn = aw0 ⋅ aw1 = 0.9 × 0.89 = 0.8 None of the dangerous pathogenic bacteria associated with food, such as Clostridium or Vibrio spp. which cause botulism and cholera, can multiply at water activity values below about 0.9. Thus, drying or providing sufficient water-binding humectants is an effective method of preventing the growth of food-poisoning bacteria. Only osmophilic yeasts and some molds can grow at water activities in the range 0.6 to 0.65. Thus, by reducing the water activity below these values, foods are microbially stable. That is, unless the packaging is such that the food becomes locally rewet, in which case local spoilage can occur, for example, when condensation occurs within a hermetically sealed package subject to rapid cooling. There are various chemical reactions that proceed, and may be accelerated, at low values of water activity. Maillard reactions leading to lysine loss and brown color development peaks at aw values around 0.5 to 0.8. Nonenzymatic lipid oxidation
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increases rapidly below aw = 0.4. Enzymic hydrolysis decreases with water activity down to aw = 0.3 and is then negligible.
3.3.3 BOTTLED WATER 3.3.3.1 Classification Water in glass or plastic bottles or cartons is becoming increasingly popular, not only in areas where the tap water supply quality may be substandard, but also where drinking bottled water is seen as culturally accepted behavior. Health benefits are claimed for some products. Marketing of bottled water has been so successful that consumers spend from 240 to over 10,000 times more per liter for bottled water than for tap water. Tap water in some municipalities may contain hazardous chemicals and microorganisms; it may also contain additives such as fluoride to provide perceived health benefits to communities. Bottled water is required by law to meet standards of chemical and biological safety. In some respects, standards for bottled water are more stringent than those for tap water because of the shelf life requirement for bottled water. There are basically three types of bottled water: 1. Natural mineral water must come from an underground source. It receives no treatment other than filtration or carbonation, and is bottled at the source. Natural mineral water must meet tight microbiological standards and be regularly tested. 2. Spring water may come from other sources. It must also be bottled at the source and meet microbiological and chemical standards. Permitted treatments are filtration and carbonation; however, spring water does not have to be stable in composition. 3. Table water is basically any water in a bottle. It could come from an underground source, but might be tap water. Table water may or may not be treated. It could have additives to change its flavor or chemical composition. 3.3.3.2 Natural Mineral Water The Codex Alimentarius Commission (CAC) has published a Standard for Natural Mineral Waters (CAC, 1981). This provides guidance for industry and regulating bodies that may choose to follow this standard or make parts of it mandatory by law. The standard defines natural mineral water as being clearly distinguishable from ordinary drinking water because it is: • •
Characterized by its content of certain mineral salts and their relative proportions and the presence of trace elements or of other constituents Obtained directly from natural or drilled sources from underground waterbearing strata for which all possible precautions should be taken within the protective perimeters to avoid any pollution of, or external influence on, the chemical and physical qualities of natural mineral water
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• • • •
51
Consistent in composition and stable in its discharge and its temperature, due account being taken of the cycles of minor natural influences Collected under conditions that guarantee the original microbiological purity and chemical composition of essential components Packaged close to the point of emergence with particular hygiene precautions Not subjected to any treatment other than those permitted by this standard
The standard also defines naturally carbonated natural mineral water, noncarbonated natural mineral water, decarbonated natural mineral water, natural mineral water fortified with carbon dioxide from the source, and carbonated natural mineral water. The standard lists permissible levels for a number of chemical substances as listed in Table 3.3. The standard requires that natural mineral water shall not pose a microbiological risk to the consumer. Escherichia coli or thermotolerant coliforms must not be present in a 250-mL sample. If no more than two total coliform bacteria, fecal streptococci, Pseudomonas aeruginosa, or sulfide-reducing anaerobes are detected in a 250-mL sample, a second examination must be carried out; if more than two are detected the batch is rejected. TABLE 3.3 Permissible Levels for Chemicals in Natural Mineral Water Chemical
Permissible level (mg/dm3 unless otherwise specified)
Antimony Arsenic Barium Borate Cadmium Chromium Copper Cyanide Fluoride Lead Manganese Mercury Nickel Nitrate Nitrite Selenium
0.05 0.01 calculated as total As 0.7 5 calculated as B 0.003 0.05 calculated as Cr 1 0.07 (See note 1 below) 0.01 0.5 0.001 0.02 50 calculated as nitrate 0.02 as nitrite (see note 2 below) 0.01
Note 1: If more than 1 mg/dm3 fluoride the product must be labeled: contains fluoride. If more than 2 mg/dm3, product must be labeled: The product is not suitable for infants and children under the age of seven years. Note 2: Set as a quality limit (except for infants). Source: From CAC (Codex Alimentarius Commission), Codex Standard for Natural Mineral Water, Codex Standard 108-1981, Food and Agriculture Organization of the United Nations (FAO), Rome, Rev. 1, 1997.
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Natural mineral water containers must be hermetically sealed and safe from possible adulteration or contamination. The label must identify the product as natural mineral water and must declare the chemical composition. No claims of medicinal effects may be shown, and any health benefit claims must be true and not misleading.
3.3.4 BOTTLED WATER OTHER
THAN
NATURAL MINERAL WATER
3.3.4.1 Definition There is a Codex standard for packaged water for drinking purposes other than natural mineral water (CAC, 2001). Such water may contain minerals and carbon dioxide naturally occurring or intentionally added, but may not contain sugars, sweeteners, flavorings, or other foodstuffs. No packaged water may contain substances that emit radioactivity in quantities that may be injurious to health. All packaged water shall comply with the Guidelines for Drinking Water Quality published by the World Health Organization (WHO, 2004). Addition of minerals must comply with the relevant Codex standards. The standard distinguishes between “waters defined by origin,” which originate from a specific underground or surface resource and do not pass through a community water system, and “prepared waters,” which may originate from any supply. Prepared waters can be subjected to antimicrobial treatments, which modify the physical and chemical properties of the original water, provided that such treatment satisfies the WHO Guidelines for Drinking Water Quality. 3.3.4.2 Water Defined by Origin Water defined by origin is limited in treatment prior to packaging to: • • •
•
• •
•
Reduction and/or elimination of dissolved gases (and resulting possible changes in pH) Addition of carbon dioxide (and resulting change in pH) or reincorporation of the original carbon dioxide present at emergence Reduction and or elimination of unstable compounds such as iron, manganese, sulfur (as So or S--) compounds and carbonates in excess, under normal conditions of temperature and pressure, of the calcocarbonate equilibrium Addition of air, oxygen, or ozone on condition that the concentration of by-products resulting from ozone treatment is below the tolerance established under the health limits for chemical and radiological substances Decrease and/or increase in temperature Reduction and/or separation of elements originally present in excess of maximum concentrations or of maximum levels of radioactivity set according to the health limits for chemical and radiological substances Antimicrobial treatments may be used to conserve the original microbial fitness for human consumption
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3.3.4.3 Hygiene, Labeling, and Health Benefits The standard recommends that packaged waters comply with the Recommended International Code of Practice—General Principles of Food Hygiene (CAC/RCP 1999), also the Code of Hygienic Practice for Bottled/Packaged Drinking Waters (Other than Natural Mineral Waters) (CAC/RCP 2001). There are requirements in the standard for approval and inspection of the source for waters defined by origin. Labeling must comply with the Codex General Standard for the Labelling of Prepackaged Foods (CODEX STAN 1-1985, Rev 1-1991) together with any additional provisions in national legislation. Where appropriate in the case of waters defined by origin, labeling may specify “naturally carbonated” or “naturally sparkling” or “fortified with carbon dioxide.” For all waters, “carbonated” or “sparkling” may be used if the carbon dioxide does not come from the same source. If there is no visible release of carbon dioxide, the words noncarbonated, nonsparkling, or still may be used. The total dissolved content of packaged waters may be shown, and in the case of waters defined by origin the chemical composition that confers the characteristics of the product may be shown. Where required by local laws, the precise location of the source of a water defined by origin must appear on the label. Water bottled from a tap water distribution system that has not undergone further treatment (e.g., the addition of carbon dioxide or fluoride) must bear the words “from a public or private distribution system.” The standard prohibits any claims for medicinal (preventive, alleviative, or curative) effects. Claims for other health benefits may only appear if true and not misleading. Any place name may not form part of the trade name unless it refers to water defined by origin collected from that place. The chemical and microbiological safeguards placed on bottled water make it a safer alternative to tap water in places where community water supplies may be substandard or compromised by drought or flood. The mineral content of bottled water may be an important and necessary supplement to dietary mineral intake. For a very exhaustive presentation of the contents of mineral components in different food sources and their role in nutrition, see Chapter 4.
3.3.5 WATER SUPPLY, QUALITY,
AND
DISPOSAL
3.3.5.1 Water Supply Just as water is an integral part of any food, the supply, quality, and disposal of water is of prime consideration in the establishment and operation of all food processing. Potable (drinkable) water may be required for addition to the product, and will certainly be necessary for cleanup. Nonpotable water may be required for heat exchangers and cooling towers. Boiler feed water must be conditioned within
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close limits of pH and hardness. Brennan et al., in their book Food Engineering Operations (1990), list four types of water used in the food and beverage industries: 1. 2. 3. 4.
General-purpose water Process water Cooling water Boiler feed water
The site and consequent viability of a food processing plant may well depend on a guaranteed, regular supply of suitable quality water and an environmentally acceptable method of disposal. Developed countries now have strict regulations for the emission of wastewater. Developing countries are becoming increasingly aware of the problems of wastewater disposal. In a recent symposium in Indonesia, a fishdrying processor was asked what his main technical problems were. He cited water pollution—not for reasons of meeting environmental control regulations, but because the fish farmers further down the river were complaining about his wastewater. 3.3.5.2 Water Quality: Standards and Treatment There are a number of international standards for potable (drinkable) water quality in existence. The World Health Organization (WHO) has a standard for potable water quality as part of the Codex Alimentarius. The standard detailed in Table 3.4 is from the United States Environmental Protection Agency. There is also a large EC directive relating to the quality of water intended for human consumption (80/778/EEC), which is contained in the Joint Circular from the Department of the Environment, Circular 20/82, London, and the Welsh Office Circular 33/82, Cardiff, issued on August 19, 1982. In most cases, water will require some treatment to assure it is of the required standard to meet food hygiene requirements and not constitute a public health hazard. Surface water from rain runoff into rivers or impoundments is likely to contain atmospheric solutes, minerals from the ground, organic matter from vegetation, microbial contamination from birds and wild and domestic animals, and human waste. Water from underground aquifers will have much of the surface contamination filtered out, but is likely to be high in dissolved mineral content. Water treatment to bring the quality to within the required standard may involve screening, sedimentation, coagulation and flocculation, filtration, and other physical or chemical treatments to remove microorganisms, organic matter, or dissolved minerals. Metal screens are used to remove particles larger than about 1 mm in size. Settling ponds remove smaller particles. Insoluble, suspended matter is usually removed by sand filters. Coagulating and flocculating agents act to bind smaller particles into clumps, which then settle or can be screened or filtered. Microorganisms can be inactivated by heat, chemical disinfection, UV radiation, or ultrasonic treatment. Most town water supplies are chlorinated or have ozone added for chemical disinfection.
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TABLE 3.4 Primary Maximum Contaminant Levels (mg/dm3 unless specified) Contaminant Arsenic Barium Cadmium Chromium Lead Mercury Nitrate (as N) Selenium Silver Fluoride Endrin Lindane Methoxychlor Toxaphene 2.4 D 2,4,5 TP Silvex Total trihalomethanes Trichloroethylene Carbon tetrachloride 1,2 dichloroethane Vinyl chloride Benzene Para-dichlorobenzene 1,1,dichloroethylene 1,1,1,trichloroethane Radium 226 and 228, combined Gross alpha particle activity Gross beta particle activity Turbidity Coliform bacteria
Level (mg/dm3 unless specified) 0.05 1 0.010 0.05 0.05 0.002 10 0.01 0.05 4.0 0.0002 0.004 0.1 0.005 0.1 0.01 0.10 0.005 0.005 0.005 0.002 0.005 0.075 0.007 0.2 5 pC/l 15 pC/l 4 millirem/year 1 tu up to 5 tu 1 per 100 ml, monthly average
Source: From the U.S. Environmental Protection Agency (EPA), The Safe Drinking Water Act, Program summary, U.S. Environmental Protection Agency, Washington, DC, October 1996.
Treatment to remove dissolved mineral matter is more complex. Dissolved bicarbonates of calcium, magnesium, sodium, and potassium cause alkalinity; soluble calcium and magnesium salts cause hardness. Alkalinity and hardness may need to be adjusted for some food processing operations. For example, the formation of a head on beer is critically dependent on water hardness. Excessively hard water may cause discoloration and toughening of certain foods. On the other hand, hardness may be required to prevent excessive foaming in cleanup operations. Iron and manganese salts may be present in water supplies forming organic slimes, which tend to clog pipes. Aeration, filtering, and settling are effective for
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the removal of iron bicarbonates. Insoluble oxides of manganese are formed through chlorination. Excessive amounts of dissolved gases, carbon dioxide, oxygen, nitrogen, and hydrogen sulfide cause problems in boiler feed water, corrosion, and bacterial formation. Treatment is by boiling and venting off the noncondensable gases, or by chemical dosing. Small amounts of hydrocarbons, such as kerosene and diesel, cause tainting in foods. Separating fuels from processing areas, personal hygiene, cleaning stations around food processing operations, and good housekeeping can prevent this problem.
3.3.6 WATER POLLUTION Polluted water is described as polluted if it poses a risk to the health of humans, fish, or other animals. Microbiologically polluted water contains bacteria and other microorganisms that may be hazardous or toxic. Human and animal wastes from sewage and farmyard runoff are the principal sources of microbiological pollution. Polluted water can be an indirect hazard as fish and shellfish may become contaminated and eaten. Water can be polluted by organic and inorganic chemicals. Domestic and industrial pesticides are a major source of chemical pollution, as are detergents. Many of these substances are slow to degrade and may be concentrated in the food chain with disastrous consequences for fish and bird life. Biodegradable substances tend to be oxygen depleting resulting in a reduction of aerobic bacteria and fish. Food waste is high in biochemical oxygen demand (BOD). Food wastes contain large quantities of organic matter, which breaks down naturally by oxidation; however, this oxygen demand is at the expense of other natural biochemical processes in waterways, which become oxygen depleted and lifeless if the BOD is too high. BOD is defined as the quantity of oxygen (in units of mg/dm3) required for microorganisms to oxidize the waste at a particular temperature (20°C) in 5 days. Food wastes can range in BOD from 500 to 4000 mg/dm3, which is higher than for domestic sewage (200 to 400 mg/dm3). An excess of nutrients, such as phosphorous and nitrogen in polluted water, will lead to an excessive growth of plant matter in waterways, and algal blooms. Besides clogging waterways and adding toxins, this extra plant material contributes to the BOD of the water. Suspended matter, even if chemically and biologically inert, can contribute to pollution. These particles will eventually settle out and cause silting, and an anaerobic environment at the bottom of waterway. Water pollution is most effectively prevented by removing the pollutants before they get into the waterways. This can be accomplished through good housekeeping practices, such as avoiding the discharge of fatty material and detergents into the domestic sewerage system, proper design of landfill areas, pretreatment of industrial wastes, separation of storm water from sewage, so as not to overload treatment plants.
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3.3.7 WASTEWATER TREATMENT
57 AND
DISPOSAL
The ultimate aim of any food processing operation is to have an environmentally neutral impact. Reuse, recycle, and sustainability are today’s catchwords. For a food operation to be truly environmentally sustainable it should recycle all water not incorporated in the product or vented to the atmosphere. The reality is that it is currently considered uneconomic to recycle wastewater from food processing operations. Current practice is to treat wastewater to limit its effect on receiving waters. Treatment of wastewater mirrors the water treatment methods described above, that is, a combination of physical, chemical, and biological unit operations and processes. A physical operation treats suspended rather than dissolved pollutants. The pollutants may be simply allowed to settle out or float to the top naturally, or the treatment may be aided mechanically, which will cause smaller particles to stick together, forming larger particles that will settle or rise faster—a phenomenon known as flocculation. Chemical flocculants may also be added to produce larger particles. Filtration through a medium such as sand as a final treatment stage can result in very clear water. Ultrafiltration, nanofiltration, and reverse osmosis force water through membranes and can remove colloidal material and even some dissolved matter. Absorption (adsorption, technically) on activated charcoal is a physical operation that can remove dissolved chemicals. Air or steam stripping can be used to remove pollutants that are gasses or low-boiling liquids from water, and the vapors that are removed in this way are also often passed through beds of activated charcoal to prevent air pollution. These last processes are used mostly in industrial treatment plants, though activated charcoal is common in municipal plants for odor control. Wastewater from food processing operations usually contains significant solid matter, which can be removed by physical operations. Fats and oils can be skimmed from the surface of settling tanks, and heavier suspended matter can be removed as sludge, which can then be dewatered, dried, and used as animal feed, fertilizer or fuel. An alternative method for the removal of oils and fats is by aeration, in which air bubbles blown from the bottom of a settling tank carry fine solids and grease to the surface. Chemical treatments of wastewater are much the same as described above for processed water. BOD can be effectively reduced by biological processes. Both aerobic and anaerobic fermentation of the organic material is used. Depending on the scale of the operation, bioreactors range in size from 7.5 m in diameter and 2 to 3 m in depth to lagoons 1 to 2 m deep covering several hectares. However, for long-term sustainable operation there must be provision for sludge removal. Where the area for treatment is not a limitation, and there is sufficient isolation for smell not to be a deterrent, wastewater is sprayed directly on the ground where it breaks down under the action of sunlight and in-ground bacteria. A typical municipal treatment plant begins with a preliminary stage in which large or hard solids are removed or crushed. The effluent then passes through a primary settling basin in which organic suspended matter will either settle out or
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float to the surface to be skimmed off. The next part of the process is usually referred to as secondary treatment wherein the remaining dissolved or colloidal organic matter is removed by aerobic biodegradation. This promotes the formation of less offensive, oxidized products. The treatment unit should be sufficiently large to remove enough of the pollutants to prevent significant oxygen demand in the receiving water after discharge. Sewage and wastewater can also be treated anaerobically. Closed reactors facilitate odor control, although anaerobic lagoons are also used. Such lagoons are deeper than for aerobic types with grease allowed to accumulate on the surface to control odor emission. Methane produced as an end product of the biochemical pathway can be used for heating the reactors in cold weather. A problem with the anaerobic digestion process is its sensitivity to pH and temperature variation, and the susceptibility of the active microorganisms to chemical disinfectants.
REFERENCES Bjerrum, N., Structure and properties of ice, Science, 115, 385, 1952. Brennan, J.G. et al., Food Engineering Operations, 3rd ed., Elsevier Applied Science, London, 1990. Brunauer, S., Emmet, P.H., and Teller, E., Adsorption of gases in multilayers, J. Am. Chem. Soc., 60, 309, 1938. CAC (Codex Alimentarius Commission), Codex Standard for Natural Mineral Water, Codex Standard 108, 1981, Rev. 1, 1997. CAC (Codex Alimentarius Commission), Code of Hygienic Practise for Bottled/Packed Drinking Water (Other than Natural Mineral Waters) Codex Standard 227, 2001. CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice) General Principles of Food Hygiene. FAO, Rome, 1-1969 Rev. 3 (1997). Amended 1999. CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice) Code of Hygienic Practice for Bottled/Packaged Drinking Waters (other than Natural Mineral Waters). FAO, Rome, 48-2001. Calabrese, E.J., Gilbert, C.E., and Pastides, H., Eds., Safe Drinking Water Act: Amendments, Regulations, and Standards, Lewis Publishers, Chelsea, MI, 1989. Chen, C.S. and Clayton, J.T., The effect of temperature on sorption isotherms of biological materials, Trans. A.S.A.E., 14, 927, 1971. Chinachoti, P., Water mobility and its relation to functionality of sucrose-containing food systems, Food Technol., 47, 134, 1993. CODEX STAN (Codex Standard) Codex General Standard for the Labelling of Packaged Foods. FAO, Rome, 1-1985 (Rev. 1-1991). Dauchez, M., Peticolas, W., Debelle, L., and Alix, A.J.P., Ab initio calculations of polyhedra liquid water, Food Chem., 82, 23, 2003. Doe, P.E., Ed., Fish Drying and Smoking Production and Quality, Technomic Publishing Co. Inc., Lancaster, PA, 1998, p. 22. Engel, A., Waltz, Th., and Agre, P., The aquaporin family of membrane water channels, Curr. Opin. Struct. Biol., 4, 545, 1994. Fennema, O.R., Water and ice, in Food Chemistry, 3rd ed., Fennema, O.R. (Ed.), Marcel Dekker, Inc., New York, 1996.
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Frank, H.S. and Quist, A.S., Pauling’s model and the thermodynamical properties of water, J. Chem. Phys., 34, 604, 1961. Frank, H.S. and Wen, W.Y., Structural aspects of ion-solvent interaction in aqueous solutions: A suggested picture of water structure, Disc. Faraday Soc., 24, 133, 1957. Giudice, E. et al., Water in biological systems, in Modern Bioelectrochemistry, Gutmann, F. and Keyzer, H., Eds., Plenum Press, New York, 1986. Haines, Th.H., Water transport across biological membranes, FEBS Lett., 346, 115, 1994. Hill, A.E., Sachar-Hill, B., and Sachar-Hill, Y., What are aquaporins for? J. Membrane Biol., 197, 1, 2004. Iglesias, H.A., Chirife, J., and Lombardi, J.L., An equation for correlating equilibrium moisture content in foods, J. Food Technol., 10, 289, 1975. Jason, A.C., A study of evaporation and diffusion processes in the drying of fish muscle, in Fundamental Aspects of the Dehydration of Foodstuffs, Soc. Chem. Ind., London, 1958, 103. Lehninger, A.L., Nelson, D.L, and Cox, M.M., Principles of Biochemistry, 2nd ed., Worth Publishers, Inc., New York, 1993. Lewicki, P., Water as the determinant of food engineering properties. A review, Journal of Food Engineering, 61, 483, 2004. Ling, G.N., A Physical Theory of the Living State, Ginn (Blaisdel), Boston, MA, 1962. Mathlouthi, M., Water content, water activity, water structure and stability of foodstuffs, Food Control, 12, 409, 2001. Mild, K. and Løvtrup, S., Movement and structure of water in animal cells. Ideas and experiments, Biochim Biophys. Acta, 822, 155, 1985. Nossal, R. and Lecar, H., Eds. Molecular and Cell Biophysics, Addison-Wesley Publishing Co., Reading, MA, 1991. Rahman, A. and Stillinger, F.H., Molecular dynamic study of liquid water, J. Chem. Phys., 55, 3336, 1971. Röntgen, W.K., Über die Konstitution des flüssigen Wassers, Ann. Physik, 45, 91, 1892. Ross, K.D., Estimation of water activity in intermediate moisture foods, Food Technol., 29, 26, 1975. Ruan, R.R. and Chen, P.L., Water in Foods and Biological Materials, Technomic Publishing Co. Inc., Lancaster, PA, 1998. Rückold, S., Isengard, H.-D., Hanss, J., and Grobecker, K., H., The energy of interaction between water and surfaces of biological reference materials, Food Chemistry, 82, 51, 2003. Starzak, M. and Mathlouthi, M., Cluster composition of liquid water derived from laserRaman spectra and molecular simulation data, Food Chemistry, 82, 3, 2003. Stillinger, F.H., Water revisited, Science, 209, 451, 1980. Subczyński, W.K. et al., Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol, Biochemistry, 33, 7670, 1994. Tomkins, R.G., Studies of the growth of moulds. 1., Proc. R. Soc. B, 105, 375, 1929. U.S. Environmental Protection Agency (EPA), The Safe Drinking Water Act, Program Summary, U.S. Environmental Protection Agency, Washington, DC, October 1987. Wallace, J.S. and Gregory, P.J., Water resources and their use in food production systems, Aquat. Sci., 64, 363, 2002. Wiggins, Ph.M., Role of water in some biological processes, Microbiol. Rev., 54, 432, 1990. Wiggins, Ph.M., Water in complex environments such as living systems, Physica A, 314, 485, 2002.
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Wolf, W., Spiess, W.E.L., and Jung, G., Sorption Isotherms and Water Activity of Food Materials, Science and Technology Publishers, Hornchurch, Essex, U.K., 1985. World Health Organization (WHO), Guidelines for Drinking Water Quality, 3rd ed. World Health Organization, Rome, 2004.
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4
Mineral Components Michał Nabrzyski
CONTENTS 4.1 4.2
The Contents and Role of Minerals Present in Foods .................................. 61 Interaction with Dietary Components ........................................................... 63 4.2.1 Introduction ........................................................................................ 63 4.2.2 Effect on Absorption.......................................................................... 63 4.2.3 Building Body Tissue and Regulating Body Processes .................... 67 4.3 Role in Food Processes.................................................................................. 67 4.3.1 Effect on Oxidation............................................................................ 67 4.3.2 Effect on Rheological Properties....................................................... 72 4.3.3 Other Effects ...................................................................................... 72 4.4 The Effect of Storage and Processing on the Mineral Components in Foods.......................................................................................................... 73 4.5 The Chemical Nature of Toxicity of Some Mineral Food Components ...... 74 4.5.1 Introduction ........................................................................................ 74 4.5.2 Aluminum........................................................................................... 76 4.5.3 Arsenic ............................................................................................... 79 4.5.4 Mercury .............................................................................................. 84 4.5.5 Cadmium ............................................................................................ 85 4.5.6 Lead.................................................................................................... 86 4.5.7 Interactions of Elements .................................................................... 88 References................................................................................................................ 90
4.1 THE CONTENTS AND ROLE OF MINERALS PRESENT IN FOODS Minerals represent from 0.2% to 0.3% of the total intake of all nutrients in the diet. They are so potent and so important that without them the organism would not be able to utilize all the other food components. The main mass of these minerals constitute the macroelements, and the trace elements constitute less than one-hundredth of one percent of the total mass of daily eaten nutrients. Foods that are good sources of some minerals are given in Table 4.1. Dietary minerals are necessary for maintenance of normal cellular metabolism and tissue function. These nutrients participate in a multitude of biochemical and physiological processes important for health. Because of their broad biochemical activity, many 61
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TABLE 4.1 The Contents of Selected Minerals in Some Foods Mineral
Range in foods (mg/100 g)
Calcium Potassium Magnesium Zinc Iron Copper Chromium
potato, 10; sardines in tomato sauce, 440; Swiss cheese, 960 cheese, >100; wheat meal, 1.30; beef, liver, >300; wheat seeds, >500 milk, 9; sardines in tomato sauce, 27 bee honey, fish, meat, 0.08–1.2; oysters, >100 egg white, 0.2; egg yolk, 7.2; pork, beef, >2; porcine liver, >20 ham, salmon, tuna meat, 0.03–0.5; wheat germ, 0.9–7; oysters, 6–17 kidney, liver, beef, <0.0015–0.004; cheese drowned, whole-meal bread, paprika, pepper, curry, 0.01–0.05; spices, >0.1–0.5 fish and its products, 0.03–0.8; black teas, 3–34 marine fish, oysters, shrimps, lobsters, 0.02–0.1; milk powder, 0.06 milk, 0.002; salmon and tuna, canned, 0.08–0.12; potato and tortilla chips, 1.0
Fluoride Iodide Selenium
of these compounds are intentionally used as functional agents in a variety of foods. On the other hand, some cations may also induce a diversity of undesirable effects that influence the nutritional quality of foods. Minerals play an important role in plant life. They function as catalysts of biochemical reactions, are responsible for changes in the state of cellular colloids, directly affect cell metabolism, and are involved in changes in protoplasm turgor and permeability. They often become the centers of electrical and radioactive phenomena in living organisms. Minerals are usually grouped in two categories: the macroelements required in human diets in amounts greater than 100 mg, and the microelements required in milligram quantities or less. The macroelements include Ca, Mg, P, Na, K, and Cl. The microelements are Fe, Zn, Cu, Mn, I, Co, Ni, Mo, Cr, F, Se, V, B, Si, and a few others whose biological functions have not yet been fully recognized. Actually, mineral deficiency states are more likely to occur than is vitamin insufficiency. At increased risk of mineral deficiencies are people who eat low-calorie diets, the elderly, pregnant women, people using certain drugs such as diuretics, vegetarians, and those living in areas where soils are deficient in certain minerals. There is increasing evidence that those humans whose nutritional status is suboptimal in certain trace elements, such as Se, may be at greater risk for some forms of cancer and heart disease. Suboptimal intake can be due to soil depletion, the effects of acid rain, the overrefining and overprocessing of foods, and other factors. Minerals occur in foods in many chemical forms. They are absorbed from the intestines as simple cations, as part of an anionic group, or in covalent or noncovalent associations with organic molecules. The chemical form of minerals in foods strongly influences their intestinal handling and biological availability. Thus, iron in the form of hemoglobin in meats is more bioavailable than inorganic iron. This may also be true for selenium in selenomethionine, and for the organic chelates of dietary chromium and zinc. Factors that affect mineral solubility or their reduction to a suitable form for cellular uptake, or those that influence the transfer through the mucosa or transport into circulation, govern the rate and efficiency of uptake of the minerals.
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For example, iron and zinc are much more bioavailable from human breast milk than from cow’s milk or comparable infant formula. The intrinsic molecular associations of these minerals with low-molecular-weight binding compounds in human mammary secretions are thought to convey this enhanced absorbability (Rosenberg and Solomons, 1984). Some minerals can produce chronic toxicity when absorbed and retained in excess of the body’s demands. Homeostatic mechanisms, often hormonally mediated, regulate the absorption of certain minerals and thereby protect against excessive accumulation. Recently developed speciation analysis makes it possible to determine the forms of minerals that are present in food and in the environment that may cause specific physiological or pharmacological effects in the human organism.
4.2 INTERACTION WITH DIETARY COMPONENTS 4.2.1 INTRODUCTION The interactions between minerals and between minerals and other substances in the diet number in the hundreds. The term interaction is used to describe interrelationships between minerals and other nutrients present in the diet, and may be defined as the effect of one element (nutrient) on one or more other elements, and thus may cause some positive or negative biochemical or physiological consequences in the organism. At the molecular level, such interactions occur at specific sites on proteins like enzymes, receptors, or ion channels.
4.2.2 EFFECT
ON
ABSORPTION
Various nutritional and nonnutritional components of the diet, other nutrients in vitamin and mineral supplements, contaminants, and also some medications can interact with minerals in the gastrointestinal tract and influence their absorption. For example, amino acids may perform as intraluminar binders for some trace minerals. Large, complex, and poorly digestible proteins, on the other hand, may bind minerals tightly and diminish their absorption. Triacylglycerols and long-chain fatty acids may form soaps with calcium and magnesium and decrease the bioavailability of these two nutrients. There are clear indications that iron deficiency promotes cadmium retention and may thus decrease the tolerance of high environmental or dietary cadmium levels. Evidence from a variety of experiments on animals suggests that iron deficiency also promotes lead uptake and retention; the evidence for humans is controversial, but in some studies substantial increases in lead uptake have occurred when dietary iron and iron status were low. Intestinal parasites, dietary fiber, phytates, and excessive sweating interfere with zinc absorption. Phytates, oxalates, and tannates can interfere with the absorption of a number of minerals. Certain medications such as tetracycline can also inhibit absorption of minerals, while others such as diiodoquin or dilantin may actually promote uptake of minerals. Apparently, chemically similar minerals share channels for absorption, and the simultaneous ingestion of two or more such minerals might result in competition for absorption.
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Minerals like Fe, Mn, Co, Cu, Cr, Ni, and Zn generally function in the organism as cations complexed with organic ligands or chelators, such as proteins, porphyrins, flavines, and pterins. These transition metals have incompletely filled the 3-dimensional orbitals (the exception is zinc). One of the characteristics of these metals is the ability to form complex ions. The electron configuration of metals at the valence shell, in the elemental as well as in the cationic state, are as listed below: Element Elemental state Divalent cationic state
Cr 3d5 4s1 3d4
Mn 3d5 4s2 3d5
Fe 3d6 4s2 3d6
Co 3d7 4s2 3d7
Ni 3d8 4s2 3d8
Cu 3d10 4s1 3d9
Zn 3d10 4s2 3d10
As can be seen from the above arrangement, copper exists in either the monovalent or divalent ion, and the sum of its biological importance is related to its ability to oscillate between the cuprous (I) or cupric (II) state, having the ability to accept or to donate electrons. Among them, the divalent zinc (3d10) is not strictly a transition metal because its orbital d is filled, but its atomic structure is very similar to the metals discussed, and thus it is ready to form complexes analogous to those formed by the transition metal ions. Some other divalent cations like cadmium Cd2+ (4d10), and lead Pb2+ (6d10 6s2), which are not transition metals, also have a pronounced tendency to form coordinate covalent bounds with ligands that contain the electron donor atoms like, such as N, O, and S, which are extensively found in proteins. There is a degree of selectivity of metal ions for electron donor atoms; copper prefers nitrogen, while zinc prefers sulfur, but several different donor atoms complex with each metal ion. The amino acid residues in proteins serve as rich sources of electron donor atoms; for example, the imidazole group of histidine supplies nitrogen, the carboxyl groups of aspartic and glutamic acids supply oxygen, and the thiol group of cysteine supplies sulfur for complexation. The interaction between a metal atom and the ligands can be thought of as a Lewis acid-base reaction. Ligands as a Lewis base are capable of donating one or more electron pairs, and a transition metal atom (either in its positively charged state) acts as a Lewis acid accepting or sharing a pair of electrons from the Lewis bases. Thus the metal-ligand bonds, sharing a pair of electrons, form coordinate covalent bond. Interesting data of competitive interaction in biological systems exist between the divalent cations such as zinc and cadmium, or zinc, copper, and iron. Zinc, cadmium, and mercury have similar tendencies to form complexes with a coordination number of 4 and a tetrahedral disposition of ligands around the metal. They have, as divalent cations, a very similar electronic structure to the monovalent cation of copper (3d10). The consequence of this is the possibility of isomorphous replacement among these elements in biological systems. The essential microelements prefer coordination numbers of 4 or 6, and they complex with four or six ligands. Copper and zinc in the cationic form have filled their orbitals (they are d10 ions) and both Cu1+ and Zn2+ form tetrahedral complexes (Cu1+ may lose one of its d orbital electrons and become a d9 ion (Cu2+), and thus form square planar complexes). On the other hand, the ferrous d6 ion (Fe2+) forms an octahedral configuration. It is possible to predict that Cu1+, having the same d orbital configuration as Zn2+ and Cd2+, will interact with both of them, and that Zn2+ will be antagonistic to Cu1+ rather than to Cu2+.
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It has been demonstrated in systematic studies of the interactions between Cd, Zn, Cu, and Fe, in chicks, rats, and mice, that an increased dietary intake of cadmium can cause increased mortality, poor growth, and hypochromic microcytic anemia. The growth rate could be restored by zinc supplementation, and the mortality and severity of anemia signs could be reduced by copper supplementation of the diet. Zinc also restores the activity of heart cytochrome oxidase. Cu prevents the degeneration of aortic elastin. Diets low in calcium promote significant increases in the absorption and retention of lead and cadmium in rat organisms, and have a marked effect on all pathologic changes ascribed to their toxicity. High calcium and high phytate in diets appear to restrict lead uptake. Low intake of phosphorus has a similar effect on Pb retention, like low intake of Ca, and the effects of Ca and P deficiency are additive (Bremner, 1974; WHO, 1996). Other dietary components such as lactose have been implicated in the enhanced absorption of calcium from milk. Pectins, cellulose, hemicellulose, and polymers produced by Maillard reactions during cooking, processing, or storage may bind minerals in the lumen and thus reduce their biological availability. Interaction between and among minerals, or with anionic species, are important determinants of mineral absorption. Absorption of iron is hindered by fiber and phosphates and promoted by ascorbic acid, copper, and meat protein. Ascorbic acid also enhances the absorption of selenium but reduces the absorption of copper. High protein intake appears to increase the excretion of calcium, whereas vitamin D ingestion promotes the retention of calcium. When imbalances that are not physiological among competitive nutrients exist as a result of leaching from water pipes, storage in unlacquered tin cans, or improper formulation of vitamin and mineral supplements, nutritionally important consequences of mineral–mineral interaction can result. To participate in a nutritionally relevant process for the organisms as a whole, a mineral must be transported away from the intestines. The concentration of circulating binding proteins, and the degree of saturation of their metallic binding sites, may influence the rate and magnitude of transport of recently absorbed minerals (Rosenberg and Solomons, 1984). Minerals require a suitable mucosal surface across which to enter the body. Resection or diversion of a large portion of the small bowel obviously affects mineral absorption. Extensive mucosal damage due to mesenteric infarction or inflammatory bowel disease or major diversion by jejunoileal bypass procedures reduces the available surface area. Minerals such as copper or iron, whose absorption primarily occurs in the proximal intestine, are affected differently from those absorbed more distally, e.g., zinc. Furthermore, the integrity of the epithelium, the uptake of fluids and electrolytes, the intracellular protein synthesis, energy-dependent pumps, and hormone receptors must be intact. Intrinsic diseases of the small intestinal mucosa may impair mineral absorption. Conditions such as celiac sprue, dermatitis herpetiformis, infiltrative lymphomas, and occasionally inflammatory bowel disease produce diffuse mucosal damage. Protein energy malnutrition causes similar damage, and tropical enteropathy affects part of the population of developing countries living under adverse nutritional and hygienic conditions. Absorption of most metals from the gastrointestinal tract is variable (see Table 4.2), and depends on many external and internal factors. Thus the quantity of metal ingested rarely reflects that which
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TABLE 4.2 Mean Daily Intake and Recommended Dietary Allowances (RDAs)1 or Safe and Adequate Intake (SAI)2 (as well as percentage of absorption of minerals from the gastrointestinal tract according to published data by U.S. National Academy Press, 1989). Milligrams per adult person Mineral
Mean daily intake
RDA1 or SAI2
Calcium Chloride Magnesium Phosphorus Potassium Sodium
960–1220 1700–5100 145–358 1670–2130 3300 3000–7000
Macroelements 800–12001 75022 280–3501 800–12001 20002 5002
Chromium
< 0.15
Microelements 0.05–0.202
Cobalt Copper Fluorine Iodine Iron Zinc Manganese Molybdenum Nickel Selenium Vanadium
Boron Silicon a b c
0.003–0.012 2.4 < 1.4 < 1.0 15 12; 18 5.6; 8 > 0.15 0.16–0.20 0.06–0.22 0.012–0.030
0.002 c1 1.5–3.02 1.5–42 0.151 10–151 12–151 2–32 0.075–0.2502 0.05; 0.3 0.055–0.070 0.01–0.025
Microelements recently considered as essential 1–3 1–2 21–46; 200 21–46
Percentage of absorption
10–50 higha 20–60 higha higha higha
< 1 or 10–25 in form of GTFb 30–50 25–60 higha 100 10–40 30–70 40 70–90 < 10 ~ 70 <1
higha 3; 40
More than 40% GTF (glucose tolerance factor) 0.002 mg of cobalt containing vitamin B12
is bioavailable. In fact, under most circumstances, only a small fraction of ingested metals are absorbed, while the great majority passes out of the gut in the feces. The Recommended Dietary Allowances (RDA) represent standards of nutrition set by the National Research Council, Food and Nutrition Board (1989). It contains the levels of essential nutrients that are adequate to meet the nutritional needs of the normal healthy population. Individuals may differ in their precise nutritional requirements. To take into account these differences among normal persons, the RDA provides a margin of safety, that is, it sets the allowances high enough to cover the
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needs of most healthy people. For additional nutrients that are necessary to keep the body in good health (for which the RDA has not yet been established) there are estimated safe and adequate daily intakes (SAI). The functions of minerals in the body involve building tissue and regulating numerous body processes. Their role in the human body is summarized in Table 4.3.
4.2.3 BUILDING BODY TISSUE AND REGULATING BODY PROCESSES Certain minerals, including calcium, phosphorus, magnesium, and fluorine, are components of bones and teeth. Deficiencies in children cause growth to be stunted and bone tissue to be of poor quality. A continual adequate intake of minerals is essential for the maintenance of skeletal tissue in adulthood. Potassium, sulfur, phosphorus, iron, and many other minerals are the structural components of soft tissue (Solomons, 1984; Eshleman, 1984). Minerals are an integral part of many hormones, enzymes, and other compounds that regulate biochemical functions in the human organism. For example, iodine is required to produce the hormone thyroxine, chromium is involved in the production of insulin, and iron is present in hemoglobin, myoglobin, and the cytochromes. Hence, the production of these substances in the organism depends on adequate intake of the involved minerals. Minerals can also act as catalysts. Calcium, for example, is a catalyst in blood clotting. Some minerals are catalysts in the absorption of nutrients from the gastrointestinal tract in the metabolism of proteins, fat, and carbohydrates, and in the utilization of nutrients by the cell. Minerals dissolved in the body fluids are responsible for nerve impulses and the contraction of muscles, as well as for water and acid-base balance. They play important roles in maintaining the respiration, heart rate, and blood pressure within normal limits. A deficiency of minerals in the diet may lead to severe, chronic, clinical signs of disease, frequently reversible after supplementation of the diet with the respective elements or following total parenteral nutrition. Their influence on biochemical reactions in living systems also makes it possible to use them intentionally in many food processes.
4.3 ROLE IN FOOD PROCESSES 4.3.1 EFFECT
ON
OXIDATION
Because minerals are an integral part of many enzymes, they also play an important role in food processing, for example, in alcoholic and lactic fermentation, meat aging, and in dairy food production. Many compounds used as food additives or for rheological modification of some foods, also contain metallic cations. A number of these compounds function as antimicrobials, sequestrants, antioxidants, flavor enhancers, and buffering agents, and sometimes even as dietary supplements. Some heavy metal ions actively catalyze lipid oxidation. Their presence, even in trace amounts, has long been recognized as potentially detrimental to the shelf life of fats, oils, and fatty foods. They can activate molecular oxygen by producing superoxide, which then, through dismutation and other biochemical changes, turns
Cobalt
Chromium
Sodium
Potassium
Phosphorus
Trivalent chromium increases glucose tolerance and plays role in lipid metabolism; useful in prevention and treatment of diabetes; hexavalent chromium is toxic Cofactor of vitamin B12 plays a role in immunity
Bone and tooth formation, blood clotting, cell permeability, nerve stimulation, muscle contraction, enzyme activation Component of bones and teeth, activation of many enzymes, nerve stimulation, muscle contraction Bone and tooth formation, energy metabolism, component of ATP and ADP, protein synthesis component of DNA and RNA, fat transport, acid–base balance, enzyme formation Osmotic pressure, water balance, acid–base balance, nerve stimulation, muscle contraction, synthesis of protein, glycogen formation Osmotic pressure, water balance, acid–base balance, nerve stimulation, muscle contraction, cell permeability
Calcium
Rarely observed; if exists, a pernicious anemia with hematological and neuralgic manifestations may be observed due to vitamin B12 deficiency
Microelements Impaired growth, glucose intolerance, elevated blood cholesterol
Rare: nausea, vomiting, giddiness, exhaustion, cramps
Nausea, vomiting, muscular weakness, rapid heart beat, heart failure
Seen in alcoholism or renal disease, tremors leading to convulsive seizures Stunted growth, rickets
Macroelements Stunted growth, rickets, osteomalacia, osteoporosis, tetany
Deficiency
Organ meats (liver, kidney), fish, dairy products, eggs
Whole-grain cereals, condiments, meat products, cheeses, and brewer’s yeast
Table salt, salted foods, MSG and other sodium additives, milk, meat, fish, poultry, eggs, bread
Meats, fish, poultry, whole grains, fruits, vegetables, legumes
Milk, hard cheese, salmon and small fish eaten with bones, some dark green vegetables, legumes Green leafy vegetables, nuts, whole grains, meat, milk, seafood Milk, meats, poultry, fish, eggs, cheese, nuts, legumes, whole grains
Sources
68
Magnesium
Function
Mineral
TABLE 4.3 The Biological Role of Some Minerals
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Cofactor of a large number of enzymes, in the aging process has a role as an antioxidant (Mn-superoxide dismutase), important for normal brain function, for reproduction, and for bone structure Cofactor of the enzymes: xantine and aldehyde oxidase. copper antagonist
Manganese
Recently considered essential; still has to be proven.
Cataract, muscular dystrophy, growth depression, liver cirrhosis, infertility, cancer, aging due to deficiency of selenoglutathione peroxidase; insufficiency of cellular immunity Probably impairs growth and development
Reduces conversion of hypoxanthine and xanthine to uric acid resulting in the development of xanthine renal calculi; deficiency state may be potentiated by high copper intake Delayed wound healing, impaired taste sensitivity, retarded growth and sexual development, dwarfism Tooth decay in young children Goiter, cretinism, if deficiency is severe.
Foods of plant origin and vegetables
Broccoli, mushrooms, radishes, cabbage, celery, onions, fish, organ meats
Drinking water rich in fluoride, seafood, tea Iodized salt, seafood, food grown near the sea
Oysters, fish, meat, liver, milk, whole grains, nuts, legumes
Grain, legumes
Liver, lean meats, legumes, dried fruits, green leafy vegetables, whole-grain and fortified cereals Tea, whole grains, nuts. Fruits and green vegetables; organ meat and shellfish contain very absorbable manganese
Anemia, decrease in oxygen transport and cellular immunity, muscle weakness In animals: chondrodystrophy, abnormal bone development, reproductive difficulties; in humans: shortage of evidence
Liver, kidney, oysters, nuts, fruits, and dried legumes
Anemia, neutropenia, leucopenia, skeletal demineralization
Mineral Components
Sources: Eschleman M.M., Introductory Nutrition and Diet Therapy, J.B. Lippincott Co., London, 1984; Hendler S.S., The Doctor’s Vitamin and Mineral Encyclopedia, Simon and Schuster, New York, 1990.
a
Borona
Prevents osteoporosis in postmenopausal women, beneficial in treatment of arthritis, builds muscle
Resistance to dental decay Synthesis of thyroid hormone that regulate basal metabolic rate Protects against a number of cancers
Fluoride Iodine
Selenium
Constituent of many enzyme systems, carbon dioxide transport; vitamin A utilization
Zinc
Molybdenum
Iron
Necessary for iron utilization and hemoglobin formation, constituent of cytochrome oxidase, and involved in bone and elastic tissue development Hemoglobin and myoglobin formation, essential component of many enzymes
Copper
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into hydroxyl free radicals. Three cations are involved in the activity of superoxide dismutase (SOD). This enzyme has been patented as an antioxidant agent for foods. Three types of metalloenzymes of SOD exist in living organisms, namely Cu ZnSOD, Fe-SOD, and Mn-SOD. All three types of SODs catalyze the dismutation of superoxide anions to produce hydrogen peroxide in vivo. There is evidence of increased lipid oxidation in apples during senescence. SOD activity may also be involved in reactions to damage induced by oxygen, free radicals, and ionizing radiation, and could help protect cells from damage by peroxidation products (Du and Bramlage, 1994). Besides SOD, catalase, ceruloplasmin, albumin and appotransferrin, and chelating agents ethylenediaminetetraacetic acid (EDTA), bathocupreine, cysteine, and purine are capable of inhibiting the oxidation of ascorbic acid induced by trace metals. Copperinduced lipid oxidation in ascorbic acid–pretreated cooked ground fish may be inhibited in the presence of natural polyphenolic compounds, the flavonoids, which are effective antioxidants and prevent the production of free radicals (Ramanathan and Das, 1993). In the presence of adenosine diphosphate-chelated (ADP) iron and traces of copper, oxygen radicals are generated in the sarcoplasmic reticulum of muscle food. Muscle contains notable amounts of iron, a known prooxidant, and trace amounts of copper also catalyzing a peroxidative reaction (Hultin, 1994; Wu and Brewer, 1994). Iron occurs associated with heme compounds and as nonheme iron complexed to proteins of low molecular weight. Reactive nonheme iron can be obtained through release of iron from heme pigments or from the iron storage protein, ferritin. Iron is part of the active site of lipoxygenase, which may participate in lipid oxidation. Reducing components of tissue such as superoxide anion, ascorbate, and thiols, can convert inactive ferric iron into active ferrous iron. There are also enzymic systems that use reducing equivalents from NADPH (nicotinamide adenine dinucleotide phosphate), to reduce ferric iron. A number of cellular components are capable of reducing ferric to ferrous iron, but under most conditions the two major reductants are superoxide and ascorbate (Hultin, 1994). In some cases the reduction of ferric iron can also be accomplished enzymically utilizing electrons from NADH (reduced nicotinamide adenine dinucleotide), and to a lesser extent, NADPH (reduced nicotinamide adenine dinucleotide phosphate) by the enzymic system associated with both the sarcoplasmic reticulum and mitochondria. Ferrous iron can activate molecular oxygen by producing superoxide. Superoxide may then undergo dismutation spontaneously or through the action of SOD, and produce hydrogen peroxide, which can interact with another atom of ferrous iron to produce the hydroxyl radical. The hydroxyl radical can initiate lipid oxidation. It is generally accepted that ferrous iron is the reactive form of iron in an oxidation reaction. Because it is likely that most iron ordinarily exists in the cell as ferric iron, the ability to reduce ferric to ferrous iron is thus critical. Development of rancidity and warmed-over flavor—a specific defect that occurs in cooked, reheated meat products following short-term refrigerated storage—has been directly linked to autooxidation of highly unsaturated, membrane-bound phospholipids and to the catalytic properties of nonheme iron (Oelinngrath and Slinde, 1988; Pearson et al., 1977; Hultin, 1994). Dietary iron may influence muscle iron stores and thus theoretically may also affect lipid oxidation in muscle food such as pork. There appears to be a threshold
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for the dietary iron level (between 130 to 210 ppm), above which the muscle and liver nonheme iron and total iron, and muscle thiobarbituric acid reactive substances begin to increase because of porcine muscle lipid oxidation (Miller et al., 1994a, b). The secondary oxidation products, mainly aldehydes, are the major contributors to warmed-over flavor and meat flavor deterioration because of their high reactivity and low flavor thresholds. Ketones and alcohols have high flavor thresholds and are lesser causes of flavor deterioration. Exogenous antioxidants can preserve the quality of meat products. Radical scavengers appear to be the most effective inhibitors of meat flavor deterioration. However, different substrates and systems respond in different ways. Active ferrous iron may be eliminated physically by chelation with EDTA or phosphates, or chemically by oxidation, to its inactive ferric form. Ferroxidases are enzymes that oxidize ferrous iron to ferric iron in the presence of oxygen: 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O Ceruloplasmin, a copper protein of blood serum, is a ferroxidase. Oxidation of ferrous iron to ferric iron tends to be favored in extracellular fluids, while chelation is more likely intracellular (Hultin, 1994). Sodium chloride has long been used for food preservation. Salt alters both the aroma and the taste of food. The addition of sodium chloride to blended cod muscle accelerates the development of rancidity. This salt-induced rancidity is inhibited by chelating agents such as EDTA, sodium oxalate, and sodium citrate, and by nordihydroguaiaretic acid and propyl gallate. Although sodium chloride and other metal salts act as prooxidants, they have a strong inhibiting effect on Cu2+-induced rancidity in the fish muscle. The most effective concentration of NaCl for this antioxidant effect is between 1% and 8% (Castell et al., 1965, Castell and Spears 1968). These authors also showed that other heavy metal ions were effective in producing rancidity when added to various fish muscles. The relative effectiveness was the following, in decreasing order: Fe2+ > V2+ > Cu2+ > Fe3+ > Cd2+ > Co2+ > Zn2+, while Ni2+, Ce2+, Cr3+, and Mn2+ had no effect in the concentrations used. Of those tested, Fe2+, V2+, and Cu2+ were by far the most active catalysts. There were, however, important exceptions. The comparative effectiveness of the metallic ions was not the same for muscle taken from all the species that were tested. EDTA is reported to be effective as a metal ion sequestrant and is approved for use in the food industry as a stabilizer and antioxidant. It acts also as an inhibitor of Staphylococcus aureus by forming stable chelates in the media with mutivalent cations, which are essential for cell growth. The effect is largely bacteriostatic and easily reversed by releasing the complexed cations with other cations for which EDTA has a higher affinity (Kraniak and Shelef, 1988). The addition of phosphate-, pyro-, tripoly-, and hexametaphosphate also protects cooked meat from autoxidation, but ortophosphate gives no protection. The mechanism by which phosphates prevent autooxidation appears to be related to their ability to sequester metal ions, particularly ferrous iron, which are the major proox-
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idants (Pearson et al., 1977). The addition of NaCl increases retention of moisture in meat and meat products.
4.3.2 EFFECT
ON
RHEOLOGICAL PROPERTIES
The interaction between metal ions and polysaccharides often affects the rheological and functional properties in food systems. In aqueous media, neutral polysaccharides have little affinity for alkali metal and alkali earth metal ions. On the other hand, anionic polysaccharides have a strong affinity for metal counterions. This association is related to the linear charge density of the polyanions. The linear charge density is expressed as the distance between the perpendicular projections of an adjacent charged group on the main axis of the molecule. The higher the linear charge density, the stronger the interaction of counterions with the anionic groups of the molecule. Such anionic hydrocolloids (0.1% solution) as alginate, karaya, arabic, and ghati have high calcium-binding affinities (Ha et al., 1989). An important functional property of alginates is their capacity to form gels with calcium ions. This makes alginates extensively suited to preparing products such as fruit and meat analogs. They are also widely used in biotechnology as immobilization agents of cells and enzymes. The method involves diffusion of calcium ions through alginate and a cross-linking reaction with the alginate carboxylic group to form the gel (Ha et al., 1989; World Health Organization [WHO], 1993a, 1993b). Carrageenans are reported to stabilize casein and several plant proteins against precipitation with calcium, and are used to prepare texturized milk products (Samant et al., 1993).
4.3.3 OTHER EFFECTS Sodium reduction in the diet is recommended as means of preventing hypertension and subsequent cardiovascular disease, stroke, and renal failure. Reducing or substituting NaCl requires an understanding of the effects caused by the new factors introduced. Several ways are proposed to reduce the sodium content in processed meat without an adverse effect on the quality, flavor, gelation, and shelf life of the products. This includes a slight sodium chloride reduction, replacing some of the NaCl with another chloride salt (KCl, MgCl2) or nonchloride salt or altering the processing methods (Barbut and Mittal, 1985). The calcium ion is a known activator of many biochemical processes. Calpain plays an important role in postmortem tenderizing of meat. The function of the metal ion in this enzyme is believed to be either neutralizaton of the charges on the surface, by preventing electrostatic repulsion of subunits, or effecting a conformational change required for association of the subunits. Thus the metal ions must be present in a specific state to perform this function. The metallic cation in solution exists as an aqua-complex ion in equilibrium with their respective hydroxy-complex: M(H2O)m+ ⇔ MOH(m–1)+ H+ aqua-complex ion hydroxy-complex (weak base)
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The acid ionization constant (pKa) of the aqua-complex ion determines whether the ion will form complexes with a protein. This depends greatly on the pH of the medium. Because the ionization constant of low charge is 12.6, they will form a stable complex only with negatively charged protein in alkaline media. They cannot bind to cationic proteins as they do not share electrons to form a covalent bond. These considerations explain why the activity of calpain is optimum in the alkaline pH range. Thus a decrease in its activity at acidic pH values may partly be due to a change in the electronic state of Ca2+ (Asghar and Bhatti, 1987; Barbut and Mittal, 1985). Generally, sodium and potassium react only to a limited extent with proteins, whereas calcium and magnesium are somewhat more reactive. The transition metals, for example, ions of Cu, Fe, Hg, and Ag, react readily with proteins, many forming stable complexes with thiol groups. Ca2+, Fe2+, and Cu2+, as well as Mg2+ cations may be an integral part of certain protein molecules or molecular associations. Their removal by dialysis or by sequestrants appreciably lowers the stability of the protein structure with respect to heat and proteases.
4.4 THE EFFECT OF STORAGE AND PROCESSING ON THE MINERAL COMPONENTS IN FOODS The effect of normal storage on mineral components is rather low, and may be connected mainly with changes of humidity or contamination. However, major changes of mineral components may occur during canning, cooking, drying, freezing, peeling, and all the other steps involved in preserving and food processing for direct consumption. The highest losses of minerals are encountered in the milling and polishing process of cereals and groats. All milled cereals undergo a significant reduction of nutrients. The extent of the loss is governed by the efficiency with which the endosperm of the seed is separated from the outer seed coat bran, and the germ. The loss of certain minerals and vitamins is deemed so relevant to health that in many countries supplementation was introduced for food products to enrich them with the lost nutrients (i.e., iron to bread). In some countries regulations have been issued concerning standards for enriched bread. If the bread is labeled “enriched,” it must meet these standards. In white flours, the losses of magnesium and manganese may reach up to 90%. These minerals remain mainly in the bran—the outer part of the grain. For this reason it seems reasonable to recommend consumption of bread baked from whole meals instead of from white meals. Although recommended, sometimes steady consumption of bran alone, for dietary purposes, should be done with great care because it may also contain many different contaminants, like toxic metals and organic pesticides. During preparation for cooking or for canning, vegetables should be thoroughly washed before cutting to remove dirt and traces of insecticide spray. Root vegetables should be scrubbed. The dark outer leaves of greens are rich in iron, calcium, and vitamins, so they should be trimmed sparingly. Peeling vegetables and fruit should be avoided whenever possible because minerals and vitamins are frequently
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concentrated just beneath the skin. Potatoes should be baked or cooked in their skins, even for hashed browns or potato salad. However, True et al. (1979) showed that cooking potatoes by boiling whole or peeled tubers, as well as microwave cooking and oven baking may have a negligible effect on the losses of Al, B, Ca, Na, K, Mg, P, Fe, Zn, Cu, Mn, Mo, J, and Se. Microwaved potatoes retain nutrients well, and contrary to popular belief, peeling potatoes does not strip away their vitamin C and minerals. Whenever practical, any remaining cooking liquid should be served with the vegetable or used in a sauce, gravy, or soup. To retain minerals in canned vegetables, the liquid from the can should be poured into a saucepan and heated at a low temperature to reduce the amount of liquid, then added to the vegetables and heated before serving. Low temperatures reduce shrinkage and loss of many other nutrients. Cooking and blanching leads to the most important nutrient losses. Cooking of vegetables results in leaching 30 to 65% of potassium, 15 to 70% of magnesium and copper, and 20 to over 40% of zinc. Thus it is reasonable to use this liquid for soup preparation. A study of changes in the cadmium content of rice during the polishing process showed only a slight decrease (~3%) of the concentration of the metal. The effects of cooking (including washing and soaking in water) showed a slight decrease (average 5%) of the cadmium content of rice. The effect of milling on wheat showed that the cadmium content in flours is about half that present in the grain. The concentration of cadmium in bran is about twice that present in whole-wheat grain, while in tofu, miso, and soy sauce it is about 65%, 80%, and 50%, respectively, of that in unprocessed soybeans. The losses depend both on the kind of food cooked and the course of the applied process. Steam blanching of vegetables generally results in smaller losses of nutrients because leaching is minimized. Frozen meat and vegetables thawed at ambient temperature lose many nutrients including minerals in the thaw drip. Frozen fruits should be eaten without delay, just after thawing, and together with the secreted juice. Foods blanched, cooked, or reheated in a microwave oven generally retain about the same or even a higher amount of nutrients as those cooked by conventional methods (WHO, 2004).
4.5 THE CHEMICAL NATURE OF TOXICITY OF SOME MINERAL FOOD COMPONENTS 4.5.1 INTRODUCTION A diet consisting of a variety of foods provides the best protection against potentially harmful chemicals in food. This is because the body tolerates very small quantities of many toxic substances, but has only a limited ability to cope with large quantities of any single one. Almost any chemical can have a harmful effect if taken in a large quantity. This is especially true for trace minerals, and to some degree, also for macroelements, as well as vitamins. For this reason, it is important to understand the difference between toxicity and hazard. Many foods contain toxic chemicals, but these chemicals do not present a hazard if consumed in allowable amounts. A number of minerals can produce chronic toxicity when absorbed and retained in excess of the body’s demands. The proportion of elements accumulated by the
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organism is different from the proportion in the environment, and this results in their concentration within the organism. Some of the elements are necessary to the organism for metabolic processes; others, however, which are accumulated in high proportions (sometimes specifically in some organs), do not have any metabolic significance for the organism (e.g., aluminum, arsenic, cadmium, mercury, and lead) and are recognized as toxic. Compounds of aluminum, arsenic, cadmium, lead, and mercury contaminate the environment and enter the food supply. However, among them, aluminum compounds are less toxic, and some of them are even used as food additives, (i.e., bread leavening, firming, emulsifying agents), and also are used in drugs (e.g., antacids), and as such all these compounds enter the gastrointestinal tract and add to the aluminum compounds naturally present in foods. Proteins, in addition to other compounds, are the most common naturally occurring chelators playing an important role in the transport, metabolism, detoxification, and retention processes in organisms exposed to higher levels of toxic metals. Particularly metallothioneins (Mts), a family of inducible cysteine-rich proteins ubiquitous in mammals, are capable of binding and storing a number of divalent and trivalent cations including Zn, Cu, Cd, and other transitional metals. These proteins are composed of about 60 amino acids of which one-third are cysteine. Their biosynthesis in organisms is induced by the above-mentioned metals. The highest concentrations of Mts are found in the kidneys, liver, and intestines. Each molecule of Mt is able to bind up to seven atoms of cadmium. Besides contributing to possible cellular defense mechanisms by sequestering potentially toxic metals, Mts are also important in overall homeostasis (WHO, 2004). Zinc metallothionein (Zn-Mt) can detoxify free radicals. Cadmium-induced Mt is able to bind cadmium intracellularly, thus protecting the organism against the toxicity of this metal. Mts occur in the organism in at least four genetic variants. The two major forms (I and II) are ubiquitous in most organs, particularly in the liver and kidney, and also in the brain. Mt isolated from adult or fetal human livers contained mainly Zn and Cu, whereas that from human kidney contained Zn, Cu, and Cd. The metals are bound to the peptide by mercaptide bonds and arranged in two distinct clusters: a four-metal cluster called the α domain and a three-metal cluster called the β cluster at the C terminal of the protein. The cluster is an obligate Zn cluster, whereas the Zn in a β cluster may be replaced by Cu or by Cd. Interaction with Mt is the basis for metabolic interaction between these metals. Metallothionein III is found in the human brain and differs from I and II by having six glutamic acid residues near the terminal part of the protein. Mt III is thought to be a growth inhibitory factor, and its expression is not regulated by metals; however, it does bind Zn. Another proposed role for Mt III is participating in the utilization of Zn as neuromodulator because it is present in the neurons that store Zn in their vesicles. Metallothionein IV occurs during differentiation of stratified squamous epithelium, but it is known to have a role in the absorption or toxicity of cadmium. Mt in the gastrointestinal mucosa might play a role in the gastrointestinal transport of Cd. Its presence in cells of the placenta impairs the transport of Cd from maternal to
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fetal blood and across the blood–brain barrier, but only when the concentration of Cd is low. In general, toxicity of metals is a function of their physical and chemical properties, their doses, and the health conditions of the organism exposed to these metals. It is also a function of the ability of the metals to accumulate in body tissues. For this reason, it is very important to have adequate information about their chemical structure. Currently this may be done by applying speciation analysis, which makes it possible to differentiate the chemical form of the examined element and better assess the safety level of its residue in foods or drinking water.
4.5.2 ALUMINUM Aluminum was in the past considered a completely indifferent substance. However, it has been discovered that the risk of Al is greatly increased in persons with impaired kidney function. Alfrey et al. (1976, 1980) (in WHO, 1989), have shown that dialysis encephalopathy in a large number of patients with renal failure (undergoing chronic dialysis) is attributable to high Al content of some water used for the preparation of dialysates. The patients on dialysis, who died of a neurologic syndrome of unknown cause (dialysis encephalopathy syndrome), had brain matter Al concentrations of 25 mg/kg dry weight, while in controls 2.2 mg/kg was measured. But whether the metal has a causative role in the pathogenesis of these diseases still remains unconfirmed. The concentration of aluminum in human tissues from different geographic regions was found to be widely varied and probably reflected the geochemical environment of individuals and locally grown food products. In healthy human tissues, the Al concentration was usually below 0.5 µ/g wet weight, but higher levels were observed in the liver (2.6 µg/g), lung (18.2 µg/g), lymph nodes (32.5 µg/g), and bone (73.4 µg/g of ash) (WHO, 1989). In plants, the negative effects of Al have been demonstrated especially on cell division and uptake, as well as on the metabolism of other favorable elements such as Mn, Mg, and P. However, in animals, encountered in natural amounts, aluminum has been reported to also have several positive physiological effects. It seems to be involved in the reactions between cytochrome c and succinyl dehydrogenase, as well as a cofactor necessary for activation of guanine nucleotide binding, which is important in protein metabolism. It has also been shown to play an important role in the development of potent immune response and in endogenous triacylglycerol metabolism. On the other hand, excess aluminum in patients with renal failure and dialysis encephalopathy may lead to skeletal defects, such as markedly reduced bone formation, resulting in osteomalacia. A further pathological manifestation of Al toxicity is a microcytic hypochromic anemia not associated with iron deficiency. Such problems have practically disappeared since the use of Al-free deionized water for dialysis became routine. Goats given low Al-semisynthetic ratios (162 µg/kg) for 4 years had significantly reduced life expectancy as compared with that of control goats receiving 25 mg/kg. In studies with mice fed diets containing bread with Al additives, a decreased number of offspring and ovarian lesions were seen. In another study with rats maintained
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on diets containing a mixture of sodium aluminum sulfate and calcium acid phosphate at different dietary levels (bred for 7 successive generations) there were no negative effects on birth weight, average weaning weight, and number of weaned animals. Histopathological examination of kidneys of surviving rats did not reveal any significant changes. Long-term medication of humans with antacids (aluminum hydroxide) may provide several grams of Al per day (WHO, 1996). This may result in inhibition of intestinal absorption and decreased plasma concentration of phosphorus and as a consequence, an increase in calcium loss. This effect is probably due to the binding of dietary phosphorus in the intestine by the aluminum, and was not observed when phosphorus-containing Al salt was used or when the interfering anion and aluminum compound were taken separately (WHO, 1989). Aluminum cations, similar to calcium and magnesium cations, are able to form poorly soluble compounds with fluoride. Thus higher intake of Al by patients using antacids may also reduce, to some extent, fluoride absorption. There are some data that Al and fluoride in animal studies (with ruminants and poultry) are mutually antagonistic in competing for absorption in the gut, and thus higher intake of Al compounds in some circumstances may prevent fluorosis. Conversely, feeding animals with higher levels of fluoride in drinking water may prevent deposition of aluminum in the brain. But still there is no sufficient evidence to conclude that consumption of drinking water containing a high but nontoxic level of fluorides will have a preventive effect on Alzheimer’s disease (Kraus and Forbes, 1992; Schenkel and Klüber, 1987). Depending on the type of environment, geographical factors, and industrialization, there can be considerable variations in levels of Al in cultivated and naturally growing plants, and consequently in animal fodder and human foods. In addition to the natural origin, some quantities (sometimes even high) of Al in the diets may be from food containing Al additives as well as from contact of food with aluminum containers, cookware, utensils, and food wrappings. Daily dietary aluminum intakes vary in different countries, but according to data published in a 1989 WHO report, ranged from about 2 to 6 mg/day for children and to about 14 mg/day for teenagers and adults. The same levels of intake are given by Pennington (1987) who compiled the data on the basis of the Total Diet Study results of teenagers and adults realized in 1984. In the same paper there are results from other countries, ranging from very low to more than 30 mg of Al per person per day. It seems that intake of Al of approximately 2 to 7 mg/day may be ingested by those consuming diets low in herbs, spices, and tea, diets that do not contain food with Al additives, and foods prepared with little contact with aluminum containers, cookware, utensils, or wrappings. In the normal daily hospital diet, mean daily intake of Al was about 21.3 ± 12.4 mg/person, (Nabrzyski and Gajewska, 1995), while in very special hospital diets (in the Pennington report, 1987), the intake was markedly lower and ranged from 1.8 to 7.33 mg/day. Certain plants were able to absorb high levels of Al, such as black tea (445.0 to 1552.0 mg/kg), while herbal or fruit teas contained about 45 mg/kg and herbal teas,
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which were partially supplemented with black teas, had up to 538.0 mg/kg (Nabrzyski and Gajewska, 1995; Fairweather-Tait et al., 1987). The major food sources of Al in daily diets are grain products, which contribute from 36.5 to 69.4%; the next are milk, yogurt, and cheese, contributing from 11.0 to 36.5%; the Al contribution of all other products was only several percent and in most cases even less than 1% (Pennington, 1987). In wild mushrooms and cultivated Agaricus bisporus, Müller et al. (1997) found a low level of Al (14 ± 7 mg/kg of dry matter). The most popular species, such as Boletus and Xerocomus, were low in Al (30 to 50 mg/kg dry matter). However, several other species of the genus Suillus, Macrolepiota rhacodes, Hyoholoma capnoides, as well as individual samples of Russula ochroleuca and Amanita rubescens contained high Al concentrations of about 100 mg/kg dry matter. It was concluded that none of the investigated species of mushrooms contributes significantly to the daily intake of aluminum by humans. In the edible parts of different seafoods such as fish, crustaceans, and molluscan shellfish, the Al level was very diversified. In the fillets of lean and fatty fish it was below 0.2 mg/kg wet weight, with the exception of the Al concentration in fillets of fish caught in coastal waters near a smelting plant, in which up to 1 mg/kg wet weight was found. The edible parts of crustacean and molluscan shellfish contained up to 5 mg/kg wet weight of Al. A comparison between fillets and different organs of cod showed higher Al concentration in organs, especially in gills, that are in continuous contact with the ambient water (above 0.6 mg/kg wet weight), and in the brain and heart (above 0.4 mg/kg wet weight). In line with the tolerable daily intake by the body of 1 mg Al/kg, the contribution of aluminum from the edible parts of aquatic food does not play a significant role in daily diets (Ranau et al., 2001). It is suggested that the additional daily intake of Al resulting from preparing all foods in uncoated Al pans is approximately 3.5 mg. Nowadays, however, most pans are made of stainless steel or Teflon®-coated aluminum, which diminishes migration of the metal into foods, and the average contribution of Al to the daily diet from the use of such utensils may be less than 0.1 mg. An additional intake of less than 0.1 mg daily could result from the occasional consumption of acid fruits and vegetables, assuming that less than 10% of these food items are prepared in aluminum pans (Müller et al., 1993). The quantities of the metal that migrate into foods depends on the acidity of the food items and can markedly increase when acidic beverages are stored or heated in aluminum cans. Generally, the quantities of Al that contribute to the daily diet from such adventitious sources are rather inconsistent and insignificant. Several of the intentional Al-containing additives used as stabilizers, buffers, anticaking and neutralizing agents, dough strengtheners, leavening agents, acid-reacting ingredients in self-rising flour or cornmeal curing agents, components in bleaching, and texturizer agents are given in Table 4.4. An example may be the acidic form of sodium aluminum phosphate that reacts with sodium bicarbonate to cause leavening action. It is used in biscuit, pancake, waffle, cake, doughnut, and muffin mixes, frozen rolls and yeast doughs, canned biscuits, and self-rising flours. Sodium aluminum sulfate is also an acidifying agent found in many household baking powders. The basic form of sodium aluminum
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phosphate is used in processed cheese and cheese foods as an emulsifying agent to give cheese products a soft texture and to allow easy melting. Sodium aluminum phosphate is also used as a meat binding agent. A slice of cake or bread made with baking powder that contains aluminum may contain 5 to 15 mg of Al. Recently, most commercial baking powders, and some that are sold for household use, contain monocalcium phosphate rather than aluminum salts. Aluminum salts used as firming agents in pickled vegetables and some pickled fruits are now replaced by calcium oxide in both industrial and home pickling, although several brands of commercially packed pickles still may contain ammonium or potassium sulfate. Aluminum silicates are found in anticaking agents, salt, nondairy creamers, and other dry, powdered products. A selected committee of the Life Sciences Research Office of the Federation of American Societies for Experimental Biology reviewed the safety of GRAS (generally recognized as safe) substances including aluminum compounds under contract with the U.S. Food and Drug Administration (FDA) and estimated that the average daily intake of emulsifying agents in processed cheese, firming agents, processing aids, stabilizers, and thickeners, containing Al compounds added to foods was about 20 mg, and that about 75% of this was in the form of sodium aluminum phosphate (Pennington, 1987). A low total body burden of Al, coupled with urinary excretion, suggests that even at high levels of consumption, thanks to regulation by homeostatic mechanisms, only small amounts—about 1% of the normally consumed dose—is absorbed by a healthy person from the gastrointestinal tract, and is then excreted by healthy kidneys, so that no accumulation occurs (except in patients with renal failure). In conclusion, there is no known risk to healthy people from the typical dietary intake of aluminum. Risk arises only from the habitual consumption of gram quantities of antacids containing Al over a long period of time, and they rise substantially in persons with impaired kidney function.
4.5.3 ARSENIC Pentavalent and trivalent arsenicals react with biological ligands in different ways. The trivalent form reacts with the thiol protein groups, resulting in enzyme inactivation, structural damage, and a number of functional alterations. The pentavalent arsenicals, however, do not react with –SH groups. Arsenate can competitively inhibit phosphate insertion into the nucleotide chains of DNA of cultured human lymphocytes, causing false formation of DNA because of the instability of the arsenate esters. Dark repair mechanisms are also inhibited, leading to persistence of these errors in the DNA molecules. Binding difference of the trivalent and pentavalent forms leads to the differences in accumulation of this element. Trivalent inorganic As is accumulated in higher levels than the pentavalent form. The organic arsenic compounds are considered less toxic or nontoxic in comparison to inorganic arsenic of which the trivalent arsenicals are the most toxic forms. Dietary As represents the major source of arsenic for most of the general population. Consumers eating large quantities of fish usually ingest significant amounts of As, primarily as organic compounds, especially those with a structure similar to
Firming, color-retention agent Anticaking and antibleaching agent Buffering, firming agent yeast food. Flavor enhancer, salt substitute
Magnesium Magnesium Magnesium Magnesium
Magnesium Magnesium Magnesium Magnesium
chloride (511) carbonate (504i) gluconate (580) glutamate DI-L- (625)
chloride hexahydrate carbonate gluconate dihydrate glutamate
Flavor enhancer, salt substitute Neutralizing agent, buffer, firming agent Surface colorant, anticaking agent, stabilizer Buffer, dough conditioner Antimicrobial, fungistatic, preservative agent
Monocalcium DI-L-glutamate Slaked lime Calcium hydrogen carbonate Calcium dilactate hydrate Calcium sorbate
ADI 0–25.0 (as sum of calcium, potassium and sodium salt) ADI not specified ADI 0–5.0 ADI not specified ADI not specified (group ADI for α glutamic acid and its monosodium, potassium, calcium, magnesium and ammonium salts)
ADI not specified ADI not limited ADI not specified
ADI 0–2.5
Antioxidant, preservative sequestrant
Calcium dihydrogen phosphate (341i)
Calcium disodium ethylenediaminetetraacetace (385) Calcium glutamate (623) Calcium hydroxide (526) Calcium hydrogen carbonate (170ii) Calcium lactate (327) Calcium sorbate (203)
not specified not specified 0–5.0 not specified not specified MTDI 70,0
ADI ADI ADI ADI ADI
ADI, TADI, PMTDI (mg/kg body weight)
Buffer, firming, raising, leavening and texturing, agent, and in fermentation processes
Thickening agent, stabilizer Antioxidant Antimicrobial, preservative Firming agent Acidity, regulator, firming agent, sequestrant
Functional class and comments
Calcium alginate Calcium ascorbate dihydrate Monocalcium benzoate Calcium chloride Tricalcium citrate, tricalcium salt of beta hydroxytricarballylic acid Calcium dihydrogen tetraoxophosphate. Monabasic calcium phosphate. Monocalcium phosphate Calcium disodium EDTA
alginate (404) ascorbate (302) benzoate (213) chloride (509) citrate (333)
Synonyms or other chemical name
80
Calcium Calcium Calcium Calcium Calcium
Chemical name of compound and (INS)
TABLE 4.4 List of Selected Mineral Compounds Used as Food Additives
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Oxidizing agent Alkali, flavor Seasoning and gelling agent, salt substitute
Color of porphyrin Buffer, sequestrant, neutralizing agent
Magnesium DI-D,L-lactate Magnesium oxide Magnesium sulfate Potassium acetate Potassium alginate
Potassium aluminosilicate Potassium ascorbate
Potassium benzoate Potassium bromate Potassium carbonate Potassium chloride, sylvine, sylvite
Potassium or sodium chlorophyllin Monopotassium dihydrogen ortophosphate, monobasic potassium phosphate
Magnesium lactate D,L- also magnesium lactate L (329) Magnesium oxide (530) Magnesium sulfate (518) Potassium acetate (261)
Potassium alginate (402)
Potassium aluminosilicate (555) Potassium ascorbate (303)
Potassium benzoate (212)
Potassium bromate (924a) Potassium carbonate (501i) Potassium chloride (508)
Potassium or sodium copper chlorophyllin (141ii) Potassium dihydrogen phosphate (340i)
Antimicrobial, preservative
Anticaking agent Antioxidant
Thickening agent, stabilizer
Alkali, color adjunct Alkali, anticaking, color retention, carrier, drying agent Buffering agent, dough conditioner, dietary supplement Anticaking, neutralizing agent Firming agent Antimicrobial, preservative, buffer
Dietary supplement
Magnesium hydrogen ortophosphate trihydrate, dimagnesium phosphate Magnesium hydroxide Magnesium carbonate hydroxide hydrated
Magnesium hydrogen phosphate (343ii) Magnesium hydroxide (528) Magnesium hydroxide carbonate (504ii)
MTDI 70.0
(continued)
ADI not limited ADI not specified ADI not specified (also includes the free acid) ADI not specified (group ADI for alginic acid and its ammonium, calcium and sodium salts) No ADI allocated ADI not specified (Group ADI for ascorbic acid and its sodium, potassium, and calcium salts) ADI 0–5.0 (expressed as benzoic acid) ADI withdrawn ADI not specified ADI not specified (group ADI for hydrochloric acid and its magnesium, potassium and ammonium salts) ADI 0–15
ADI not limited
MTDI 70 (expressed as phosphorus from all sources) ADI not limited ADI not specified
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L-Monopotassium L-glutamate Sodium alginate Salp, sodium trialuminium tetradecahydrogen octaphosphate tetrahydrate (A), trisodium dialuminium pentadecahydrogen octaphosphate (B) Kasal, autogenous mixture of an alkaline sodium aluminum phosphate Sodium L-ascorbate Sodium salt of benzenecarboxylic acid Monosodium dihydrogen monophosphate (orthophosphate) Disodium EDTA, disodium edetate
Potassium glutamate (622) Sodium alginate (401) Sodium aluminium phosphate acidic (541i)
Sodium iron III-ethylenediamine tetraacetatetrihydrate
Disodium ethylenediaminetetraacetate (386) Sodium glutamate (621) Monosodium L-glutamate (MSG), glutamic acid monosodium salt monohydrate Ferric sodium edeteate, sodium iron EDTA, sodium feredetate
Potassium bicarbonate Potassium hydrogen sulfite
Potassium hydrogen carbonate (501ii) Potassium hydrogen sulfite (228)
Nutrient supplement (provisionally considered to be safe in food fortification programs)
ADI not specified ADI 0–5.0 MTDI 70.0
Antioxidant Antimicrobial, preservative Buffer, neutralizing agent, sequestrant in cheese, milk, fish, and meat products Antioxidant, sequestrant, preservative, synergist Flavor enhancer
ADI acceptable
ADI 0–2.5 (as calcium disodium EDTA) ADI not specified
ADI 0–0.6
ADI not specified ADI 0–0.7 (Group ADI for sulfur dioxide and sulfites, expressed as sulfur dioxide, covering sodium and potassium metabisulfite, potassium and sodium hydrogen sulfite and sodium thiosulfate) ADI not specified ADI not specified ADI 0–0.6
ADI, TADI, PMTDI (mg/kg body weight)
Emulsifier
Flavor enhancer, salt substitute Thickening agent, stabilizer Raising agent
Alkali, leavening agent, buffer Preservative, antioxidant
Functional class and comments
82
Sodium aluminum phosphate basic (541ii) Sodium ascorbate (301) Sodium benzoate (211) Sodium dihydrogen phosphate (339i)
Synonyms or other chemical name
Chemical name of compound and (INS)
TABLE 4.4 (continued) List of Selected Mineral Compounds Used as Food Additives
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Antimicrobial, fungistatic agent
ADI 0–25.0
MTDI 70.0
ADI 0–0.7 (Group ADI for sulfur dioxide and sulfites expressed as SO2, covering sodium and potassium salt) ADI 0–0.1 ADI 0–5.0
Mineral Components
Source: WHO (World Health Organization), Food Additives Series, 35, Toxicological evaluation of certain food additives and contaminants, paper presented at 44th Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1994, 1996.
MTDI: Maximum Tolerable Daily Intake (or Provisional Maximum Tolerable Daily Intake [PMTDI]), is a term used to describe the endpoint of contaminants with no cumulative properties. Its value represents permissible human exposure as a result of the natural occurrence of the substance in food or drinking water. In the case of trace elements that are both essential nutrients and unavoidable constituents of food, a range is expressed; the lower value represents the level of essentiality and the upper value the PMTDI.
TADI: Temporary ADI: term established by the JECFA for substances for which toxicological data are sufficient to conclude that use of the substance is safe over the relatively short period of time required to evaluate further safety data, but are insufficient to conclude that use of the substance is safe over a lifetime. A higher-than-normal safety factor is used when establishing a TADI, and an expiration date is established by which time appropriate data to resolve the safety issue should be submitted to JECFA.
ADI not specified or ADI not limited: terms applicable to a food substance of very low toxicity which, on the basis of the available data (chemical, biochemical, toxicological, and other), as well as the total dietary intake of the substance arising from its use at levels to achieve the desired effect and from its acceptable background in food does not, in the opinion of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), represent a hazard to health. For that reason, and for reasons stated in individual evaluations, the establishment of ADI in numerical form is not deemed necessary. An additive meeting this criterion must be used within the bound of good manufacturing practice, that is, it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect, it should not conceal inferior food quality or adulteration, and it should not create a nutritional imbalance.
ADI: Acceptable Daily Intake, an estimate of the amount of a substance in food or drinking water that can be ingested daily over a lifetime without appreciable risk to health. Values are expressed on a body-weight basis for a standard human weighing 60 kg.
Notes: INS: International numbering system (parenthetical values), prepared by the Codex Committee for Food Additives for the purpose of providing an agreed-upon international numerical system for identifying food additives in ingredient lists, as an alternative to the declaration of the specific name (Codex Alimentarius, vol. 1, 2nd ed., FAO/WHO, Geneva, 1992, Section 5.1).
Sodium or potassium sorbate (201, 202)
Sequestrant, emulsion stabilizer, buffer
Antimicrobial, color fixative Antimicrobial, color fixative
Sodium nitrite Sodium nitrate, cubic or soda nitre, chile salpetre Trisodium phosphate, trisodium monophosphate, ortophosphate, sodium phosphate. Sodium or potassium sorbate
Sodium nitrite (250) Sodium nitrate (251)
Sodium phosphate (339iii)
Antimicrobial, preservative, bleaching agent, antibrowning agent
Disodium or potassium pentaoxodisulfate
Sodium or potassium metabisulfite (223, 224)
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arsenobetaine and arsenocholine, as well as various other arsenic derivates. Fish of many species contain arsenic between 1 to 10 mg/kg. Arsenic levels at or above 100 mg/kg have been found in bottom-feeders and shellfish. Both, lipid and water-soluble organoarsenic compounds have been found, but the water-soluble forms, mainly the quartenary arsonium derivates, constitute the larger portion of the total arsenic in marine animals (Vaessen and van Ooik, 1989; WHO, 1989). Studies in mice have demonstrated that over 90% of arsenobetaine and arsenocholine were absorbed and about 98% of the administered dose of arsenobetaine was excreted unchanged in the urine, whereas 66% and 9% of single oral doses of arsenocholine was excreted in the urine and feces, respectively, within 3 days. The majority of arsenocholine was oxidized in animal organisms to arsenobetaine, and excreted in this form in the urine. The retention of arsenocholine in the animal body was greater than the retention of arsenobetaine. The fate of organic arsenicals in man still has not been fully clarified. The minimal available information on the organoarsenicals present in fish and other seafood indicates that these compounds appear to be readily excreted unchanged in the urine, with most of the excretion occurring within two days of ingestion.Volunteers who consumed flounder excreted 75% of the ingested arsenic unchanged in urine within 8 days after eating the fish. Less than 0.35% was excreted in the feces. There are no data on tissue distribution of arsenic in humans following ingestion of arsenic present in fish and seafood. Also there have been no reports of ill effects among ethnic populations consuming large quantities of fish resulting in organoarsenic intakes of about 0.05 mg/kg body weight per day (WHO, 1989). Inorganic tri- and pentavalent arsenicals are metabolized in man, dog, and cow to less toxic methylated forms such as monomethylarsonic and dimethylarsinic acids (Peoples, 1983).
4.5.4 MERCURY Organic mercury compounds, especially methylmercury, are recognized as more dangerous for man than the inorganic ones. Most foods (except fish) contain very low amounts of total mercury (<0.01 mg/kg), which is almost entirely in the form of inorganic compounds. Over 90% of mercury in fish and shellfish appears as methylmercury. This is because fish feed on aquatic organisms that contain this compound, originating from microorganisms that contain biomethylated inorganic mercury. Marlin is the only pelagic fish known to have more than 80% of the total muscle mercury present as inorganic mercury (Cappon and Smith, 1982). The amount of methylmercury is especially high in large, old fish of predatory species like shark and swordfish. In fish of freshwater species, the mercury content depends on the concentration of mercury in the water and sediment, and on the pH of the water. The concentration of methylmercury in most fish is generally less than 0.4 mg/kg, although predators such as swordfish, shark, and pike may contain up to several milligrams of methylmercury per kg in the muscle. The intake of methylmercury depends on fish consumption and the concentration of methylmercury in the fish consumed. Many people eat about 20 to 30 g
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of fish per day, but certain groups eat 400 to 500 g per day. Thus the daily dietary intake of methylmercury can range from about 0.2 to 3 to 4 µg/kg body mass. Studies of the kinetics of methylmercury after ingestion show that its distribution in the tissues is more homogenous than that of other mercury compounds, with the exception of elemental mercury. The most important features of the distribution pattern of methylmercury are high blood concentration, a high ratio of erythrocyteto-plasma concentration (about 20), and high deposition in the brain. Another important characteristic is slow demethylation, which is a critical detoxification step. Methylmercury and other mercury compounds have a strong affinity for sulfur and selenium. Although selenium has been suggested as providing protection against the toxic effects of methylmercury, no such effect has been demonstrated. Various effects have been observed in animals treated with toxic doses, but some of these such as renal damage and anorexia, have not been observed in humans exposed to high doses. The primary tissues of concern in humans are the nervous system and particularly the developing brain, and this has been the main reason for the wide range of epidemiological studies. Neurotoxicological effects induced by methylmercury in nonhuman primates resemble those seen in humans and support the concept that the developing fetus is more sensitive than an adult. Methylmercury is readily absorbed (up to 95%) after oral exposure, and passes about 10 times more readily through the placenta and blood–brain barrier, resulting in higher concentrations of this compound in the brain of the fetus than of the mother. It is eliminated from the organism mainly via the bile and feces. Neonatal animals have a lower excretory capacity than adults. The dermal absorption of methylmercury is similar to that of inorganic mercury salts. The LD50 values after oral administration are 25 mg/kg body weight in old rats, and 40 mg/kg in young rats. Clearance half-life of methylmercury for the whole human body is about 74 days, and for blood is 52 days (WHO, 2000).
4.5.5 CADMIUM Cadmium shares chemical properties with zinc and mercury, but in contrast to mercury, it is incapable of environmental methylation, due to the instability of the monoalkyl derivate. Similarities and differences also exist in the metabolism of Zn, Cd, and Hg. Cadmium retention in body tissues is related to the formation of Cdmetallothionein, a Cd-protein complex of low molecular mass. Metallothionein (Mt) in the gastrointestinal (GI) mucosa plays a role in the GI transport of Cd. Its presence in cells of the placenta impairs the transport of Cd from maternal to fetal blood and across blood–brain barriers, but only when the concentration of Cd is low. Newborns are virtually cadmium free, whereas zinc and copper are readily supplied to the fetus. Rapid renal concentration occurs mainly during the early years of life. In plasma, Cd is transported as a complex with Mt, and may be toxic to the kidneys when excreted in the glomerular filtrate. Most Cd in urine is bound to Mt. Cadmium bound to Mt in food does not appear to be absorbed or distributed in the same way as its inorganic compounds. Low dietary concentrations of calcium promote absorption of Cd from the intestinal tract in experimental animals. A low iron status in laboratory animals and humans has also been shown to result in greater absorption of Cd. In
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particular, women with low body-iron stores, as reflected by low serum ferritin concentrations, had an average GI absorption rate twice as high as that of a control group of women having about 5%. High iron levels result in decreasing total and fractional Cd accumulation from diets, whereas low iron levels promote accumulation of Cd. Studies in rats with reduced iron levels showed that the inclusion of wheat bran-containing phytate hindered absorption of iron, calcium, and other minerals in their diets, but increased the uptake of Cd. After a single dose of cadmium chloride, the LD50 value for rats and mice treated orally ranges from about 100 to 3000 mg/kg body mass. The high affinity of Cd for –SH groups and the ability of imparting moderate covalency in bonds result in increased lipid solubility, bioaccumulation, and toxicity. In humans with normal levels of exposure, about 50% of the body burden is found in the kidneys, about 15% in the liver, and about 20% in the muscles. The proportion of Cd in the kidney decreases as liver concentration increases. The lowest concentrations of Cd are found in brain, bone, and fat. Accumulation in the kidney continues up to 50 to 60 years of age in humans and falls thereafter, possibly due to age-related changes in the functional integrity of the kidney. In contrast, Cd levels in the muscle continue to increase over the course of life. The average concentration in the renal cortex of nonoccupationally exposed persons aged 50 varies between 11 and 100 mg/kg in different regions. The diet is the major route of human exposure to cadmium. Contamination of foods with Cd results from its presence in soil and water. The concentration of cadmium in foods ranges widely; the highest average concentrations were found in mollusks, kidney, liver, cereals, cocoa, and leafy vegetables. A daily intake of about 60 µg would be required to reach a concentration of 50 mg/kg in the renal cortex at the age of 50, assuming an absorption rate of 5%. About 10% of the absorbed daily dose is rapidly excreted (WHO, 1989, 2001).
4.5.6 LEAD The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and other WHO committees have recognized that infants and children are the groups at highest risk to Pb exposure from food and drinking water. As an anthropogenic contaminant, Pb finds its way into the air, water, and surface soil. Pb-containing products also contribute to the Pb body burden. The domestic environment, in which infants and children spend the greater part of their time, is of particular importance as a source of Pb intake. In addition to exposure from general environmental sources, some infants and young children, as a result of normal, typical behavior, can receive high doses of Pb through mouthing or swallowing of nonfood items. Pica, the habitual ingestion of nonfood substances, which occurs among many young children, has frequently been implicated in the etiology of Pb toxicity. In the United States, on average, 2-year-old children may receive about 45% of their daily Pb intake from dust, 40% from food, 15% from water and beverages, and 1% from inhaled air (WHO, 1986). Pb absorption is heavily influenced by food intake, much higher rates occurring after fasting than when Pb is ingested with a meal. This effect may be due mainly
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to competition from other ions, particularly iron and calcium, for intestinal transport pathways. Absorption is also affected by age; the typical absorption rates in adults and infants are 5 to 10% and about 50%, respectively. Children absorb Pb from the diet with greater efficiency than do adults (WHO, 2000). After absorption and distribution in the blood, where most Pb is found in erythrocytes, it is initially distributed to soft tissues throughout the body. Subsequently, it is deposited in the bone, where it eventually accumulates. The half-life of Pb in blood and other soft tissues is 28 to 36 days. Pb that is deposited in physiologically inactive cortical bone may persist for decades without substantially influencing its concentration in blood and other tissues. On the other hand, Pb that is accumulated early in life may be released later when bone resorption is increased as result of, for example, calcium deficiency or osteoporosis. Pb that is deposited in physiologically active trabecular bone is in equilibrium with blood. The accumulation of high concentrations of Pb in blood when exposure is reduced may be due to the ability of bone to store and release this element. Dietary Pb that is not absorbed in the gastrointestinal tract is excreted in the feces. Pb that is not distributed to other tissues is excreted through the kidney, and to a lesser extent by biliary clearance (WHO, 2000). The biochemical basis of Pb toxicity is its ability to bind to biologically important molecules, thereby interfering with their function through a number of mechanisms. At the subcellular level, the mitochondrion appears to be the main target organelle for the toxic effects of Pb in many tissues. Pb has been shown to selectively accumulate in the mitochondria. There is evidence that it causes structural injury to these organelles and impairs basic cellular energetics and other mitochodrial functions. It is a cumulative poison, producing a continuum of effects, primarily on the hematopoietic system, the nervous system, and the kidneys. At very low blood levels, it may impair normal metabolic pathways in children. At least three enzymes of heme biosynthetic pathways are affected. At about 10 µg/100 cm3 in blood, Pb interferes with δ-aminolevulinic acid dehydratase (WHO, 1986). Alteration in the activity of the enzymes of the heme synthetic pathway leads to accumulation of the intermediates of the pathway. There is some evidence that accumulation of δ-aminolevulinic acid exerts toxic effects on neural tissues through interference with the activity of the neurotransmitter γ-amino-butyric acid. The reduction in heme production per se has also been reported to adversely affect nervous tissue by reducing the activity of tryptophan pyrollase, a heme-requiring enzyme. This results in increased metabolism of tryptophan via a second pathway, which produces high blood and brain levels of the neurotransmitter serotonin. Pb interferes with vitamin D metabolism because it inhibits hydroxylation of 25hydroxy-vitamin D to produce the active form of vitamin D. The effect has been reported in children at blood levels as low as 10 to 15 µg/ 100 cm3 (WHO, 1986). Measurements of the inhibitory effects of Pb on heme synthesis is widely used as a screening test to determine whether medical treatment for Pb toxicity is needed for children in high-risk populations who have not yet developed overt symptoms of poisoning.
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4.5.7 INTERACTIONS
Chemical and Functional Properties of Food Components OF
ELEMENTS
All minerals are toxic at some dose, and the range of safety versus toxicity is highly variable. Data concerning the toxicity of the five discussed toxic minerals are presented in Table 4.5 and Table 4.6. The uptake of elements is not entirely independent of one another. Elements of similar chemical properties tend to be taken up together. Sometimes one element has an inhibiting effect on another, or there can be synergistic effects, such as enhancement of the absorption of calcium in the presence of adequate amounts of phosphorus; cadmium and lead hindering calcium and iron absorption; zinc and copper antagonism and their influence on the ratio of Zn to Cu on copper deficiency; aluminum and phosphorus or fluoride antagonism, which may result in decreased plasma concentrations of phosphorus; or reduced fluoride absorption by mutually antagonistic competition for absorption in the gut. Interaction with other substances, such as drugs as well as food ingredients, should also be taken into account when healthy nutrition is considered.
TABLE 4.5 The Provisional Tolerable Weekly Intake (PTWI) of Toxic Elements According to the Joint FAO/WHO Expert Committee on Food Additives for Man Element Aluminum mg/kg body weight Arsenic µg/kg body weight Cadmium µg/kg body weight Lead µg/kg body weight
Mercury µg/kg body weight
PTWI 0–7.0 15.0 7.0 25.0
3.3 as methylmercury and 5.0 as total mercury
Comments
For inorganic arsenic — When blood lead levels in children exceed 25 µg/ 100 cm3 ( in whole blood), investigations should be carried out to determine the major sources of exposure, and all possible steps should be taken to ensure that lead levels in food are as low as possible With the exception of pregnant and nursing women who are at qreater risk of adverse effects from methylmercury; life in utero is the critical period for the occurrence of neurodevelopmental toxicity; PTWI of 1.6 µg/kg bw is considered sufficient to protect the developing fetuses
Note: PTWI: For definition, see notes in Table 4.4. Sources: WHO (World Health Organization), Food Additives Series 21, 24, 44, 46, and 52. Toxicological evaluation of certain food additives and contaminants; Safety evaluation of certain food additives and contaminants. Papers presented at meetings of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1986, 1989, 2000, 2001, and 2004.
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TABLE 4.6 Metal Toxicity in Man
Metal Aluminum
Arsenic
Cadmium
Daily intake (mg per adult person)/ Toxic effects Few to more than 30 Neurotoxicity. It is supposed that Al may be implicated in the development of an encephalopathy in patients receiving dialyses, in Alzheimer’s disease, and in Parkinsonism dementia. Signs of neurotoxicity may be manifested when the brain concentration exceeds 10 to 20 times the normal level (>2 mg/kg dry weight of the grey matter). A further toxicological manifestation of Al toxicity is a microcytic hypochromic anemia not associated with Fe deficiency. Oral Al has not been associated with Al-induced encephalopathy. There is no risk of toxicity to healthy people from typical dietary intakes. 0.0–0.29 Inorganic compounds cause abnormal skin hyperpigmentation, hyperkeratosis, skin and lung cancer. Organoarsenic compounds present in fish are less toxic or nontoxic <0.024– 0.036 Accumulates mainly in liver and renal cortex. Nephrotoxicity, decalcification, osteoporosis, osteomalacia, and Itai-itai disease. In early gestation, embryotoxic. Impairs the immune system, and Ca and Fe absorption. Hypertension and cardio-vascular disease. Kidney is the critical organ.
Source of exposure
Absorption (percent)
Grain, some bakery, dairy products made with additives such as buffers, dough strengtheners and leavening agents, acid reacting ingredients in self-rising flour or cornmeal, emulsifying agents for processed cheeses, stabilizers, texturizers, and anticaking agents, thickeners, and curing agents. High levels in black and herbal teas and spices. Al naturally present in water is generally low (0.001–1 mg/dm3), but when water is treated with aluminum sulfate salts (as coagulating agents) in water supplies, the content may rise up to threefold of the normal accepted level (0.2 mg/dm3).
<1–7 %
Contaminated water, food containing residue of arsenic pesticides, and veterinary drugs. Fish and shellfish are the richest sources of organic compounds: arsenobetaine and arsenocholine.
Organoarsenic compounds >90%, and inorganic trivalent compounds high 3–10% Cd bound to metallothionein is well absorbed.
Oysters, cephalopods, crops growing on land fertilized with high contaminated phosphate and sewage sludge, Cd leaching from enamel and pottery glazes, contaminated water.
(continued)
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TABLE 4.6 (continued) Metal Toxicity in Man
Metal Lead
Mercury
Daily intake (mg per adult person)/ Toxic effects <0.1–0.2 At blood levels greater than 40 µg/100 cm3, exerts a significant effect on hemopoietic system resulting in anemia; affects the central nervous system. <0.02–0.1 Methylmercury compounds easily pass the blood–brain and placental barriers; causes severe neurological damage, greater in young children; in animals, also renal damage and anorexia.
Source of exposure Food contaminated from leaching of glazes of ceramic foodware, as well as from motor-vehicle exhaust, atmospheric deposits, canned food, water supply from plumbing system. Fish and shellfish, meat from animals fed with mercury-dressed grains.
Absorption (percent) 5–10% in adults and 40–50% in children.
>90% as methylmercury compounds, and 15% as inorganic mercuric compounds.
REFERENCES Asghar, A. and Bhatti, A.R., Endogenous proteolytic enzymes in skeletal muscle: their significance in muscle physiology and during postmortem aging events in carcasses, Adv. Food Res., 31, 343, 1987. Barbut, S. and Mittal, G.S., Rheological and gelation properties of meat batters prepared with three chloride salts, J. Food Sci., 53, 1296, 1985. Bremner, I., Heavy metal toxicities, Quarterly Rev. of Biophysics, 7, 75, 1974. Cappon, C.J. and Smith, J.C., Chemical form and distribution of mercury and selenium in edible seafood, J. Anal.. Toxicol., 6, 10, 1982. Castell, C.H. and Spears, D.M., Heavy metal ions and the development of rancidity in blended fish muscle, J. Fish. Res. Bd. Can., 25, 639, 1968. Castell,C.H., MacLeam, J., and Moore, B., Rancidity in lean fish muscle. IV Effect of sodium chloride and other salts, J. Fish. Res. Bd. Can., 22, 929, 1965. Du, Z. and Bramlage, W.J., Superoxide dismutase activities in senescin apple fruit (Malus domestica borkh), J. Food Sci., 59, 581, 1994. Eschleman, M., Ed., Introductory Nutriiton and Diet Therapy, J.B. Lippincott, London, 1984. Fairweather-Tait, S.J. et al., Aluminium in the diet, Human Nutrition: Food Sc. and Nutrition, 41 F, 183, 1987. FAO/WHO (Food and Agriculture Organization/World Health Organization), Summary of Evaluations Performed by the Joint FAO/WHO Expert Committee on Food Additives 1956–1993, International Life Science Inst. Press, Geneva, 1994. Ha, Y.W. et al., Calcium binding of two microalgal polysaccharides and selected industrial hydrocolloids, J. Food Sci., 54, 1336, 1989. Hendler, S.S., Ed., The Doctor’s Vitamin and Mineral Encyclopedia, Simon and Schuster, New York, 1990.
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Hultin, H.O., Oxidation of lipids in seafoods, in Seafoods: Chermistry, Processing Technology and Quality, Shahidi, F. and Botta, J.R., Eds., Chapman and Hall, London, 1994, p. 49. Kraniak, J.M. and Shelef, L.A., Effect of ethylenediaminetetraacetic acid (EDTA) and metal ions on growth of Staphylococcus aureus 196 E in culture media, J. Food Sci., 53, 910, 1988. Kraus, A.S. and Forbes, W.F., Aluminium, fluoride and the prevention of Alzheimer’s disease, Can. J. Publ. Health, 83, 97, 1992. Miller, D.K. et al., Dietary iron in swine affects nonheme iron and TBARs in pork skeletal muscles, J. Food Sci. 59, 747, 1994a. Miller, D.K. et al., Lipid oxidation and warmed-over aroma in cooked ground pork from swine fed increasing levels of iron, J. Food Sci., 59, 751, 1994b. Müller, J.P., Steinegger, A., and Schlater, C., Contribution of aluminium from packaging materials and cooking utensils to the daily aluminium intake, Z. Lebensm. Unters. Forsch., 197, 332, 1993. Müller, M., Anke M., and Illing-Gunter, H., Aluminium in wild mushrooms and cultivated Agaricus bisporus, Z. Lebensm. Unters. Forsch. 22205, 242, 1997. Nabrzyski, M. and Gajewska, R., Aluminium and fluoride in hospital daily diets and teas, Z. Lebensm. Unters. Forsch., 201, 307, 1995. National Research Council, Food and Nutrition Board, Recommended Dietary Allowances, 10th ed., National Academy Press, Washington, DC, 1989. Oelingrath, I.M. and Slinde, E., Sensory evaluation of rancidity and off-flavor in frozen stored meat loaves fortified with blood, J. Food Sci., 53, 967, 1988. Pearson, A.M., Love, J.D., and Shorland, F.B., “Warmed over” flavor in meat, poultry, and fish, Adv. Food Chem., 23, 2, 1977. Pennington, J.A.T., Aluminium content of foods and diets, Food Additives Contam., 5, 161, 1987. Peoples, S.A., The metabolism of arsenic in man and animals, in Arsenic Industrial, Biomedical, Environmental Perspectives (Proceedings of the Arsenic Symposium), Lederer, W.H. and. Fensterheim R.J., Eds.,Van Nostrand Reinhold Co., New York, 1983, p. 125. Ramanathan, L. and Das, N.P., Effect of natural copper chelating compounds on the prooxidant activity of ascorbic acid in steam-cooked ground fish, Int. J. Food Sci. Technol., 28, 279, 1993. Ranau, R., Oelhlenschlager, J., and Steinhart, H., Aluminium content in edible parts of seafood, Eur. Food Res. Techn., 212, 431, 2001. Rosenberg, J.H. and Solomons, N.W., Physiological and pathophysiological mechanism in mineral absorption, in Absorption and Malabsorption of Minerals, Vol. 12., Solomons, N.W., and Rosenberg, J.H, Eds., Alan R. Liss, Inc., New York, 1984, p. 1. Samant, S.K. et al., Protein-polysaccharide interactions: a new approach in food formulation, Int. J. Food Sci. Technol., 28, 547, 1993. Schenkel, H. and Kluber, J., Mogliche Auswirkungen einer erhöhten Aluminiumaufnahme auf Landwirtschaftliche Nutztiere, Obers. Tierernährung, 15, 273, 1987. Solomons, N.W., Ed., Absorption and Malabsorption of Mineral Nutrients, Alan R. Liss, New York, 1984, p. 269. True, R.H. et al., Changes in the nutrient composition of potatoes during home preparation, III, Min. Am. Potato J., 56, 339, 1979. Vaessen, H.A.M.G. and van Ooik, A., Speciation of arsenic in Dutch total diets; methodology and results, Z. Lebensm. Unters. Forsch., 189, 232, 1989. WHO (World Health Organization), Environmental health criteria, 18, in Arsenic, WHO, Geneva, 1981.
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WHO (World Health Organization), Food Additives Series, 21, Toxicological evaluation of certain food additives and contaminants, paper presented at 30th Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Cambridge, U.K., 1986. WHO (World Health Organization). Food Additives Series, 24, Toxicological evaluation of certain food additives and contaminants, paper presented at 33rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Cambridge, U.K., 1989. WHO (World Health Organization). Food Additives Series, 30, Toxicological evaluation of certain food additives and naturally occurring toxicants, paper presented at 39th Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1993a. WHO (World Health Organization). Food Additives Series, 32, Toxicological evaluation of certain food additives and contaminants, paper presented at 41st meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1993b. WHO (World Health Organization), Food Additives Series, 35, Toxicological evaluation of certain food additives and contaminants, paper presented at 44th Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1996. WHO (World Health Organization), Food Additives Series, 44, Safety evaluation of certain food additives and contaminants, paper presented at 53rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 2000. WHO (World Health Organization), Food Additives Series, 46, Safety evaluation of certain food additives and contaminants, paper presented at 55th Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 2001. WHO (World Health Organization), Food Additives Series, 52, Safety evaluation of certain food additives and contaminants, paper presented at 61st Meeting of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 2004. Wu, S.Y. and Brewer, M.S., Soy protein isolate antioxidant effect on lipid peroxidation of ground beef and microsomal lipids, J. Food Sci., 59, 702. 1994.
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5
Saccharides Piotr Tomasik
CONTENTS 5.1 5.2 5.3 5.4
Natural Food Saccharides, Occurrence, Role, and Applications .................. 93 Carbohydrate Structure .................................................................................. 94 Carbohydrate Chirality................................................................................. 100 Carbohydrate Reactivity............................................................................... 101 5.4.1 Chemical and Physical Transformations of Mono-, Di-, and Oligosaccharides Essential in Food Chemistry........................ 101 5.4.1.1 Reactions of Aldehyde and Ketone Function .................. 101 5.4.1.2 Reactions of the Hydroxyl Groups .................................. 102 5.4.1.3 Reactions of the Glycosidic Bond ................................... 107 5.4.1.4 Specific Reactions of Saccharides.................................... 108 5.4.2 Chemical and Physical Transformations of Polysaccharides.......... 109 5.4.2.1 Depolymerization of Carbohydrates ................................ 113 5.4.2.2 Chemical Modification of Polysaccharides without Attempted Depolymerization ........................................... 114 5.4.2.3 Retrograded, Cross-Linked, and Graft Polysaccharides ..... 116 5.4.3 Enzymatic Conversions of Carbohydrates....................................... 117 5.4.4 Cereal and Tuber Starches ............................................................... 118 5.5 Functional Properties of Carbohydrates ...................................................... 119 5.5.1 Taste ................................................................................................. 119 5.5.2 Colorants .......................................................................................... 121 5.5.3 Flavor and Aroma ............................................................................ 122 5.5.4 Texture.............................................................................................. 122 5.5.5 Encapsulation ................................................................................... 123 5.5.6 Polysaccharide-Containing Biodegradable Materials...................... 124 References.............................................................................................................. 125
5.1 NATURAL FOOD SACCHARIDES, OCCURRENCE, ROLE, AND APPLICATIONS Nature commonly utilizes saccharides as a source of energy, structure-forming material, water-maintaining hydrocolloids, and even sex attractants. Amino acids synthesize in the concentrated space and polymerize into proteins on already-available polysaccharide matrices. All organism cells, including those of animals, contain 93
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saccharide components in their membranes. Frequently, saccharides exist in naturally derivatized forms, including aminated forms, as in chitin and chitosan; esterified; alkylated as in glycosides; oxidized; reduced; or linked to proteins, lipids, and other structures, such as glycoproteins. Lower monosaccharides, such as aldotrioses and aldo- and ketotetroses, do not exist naturally in a free state. Glyceroaldehyde in phosphorylated form is the product of alcoholic fermentation and glycolic sequence. Erythrose, an aldotetrose, and erythrulose, a ketotetrose, also appear in phosphorylated forms in the pentose cycle of glucose, while ketopentose-ribulose can be found as its phosphate ester. Several common and uncommon saccharides (erlose, turanose, trehalose, isomaltose, melecitose) have been found in honey (Rybak-Chmielewska, 2004). In nature, various saccharide derivatives are also found. Among them are so-called sugar alcohols (alditols). They are the natural products of the reduction of saccharides. The occurrence and applications of common saccharides, oligosaccharides, polysaccharides, and sugar alcohols are listed in Table 5.1. Algal gums and mucilages (Ramsden, 2004) constitute an abundant group of polysaccharides in plants.
5.2 CARBOHYDRATE STRUCTURE Carbohydrates are either polyhydroxyaldehydes (aldoses, oses) or polyhydroxyketones (ketoses, uloses); there is an electron gap at their carbonyl carbon atom. Typically, aldehydes and ketones accept nucleophiles, such as alcohols, to form hemiacetals and hemiketals, respectively. In pentoses, pentuloses, hexoses, hexuloses, and higher carbohydrates, one of the hydroxyl groups can play the role of internal nucleophile. Thus, open-chain structures (Structures 5.2 and 5.5) cyclize into internal hemiacetals (Structures 5.4 and 5.6), and hemiketals (Structures 5.1 and 5.3) with either five- or six-membered cycles. Formation of such rings is thermodynamically favored. Because of the oxygen atom in the rings, these rings are called furanoses and pyranoses, respectively. Because their carbon atoms are sp3–hybridized, the rings cannot be planar. They preferably take the chair conformation (Siemion, 1985). In some cases, the pyranose ring formation can be either obstructed or blocked, and a furanose ring dominates for the given sugar. The molecular structure of disaccharides and higher saccharides is additionally controlled by a potential energy benefit resulting from the formation of intramolecular hydrogen bonds, as in cellobiose (Structure 5.7a), lactose (Structure 5.7b), maltose (Structures 5.8 and 5.9) and sucrose (Structure 5.10). Both maltose structures correspond to two energy minima. Two hydrogen bonds stabilize the sucrose molecule. In order to achieve such stabilization, the fructosyl moiety must take the furanosyl structure. In polysaccharides, the structural factors are even more important. A number of different saccharide units in the chain, the branching of the chain, and the presence of more polar groups (COOH, PO3H2, and SO3H) or less polar groups (OCH3 and NHCOCH3) than the OH group are crucial for the overall macrostructure of the polysaccharide. Amylose and cellulose are the most regularly built; they form polymer chains of α-D- and β-D-glucose units, respectively. In very random cases,
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TABLE 5.1 Essential Natural Saccharides in Food, Their Occurrence and Applications Saccharide
Occurrence
Applications
Monosaccharides and Their Natural Derivatives Pentoses D-Apiose L-Arabinose D-Xylose
Hexoses L-Fucose D-Galactose
D-Glucose
D-Mannose L-Rhamnose
Parsley and celery Plant gums, hemicelluloses, saponins, protopectin Accompanies L-arabinose
Mother’s milk, algae, plant mucus and gums Oligo- and polysaccharides, plant mucus and gums, saponins, glycosides Plants and animals, honey, inverted sugar, saponins
Alcoholic fermentation, furan-2aldehyde production Reduction to xylitol; sucrose substitute; alcoholic fermentation; production of furan-2-aldehyde
Diagnostic in liver tests
Alcoholic fermentation; sweetener; energy pharmacopeial material; nutrient; food preservative
Algae, plant mucus, oranges Plant mucus and gums, pectins saponins, glycosides
Hexuloses D-Fructose
Fruits, honey, inverted sugar
D-Glucosylamine
Chitin, chitosan
L-Sorbose
Rowan berries
Lactose
Disaccharides (oses) Mammalian milk
Maltose Sucrose
Starch, sugar beet, honey Sugar beet, sugar cane
Agar
Red algae
Alginates
Brown algae
Noncavity-causing sweetener; sweetener for diabetics; food humidifier and preservative Pharmaceutical aid; antiarthritic drugs; ion exchanger Synthesis of ascorbic acid
Dairy product taste improver; fermenting component of milk Alcoholic fermentation; common sweetener; caramel production; food preservation
Polysaccharides Microbiological nutrient; gelforming agent; emulsifier; bread staling retardant; meat texturizer; meat substitute Thickener; gel-forming agent; food and beer foam stabilizer (continued)
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TABLE 5.1 (continued) Essential Natural Saccharides in Food, Their Occurrence and Applications Saccharide
Occurrence
Applications
Carrageenans ι, κ, λ, µ, ν
Red seaweed
Gel-forming agent; stabilizer; protein fiber texturizer; milk fat anticoagulant; milk clarifying agent Saccharification to glucose; dietary fiber; chromatographic sorbent Chromatographic sorbent (Sephadex); blood substitute Gel-forming agent; filler; marmalade stabilizer; protein precipitant Glucose reservoir for organisms
Cellulose
Plants
Dextran
Frozen sugar beet
Furcellaran
Red seaweed
Glycogen Gums: Arabic
Liver, muscles
Gatti Guaran Karaya Locust bean Tragacanth Hemicelluloses: Arabinogalactan Galactan, mannans, xylans Heparin Hialuronic acid Inulin Pectins
Senegal acacia Anageissus latifolia tree Leguminous plants Sterculiacea tree (India) Locust bean Astragalus species (Middle East) Larch Plants
Protopectin
Liver, lungs Connective tissues Endive, Jerusalem artichoke Plants, mainly apples, citrus, sugar beet Plants and nonmatured fruits
Starch Amylose, amylopectin
Tubers, grains, some fruits, sago palm
Tamarind flour
Tamarind tree (India)
Xanthan gum
Semiartificial gum
Alditols D-Glucitol (sorbitol)
Apples, pears, cherries, aprocits
D-Mannitol D-Ribitol D-Xylitol
Plant exudates, olives, palm trees, marine algae, mushrooms, onion Plants, riboflavin Corncobs, mushrooms
Emulsifier; antistaling stabilizer; flavor fixative Emulsifier; stabilizer Food; cosmetics; pharmaceutical thickener and stabilizer Foam stabilizer; thickener Thickener; adhesive Thickener; stabilizer Emulsifier; stabilizer Alcoholic fermentation; reduction to alcohol Blood anticoagulant Water absorbent Prebiotic Gel-forming agent; beer stabilizer Decomposes to pectins on plant maturation and cooking Food filler; thickener; gel-forming agent; bakery products; saccharification to syrups Thickener; marmalade; jelly; ice cream; mayonnaise stabilizer Hydrocolloid stabilizer
Sweetener; softener; crystallization inhibitor Energy storage in organisms
Sweetener
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O
HOH2C
HO
O
O H
HOH2C
H
HO
CH2OH
HO
CH2OH
CH2OH O
HO
HO
HO OH
CH2OH O O H
HO OH
O H
4
OH
O
6
5
STRUCTURES 5.1–5.6 CH2OH O
HO
H
OH
OH O
HO
OH
O
HO
O
OH
CH2OH 7a
7
7b
OH H O
OH
HO
OH O
O O
O
HO
OH
OH
O
HO
O
OH
HO 8
9 Maltose
HOCH2 HO HO
O
O
O
O H
H O
H O OH 10
STRUCTURES 5.7–5.10
Sucrose OH OH
OH OH
OH O H
H
3
CH2OH O H
HO
O
OH
2
1
CH2OH
HO
OH
OH
O
HOH2C
O
OH
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amylose is branched with short chains (Ball et al., 1998). The amylose chain is originally long and randomly coiled (Figure 5.1), but it turns into a more ordered, helical structure. Helical complexes are formed if hydrocarbons, alcohols, lipids, fatty acids, and barlike anions, such as I5– and OCN– are present in the amylose environment (Tomasik and Schilling, 1998a, b). Such compounds and anions, potential guests of the complex, either have hydrophobic fragments or are fully hydrophobic. The possibility of reduction of the energy in the system by interactions of hydrophobic sides of amylose and the potential guest is the driving force of the formation of a helical complex. Thus, its cavity is hydrophobic and all hydroxyl groups of the D-glucose units are situated on the external surface of the helix, as well as on the edges of its cavity (the V-type amylose). The number of glucose units in one helix turn depends on the size of the guest molecule inside the helical complex. With KOH as a guest, one turn involves six glucose units. Inclusion of KBr reduces the turn to four glucose units, whereas inclusion of tert-butanol and α-naphtol requires the turns of seven and eight glucose units, respectively (Tomasik and Schilling, 1998a). An additional stabilization of the amylose helix comes from the double-helix formation (Imberty et al., 1991). Depending on the helix–helix interactions, and in consequence, their mutual agreement, Aand B-type amyloses are formed (Figure 5.2). Distinguished C-type amylose is a combination of both A and B patterns. The conformation of β-D-glucose units bound in cellulose in the 1→ 4′ manner offers a particularly strong hydrogen-bonded, crosslinked macrostructure of this polysaccharide.
X
X FIGURE 5.1 Randomly coiled amylose chain and its helical complex, formed as a result of its interaction with a nonpolar chain fragment of a molecule.
FIGURE 5.2 The crystallographic A- and B-types of amylose depend on the structure of double amylose helices.
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Glycans with 1,2-, 1,3-, and particularly 1,6-linked units have a more irregular, loosely joined structure. The heterogenicity of the polysaccharide structural units and their volumes introduce further regularities or irregularities in the macrostructure. A decrease in the group polarity, for example, by methylation, and of the number of units, results in a more irregular polysaccharide structure. An opposite effect can be achieved in the presence of more polar groups, or even less polar but suitably oriented groups. For instance, chitin, a polysaccharide with acetylamino groups, has a very regular, compact structure that provides insolubility of chitin in water. This polysaccharide is a common insect carapace-forming material. Because of this property, chitin has found several technical applications. Amylopectin (Figure 5.3) presents a special case. It is a highly branched homopolymer of α-D-glucose units. The 1→ 6-linked terminal branches, which occur at about every 8th glucose unit, contain 15 to 30 glucose units. These branches can also participate in the formation of helical complexes. Some guest molecules may situate in areas around the branching sites in the amylopectin molecule. In spite of the irregular, bulky structure, amylopectin also forms a double helix (Imberty et al., 1991). Because of functional properties, the structure of the polysaccharide matrix, the tertiary structure, is also essential. In solution, polysaccharides such as amylose form separate fibrils that, upon coiling, turn into either micelles at a low-temperature gradient or gels at a high-temperature gradient. Both amylose and amylopectin participate in the starch granule organization (Gallant et al., 1997). Their seminative mixture has specific functional properties. It strongly depends on the amylose-to-amylopectin ratio, size of granules (Table 5.2), content of residual components of native starch, such as lipids, proteins, and mineral salts, and random esterification with phosphoric acid, the latter exclusively in the case of potato amylopectin. Phosphorus present in other starches, resides as a component of phospholipids and glycoproteins. The amylose-to-amylopectin ratio determines the aqueous solubility of starch and the texture of its gels, resulting from the penetration of water into the starch granules (swelling) and from pushing the granule interior into a solution where the gel network is formed. Empty domains inside the starch granules can be utilized as
FIGURE 5.3 Scheme of the amylopectin molecule.
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TABLE 5.2 Selected Properties of Starches of Various Botanical Origin
Starch origin Barley Maize Oat Potato Rice Rye Triticale Waxy maize Wheat a
Amylose content (%)
Granule size (µm)
Gelation temperature (°C)
Protein content (%)a
Lipid content (%)a
19.22 21–24 23–30 18 –23 8 –37 24 –30 24–34 1–2 24–29
5–40 10–30 5–15 10–100 2–10 8 –60 2–40 10–30 2–36
51–59 67–100 87–90 59–68 68–78 55–70 55–62 62–72 59–64
up to 12.2 3.2 up to 3.9 1.5 up to 28.2 3.2 2.7 up to 3.1 2.4
up to 2.0 6.0 up to 6.2 0.6 up to 10.0 2.2 3.9 up to 2.0 3.6
Average values on a dry basis.
natural microcapsules for volatile and air-sensitive compounds (Korus et al., 2003). The size of the granules is essential for the smoothness of products prepared from starch (e.g., puddings, gels). In practice, not all starch granules swell and participate in gel formation. Larger granules are more susceptible to gelation and chemical modification. Also, among several starch varieties, potato starch gels significantly more readily than others (Lii et al., 2003a). The nutritional value of starches usually increases with the content of residual components. However, in several cases, when starch is subjected to chemical modifications or is used for specific nonnutritional purposes, such “contaminants” are not beneficial. Cellulose that is completely insoluble in water forms microfibrils, which are composed of crystallites and amorphous regions. Such regions may also be distinguished inside of starch granules. Roughly, they form concentric crystalline and amorphous layers surrounding the hilum, the origin of the granule growth (Gallant et al., 1997). The amorphous regions contain amylopectin (Szymońska et al., 2000). The structure of granules is developed on plant vegetation by enzymatic debranching of so-called plant glycogen (Erlander, 1998). These enzymes reside inside starch granules and can be activated on starch processing.
5.3 CARBOHYDRATE CHIRALITY Chirality is a property resulting from a lack of symmetry of molecules. All carbohydrates, including polysaccharides, have centers of asymmetry and, therefore, are chiral. Chirality is expressed as the concentration-independent specific rotation, [α]o: [α]o = (α ⋅ 100)/lc
(5.1)
where α is the angle of twist determined polarimetrically, l is the length of the polarimetric tube, and c is the concentration of the saccharide in g/100 cm3. Chirality
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of freshly prepared aqueous solutions of saccharides is either variable or constant in time, depending on mutarotation (see below).
5.4 CARBOHYDRATE REACTIVITY 5.4.1 CHEMICAL AND PHYSICAL TRANSFORMATIONS OF MONO-, DI-, AND OLIGOSACCHARIDES ESSENTIAL IN FOOD CHEMISTRY 5.4.1.1 Reactions of Aldehyde and Ketone Function Mutarotation: This transformation concerns only reducing sugars, such as those with a hydroxyl group at the anomeric carbon atom. The molecules transform reversibly, either spontaneously or on acid or base catalysis, through the pyranose and furanose ring opening followed by ring closure (Structures 5.11a and 5.14 into 5.12 and 5.11b). An open-chain structure is not typical for saccharides. Aqueous D-glucose at 25°C has approximately 0.003% of the open-chain compound. Mutarotation has limited rather diagnostic significance in food chemistry and technology. Practical use of this reaction is demonstrated in milk powder manufacturing. Evaporation of milk at a rate lower than that of mutarotation of lactose yields a product with less α-lactose isomer, which crystallizes in prism- or pyramid-like form. Fast milk evaporation gives an amorphous mixture of α- and β-lactose (Structure 5.7b). Isomerization: This process involves mainly D-glucose, which is isomerized into D-fructose, which is important for food technology. Some oligosaccharides CH2OH O HO OH OH
HO
HO
D-Mannose
D-Glucose
11b
11
11a
Reduction
Reduction
CH2OH
CH2OH
OH O
O
HOH2C
O OH
OH OH
OH
OH
HO OH 12 L-Sorbitol (D-Glucitol)
STRUCTURES 5.11A–5.14
CH2OH
OH
OH
HO
OH 14
13 D-Mannitol
OH
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Chemical and Functional Properties of Food Components
can also be isomerized (Boruch and Nebesny, 1979). This process is catalyzed by glucose isomerase. Reduction to alcohols: The industrial scale reduction involves either NaBH4 or electrochemical and catalytic (Raney nickel) hydrogenation. The resulting openchain polyols, the sugar alcohols (Structures 5.12 and 5.13), have a new chiral center. In consequence, each ketose (Structure 5.14) yields two alcohols, whereas aldoses (Structures 5.11a and 5.11b) yield only one alcohol. Addition to the carbonyl group: The internal, cyclic hemiacetal formation is one of the illustrations of this type of addition. The H2N–X nucleophiles, with X being NH2 (hydrazine), NHAr (arylhydrazines), OH (hydroxylamine), NHCONH2 (semicarbazide), NHCSNH2 (thiosemicarbazide), or alkyl (primary amine), produce hydrazones, arylhydrazones, oximes, semicarbazones, thiosemicarbazones, and alkyl amines (Schiff bases), respectively, according to the following path: Structures 5.4 + 5.15 → 5.16 → … → 5.21. Hydrazones and arylhydrazones (Structure 5.20) react with the second molecule of the corresponding reagent into osazones (Structure 5.21). The reaction with mono-, di-, and lower oligosaccharides has some analytical value. Reaction with H2N–X nucleophiles in which X = CH3COOH, amino acids, nucleotides, and proteins, as well as NH3+ (ammonia) produce aldosylamino acids and aldosylamines, respectively (Structure 5.15 with X = CH3COOH or H). Aldoses undergo the Amadori rearrangement and subsequently turn into caramels, the natural brown food colorants, and/or heteroaromatic compounds—derivatives of pyrrole, imidazole, and pyrazine. Ketones react similarly into ketosylamino acids or ketosylamines. This reaction is the first step of either thermal or enzymatic changes (the Maillard reaction) (see Davidek and Davidek, 2004) resulting in the browning of food and the development of the aroma of roasted, baked, or fried foodstuffs. Oxidation: The oxidation of aldoses (Structure 5.24) with bromine or chlorine in alkaline solution (hypobromite and hypochlorite, respectively) leads to aldonic acids that readily self-esterify (lactonize) into δ- (Structure 5.25) and γ- (Structure 5.26) lactones residing with free acid (Structure 5.27) in equilibrium. β-Conformers oxidize more readily than α-conformers. Glucono-δ-lactone (Structure 5.25) has found its application in baking powders, raw fermented sausages, and dairy products, where the slow release of acid is required. The oxidation with Cu2+ (the Fehling, Benedict, and Barfoed tests) and Bi3+ (the Nylander test) are the only analytical reactions for reducing sugars. 5.4.1.2 Reactions of the Hydroxyl Groups Esterification: Saccharides are commonly esterified with acyl chlorides, as well as organic and inorganic acid anhydrides. These reactions can be run either exhaustively with involvement of all hydroxyl groups of the saccharide molecules or selectively. In the latter case, a protection (blocking) of certain hydroxyl groups is required. For instance, all hydroxyl groups of D-glucose, except that at C3, can be protected in reaction with acetone in an acidic medium. The resulting 1,2,5,6-di-O-isopropylidene-α-D-glucofuranose (Structure 5.28), after acylation at a nonprotected
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103
CH2OH
CH2OH
OH
O
H
H +
OH OH
HO
N
X
H
N
OH
X
OH
HO OH
OH 4
16
15 CH2OH OH N
OH
X
HO OH 17
CH2OH
CH2OH OH
OH N
OH
X
N
OH
H
HO
X
H
HO
H O
O 19
18 Amadori rearrangement
+ 2 NH2NH2
CH2OH
CH2OH
OH
OH N
OH
H
HO
X
+ 2 NH2NH2
N
OH HO N NH2
O 20
NH2
H
21
STRUCTURES 5.15–5.21
hydroxyl group to give monoacylated diketal (Structure 5.29), is then decomposed with carboxylic acid into a 3-monoacylated saccharide (Structure 5.30). Such reactions can be applied to polysaccharides, although polysaccharides readily esterify with carboxylic acids simply on heating of their blends (Tomasik and Schilling, 2004). The acylation of mono- and oligosaccharides and their derivatives, mainly sorbitol and sucrose, with higher fatty acids yields surface-active agents and fat replacers (Wang, 2004). Hydrolysis of the acyl group can be achieved by either transesterification (water or alcohol) or ammonolysis.
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Chemical and Functional Properties of Food Components CH2OH
CH2OH
CH2OH
OH
OH N
OH
R
OH
N
OH
HO
R
H
HO
H O
OH
18
O
22
CH2OH
23
CH2OH
O
CH2OH
O Br2/OH
O
-
O
Br - HBr
OH
OH
OH
HO
N
OH
H
HO
O
OH
HO
HO
OH
OH
OH
25
24
4
CH2OH
CH2OH
OH
O
HO
O
OH
OH
C
OH
O
HO OH
OH
27
26
D-Gluconic acid
STRUCTURES 5.22–5.27 CH2OH O
Me2C OH
O
Me
O CH2
Me
O
Ac2O
+
OH
H
OH
HO
O
OH
Me
Me
O
4
Me
Me
28
CH2OH
O CH2 O
Py
O
O
O
AcOH OAc O
OH
OH
O
HO Me
OH Me
29
STRUCTURES 5.28–5.30
30
R
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Etherification: This reaction may involve hydrochloride catalyzed glycosidation with alcohols, exhaustive methylation with dimethyl sulfate in an alkaline medium, exhaustive methylation with methyl iodide in the presence of Ag2O, and glycosidation, which is the substitution of an α-halo atom of α-halopentadecyl sugars with methanol in the presence of Ag2CO3. Commonly, polysaccharides are etherified with alkyl halides and their derivatives with substituents in the alkyl groups such as the carboxylic, amino, and quarternized amino groups. Reaction with epoxy compounds also results in etherification. Such reactions with small alkyl groups are important for saccharide structural analysis. Reactions with long-chain alkyl halides and epoxides provide products useful as biodegradable detergents (Wang, 2004). Reactions with halocarboxylic acids and haloalkyl amines lead to anionic and cationic starches. Anionic starches are required components of polysaccharide—protein complexes, while cationic starches are designed as components of cellulose pulp. Halogenation: The hydroxyl group at C1 is readily substituted with a halide atom (X) when a pentacetylated saccharide is treated with HX in acetic acid. Such saccharides are suitable for synthesis of desoxysaccharides. Exhaustive replacement of all hydroxyl groups in lower saccharides and polysaccharides can be achieved with reagents suitable for such reaction with simple alcohols, such as COCl2, SOCl2, POCl3, PCl3, and PCl5. Chlorination of some sugars results in products of increased sweetness (Table 5.3). Dehydration: It is an intramolecular elimination of one water molecule producing 1,6- (Structure 5.31), 3,6- (Structure 5.32), or 1,2- (Structure 5.34) anhydrosugars, as well as saccharide enol (Structure 5.33), and ketone (Structure 5.35). Anhydrosugars can be utilized for the synthesis of some derivatives, such as amino sugars and others. Dehydration is the first step of sugar caramelization. Further
TABLE 5.3 Relative Sweetness (RS) of Various Substances in 10% Aqueous Solutions (RS of Sucrose = 1.0) Substance Sucrose β-D-Fructopyranose Inverted sugar D-Glucose αβD-Mannopyranose αβD-Galactopyranose Maltose D-Lactose αβD-Galactosucrose Raffinose
RS 1.00 1.80 1.30 0.70 0.80 0.30 Bitter 0.32 0.32 0.20 0.30 Tasteless 0.01
Substance Stachyose 1′-Chloro-1′-desoxysucrose 4-Chloro-6-desoxysucrose 6-Chloro-6-desoxysucrose 1,4,6′-Trichloro-1,4,6-tridesoxygalactosucrose Mannitol Sorbitol Xylitol Honey Molasses Saccharin Cyclamates Aspartame Neohesperidin dihydrochalcone
RS 0.10 0.20 0.05 Bitter 20.00 0.40 0.60 0.85–1.2 0.97 0.74 200–700 30–140 200 2000
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Chemical and Functional Properties of Food Components
heating of dehydrated saccharides results in the formation of three subsequent compounds called caramelan, caramelen, and caramelin (Tomasik et al., 1989). 6 C12H22O11 – 12 H2O = 6 C12H12O9 (caramelan) 6 C12H22O11 – 18 H2O = 2 C36H18O24 (caramelen) 6 C12H22O11 – 27 H2O = 3 C24H26O13 (caramelin) b
O CH2O
O
a
H
H2O
O
HO
O
HO
+
HO
HO OH b
OH
OH
a
OH
31 Product a
4
32 Product b
- H2O
+ H2O
HO
OH
HO
CH2OH O
HO
CH2OH O
HO
HO O
O
H
33
34
CH2OH O
HO HO
O 35
AcO
CH2OAc O
AcO
AcO
CH2OAc O
HO
AcO
CH2OH O
HO
AcO Br 36
HO
38
37
CH2OH O
H2O
HO
CH2OH O
H2SO 4
STRUCTURES 5.31–5.41
-
OSO3H
OH 41
CH2OH + O
HO
HO
HO
HO
40
39
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Reduction: A multistep reaction leads to desoxysaccharides. It involves 1-halopentacetylated saccharide, which is dehalogenated with zinc into acetylated glucal (Structure 5.35). Hydrolyzed glucal accepts sulfuric acid, the SO3H residue, which is readily hydrolyzed into desoxysugar (Structure 5.41). Oxidation: Apart from CO2 and H2O, there are three series of products that result from the oxidation of saccharides. They are 2,3-dialdehydes (Structures 5.43 and 5.49) formed on the oxidative cleavage of saccharides (Structures 5.42 and 5.48) with periodates, the sole oxidants providing this course of oxidation [except Pb(IV)]. Such dialdehydes are considered toxic. Further oxidation of dialdehydes leads to glyceric acid (Structure 5.45), glyoxalic acid (Structure 5.47), hydropyruvic acid (Structure 5.46), and erythronic acid (Structure 5.51), as shown below for the oxidative cleavage of sucrose (Structure 5.42) and maltose (Structure 5.48). The oxidation of monosaccharides, such as D-galactose (Structure 5.52) with strong oxidants proceeds at both C1 and C6 atoms, leaving dicarboxylic, aldaric acids (Structure 5.53). Aldaric can lactonize into lactones (Structure 5.54). Lactones produce uronic acids (Structure 5.54) when reduced with sodium amalgam. The oxidation at C1 can be prevented by protection of the 1–OH group. The glycosidic bond in oligosaccharides offers sufficient protection. Application of weak oxidants offers a direct route to uronic acids. Complex formation: The hydroxyl groups offer two types of interactions with molecules having either a clearly dipole character or charge, that is, ions. The hydrogen atoms of these groups are capable of interactions with electron-excessive sites of dipoles and anions, whereas lone electron pairs of the oxygen atom are electron donors for cations and the positive side of dipolar molecules. The complex formation is a general ability of saccharides. Fruitful results were noted in the case of Ca2+ salts, preferably chloride, and hydrogen carbonate. The cation forms fairly stable compounds. This property was widely utilized in sugar manufacture for the separation of sucrose from its syrup. Saccharide alcohols also coordinate metal ions. Depending on the cations, their optical rotation is affected to a different extent, but always in the order of Na+ < Mg2+ < Zn2+ < Ba2+ < Sr2+ < Ca2+. Complexes of saccharides with a wide variety of cations were prepared and characterized (Angyal, 1989). Saccharides also complex to other, nonmetalic compounds, including organic food components such as polysaccharides and proteins. The products of the interactions are sorption complexes with involvement of hydrogen bonds. The energies of the complex formation are low and do not exceed 4 kJ/mole (Tomasik et al., 1995). Nevertheless, they are essential in food texturization and thermal stability (Ciesielski and Tomasik, 1996; 1998; Ciesielski et al., 1998). The complexation itself can seriously affect sugar metabolism in the organism. 5.4.1.3 Reactions of the Glycosidic Bond As a typical acetal bond, a glycosidic bond readily hydrolyzes in an acid-catalyzed reaction. In this manner, di- and oligosaccharides can be split into monosaccharides. It is a common method for the manufacturing of invert sugar, a mixture of α-D-glucose and β-D-fructose, from sucrose.
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Chemical and Functional Properties of Food Components
CH2OH O
HOCH2 O
OH
CH2OH
O
O
CH2OH
OH
O
HO
CH2OH
OH
OH O
HO
OH
OH
OH
OH
OH 48
42
+ 4 IO4-, - 2HCOOH
+ 3 IO4-, -HCOOH CH2OH
CH2OH
CH2OH
O
O
O
HOCH2 O
HC
HC
O
O
HC
O
CH HC
O
O
CH2OH
CH O
HC
O 49
43
+ Br2 , H2O
+ Br2 , H2O CH2OH O
CH2OH
CH2OH
O
O
HOCH2 O
COOH
COOH
COOH HOOC
O HOOC
CH2OH CO OH
O
HOOC
+ H+
+ H+
OH CH2OH 45
COOH
50
44
COOH
CH
O
O
O
CH2OH O
CHO
CO 2
COOH
OH OH
COOH 46
COOH
47
D-Glyceric Hydroxypyruvic Glyoxalic acid acid acid
CH2OH 51
CH2OH O
CHO COOH
COOH 46
47
Erythronic acid
STRUCTURES 5.42–5.51
5.4.1.4 Specific Reactions of Saccharides In strongly acidic media, saccharides produce furan derivatives in a sequence of reactions that are rearrangements and dehydrations followed by cyclization. Similar products are available thermally. Pentoses and hexoses give furan-2-aldehyde and 5-hydroxymethylfuran-2-aldehyde, respectively. Both products are responsible for the specific aroma of caramel and burnt sugar.
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109
OH CH2OH O
HNO3 OH
HO
OH COOH O H HO
C
OH
OH
OH
O 53 Mucic acid
52 D-Galactose OH COOH O
Na/Hg +
H
HO OH
OH COOH O OH
O
54 D-Galactaric acid monolactone
OH
HO
55 D-Galacturonic acid
STRUCTURES 5.52–5.55
In weakly acidic and neutral media, the reactions proceed at a lower rate. Reductones (Structures 5.55 through 5.57), compounds with the carbonyl groups vicinal to an endiol moiety, which are formed, are stable at pH < 6, and act as natural antioxidants. They transform into desoxysugars, uloses (Structure 5.58). The latter undergo cyclization into 5-hydroxymethylfuran-2-aldehyde (Structure 5.62). Corresponding osuloses (Structure 5.63) in similar sequences of transformations yield diacetylformosine (Structure 5.69). In acid-based reactions, 2-acetyl-3-hydroxyfuran (isomaltol) (Structure 5.76), 3hydroxy-2-methylpyran-4-one (Structure 5.77), and maltol (Structure 5.79) are formed. They are responsible for the aroma of baked bread. Endiols (Structure 5.80) can isomerize into other saccharides in the Lobry de Bruyn-van Ekenstein rearrangement, Thus, D-glucose (Structure 5.4) can isomerize into mannose (Structure 5.81) and fructose (Structure 5.1) accompanied by a small amount of D-psicose (Structure 5.83). An alkaline medium provides isomerization to disaccharides, which turn from aldoses into ketoses, as shown for lactose (Structure 5.7b) isomerized to lactulose (Structure 5.84). Because the enolization is not restricted to the 2 and 3 positions, a number of products are formed that undergo subsequent aldol condensations and the Cannizzaro oxidation. They are all 2-hydroxy-3-methyl, 3,4-dimethyl-2-hydroxy, 3,5-dimethyl2-hydroxy, and 3-ethyl-2-hydroxy-2-cyclopenten-1-ones; γ−butyrolactone; and such furan derivatives as furyl alcohol, 5-methyl-2-furyl alcohol, and 2,5-dimethyl-4hydroxy-3(2H)-furanone. These are food flavoring agents.
5.4.2 CHEMICAL AND PHYSICAL TRANSFORMATIONS OF POLYSACCHARIDES Although starch is the most widely and massively studied, and has found numerous large-scale applications, pectic polysaccharides (MacDougall and Ring, 2004), fructans (Praznik et al., 2004), chitin and chitosan (Einbu and Vaarum, 2004), and hemicelluloses,
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Chemical and Functional Properties of Food Components
CH2OH O
HO
CH2OH O H
HO OH
OH
HO
4
- H2O
O H
HO OH
O
HOH2C
+
OH
H
56
CH2OH
OH OH 1
CH2OH O H
HO
C O +
O
CH2OH O H
HO
C
H
- H2O
O
H
O
57
58 3-Desoxy-D-glucosulose
H
H
HOH2C
CH2OH O H C O
HO
O
C O H O 60
59
H CO HOH2C
O 61
OH
H
- H2O HOH2C
O
C
O H
62
STRUCTURES 5.56–5.62
are also modified on an industrial scale. The products of their modifications are widely utilized in food technology and everyday food preparations. Food processing, such as cooking, baking, frying, and pickling, usually induce food carbohydrate transformations. The functional group reactivity in polysaccharides is, to a great extent, obstructed by their macrostructure. These potential reaction sites, which do not reside on the surface, may be unavailable for many reagents, as they are either hidden inside the macrostructure or involved in the formation of intra- and intermolecular hydrogen bonds, crucial for macrostructure properties. Many reactions of polysaccharides are
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111
OH OH HO
HO
O H
O
O O
OH
O H
CH3
63
CH3 64 OH
OH HO
HO
OH
O
O O
OH O H
CH3
CH3 66
65
O H O
O
OH
CH3 OH
O O
CH3 67
O
HO H3C
O
CH3 68
OH
O
CH3
69
STRUCTURES 5.63–5.69
governed by the heterogenicity of the reaction system. Only randomly reacting polysaccharides are solvent soluble. Among polysaccharides utilized either on or after transformation, hemicelluloses are exclusive. They are water soluble or they swell. Problems of solubility and compactness are encountered, particularly in cellulose and starch, which are fibrillar and granular, respectively. The reactivity in terms of the rate and degree of transformation can be controlled by either application of suitable reagents or loosening of the compact structure with involvement of a physical action. The solvent effect (water in the case of starch and higher alcohols [Ruck, 1996] in case of cellulose) is the most commonly used tool. But high pressure; sonication with ultrasounds; ultraviolet, microwave, and polarized light (Fiedorowicz et al., 2001), glow plasma (Lii et al., 2002a), corona discharges (Lii et al., 2003b), ionizing radiation, thermolysis; and deep freezing and thawing (Szymońska et al., 2000) might also be suitable for loosening a compact polysaccharide structure (Tomasik and Zaranyika, 1995). Pasting and gelatinization of starch by heating it in water or immersing it in aqueous alkali delivers pregelatinized starch. Because such processing breaks several intra- and intermolecular hydrogen bonds, pregelatinized starch is easily water soluble and chemically more active. When granular starch is passed under pressure through narrow nozzles, so-called α-starch (nanostarch) with improved solubility in water is formed. Physical modifications of macrostructure frequently result in depolymerization of the polysaccharide. Starch granules are composed of amylose and amylopectin, together forming crystalline and amorphous regions of the granule. The polysaccharides in an amorphous region are more susceptible to enzymatic digestion than in crystalline regions.
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Chemical and Functional Properties of Food Components
OH
O
HOH2C
OH
HO
O H
CH2OH
OH
- H2O O H
OH
CH2OH
OH
+
70
1
H OH
OH HO
HO
O
O O
O H OH
OH
CH3
CH2 72
71 OH HO
O H
O H
O O
OH
CH3
73
O OH
74
a
b
HO
OH
O H3C
C HO
O O 75
H3C HO
O
O C
O 77
HO
H3C
CH3
O
HO
O H HO
76 H3C
Isomaltol H3C
O 79
HO
O 78
Maltol
STRUCTURES 5.70–5.79
Starch in the latter region is one of four forms of so-called resistant starch (RS). Resistant starch is used as a prebiotic—a nutrient for probiotic bacteria colonizing the human intestine. Another type of resistant starch is available by chemical modification of starch (Fiedorowicz et al. 2004; Kapuśniak and Marczak, 2005). Controlled
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113
CH2OH HO
OH
O
HO
OH
HO HO
OH
OH
OH
81
80
CH2O
O
HOH2C
OH
HO
CH2OH
OH
H
HO
OH
O
HO
CH2OH
HO O
OH
OH
OH
OH 82
1
CH2OH O
HO
83 D-Psicose
OH O
OH
O
HOH2C
CH2OH OH
OH
OH O OH
OH
HOH2C
OH
HO
O
O
OH
HOH2C
7b
84 Lactulose
STRUCTURES 5.80–5.84
swelling of starch granules in water can remove a considerable part of the amorphous interior of the starch granule, producing empty domains within the granules utilized in microcapsulation (Korus et al., 2003). Such exudation of the amorphous content of granules begins in the process of starch isolation (Starzyk et al., 2001). The functional properties of starch depend on the amylose-to-amylopectin ratio, For some purposes, amylose-rich starch (Hylon starch) is more beneficial and, for the others, application of amylopectin-rich (waxy) starch is advantageous. Such starches are usually genetically engineered. However, recently enrichment of granular starches in linear, amyloselike polysaccharides with illumination of starch by linearly polarized light was described (Fiedorowicz et al., 2001). 5.4.2.1 Depolymerization of Carbohydrates If not utilized in the pulp industry, hemicelluloses are hydrolyzed in the acidcatalyzed process, mainly to monosaccharides and to furan-2-aldehyde (pentosanes) and 5-hydroxymethylfuran-2-aldehyde (Structure 5.62) (hexosanes). Monosaccharide-containing syrups, after purification, are either fermented or utilized as wood molasses for feeding ruminants. In another approach, xylose, the least soluble
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component of the syrup, is allowed to crystallize. The separated xylose is then hydrogenated over an Ni/Al catalyst at 120°C under 6 × 106 Pa into xylitol. Hemicelluloses, together with proteins, participate in the Maillard reaction and may contribute to the overall secondary food aroma of processed foodstuffs (Tomasik and Zawadzki, 1998). The acid-catalyzed hydrolysis of cellulose, the saccharification, results in splitting the terminal D-glucose unit of the fibril. Thus, under thorough control of the reaction conditions, D-glucose is the sole product. Glucose syrup is either a source of pharmaceutical-grade D-glucose or is fermented to ethanol. The β-glycosidic bond of cellulose can be split thermally. Perhaps the most ancient polysaccharide processing—dry wood distillation—delivers charcoal, water, tar, methanol, acetone, acetic acid, and gases. The liquid and gaseous fractions result from the thermolysis of thermally split D-glucose. Thermolysis of cellulose with proteogenic α-amino and α-hydroxy acids produces several aromas potentially interesting for the food and cosmetic industries. Starch and pectins also generate aromas on heating with these acids (Bączkowicz et al., 1991; Sikora et al., 1998). Depolymerization of starch yields dextrins—one of the most useful products of the food industry. The most frequent dextrinization involves proton catalysis or heat. Hydrochloric acid is a superior catalyzing proton donor, but organic acids are also capable of starch dextrinization. Such processes extended in time can lead to the saccharification of starch into oligosaccharides and, finally, to D-glucose, maltose, and glucose syrups, which are used directly as sweeteners or are fermented. Thermal dextrinization of starch up to 260°C, produces canary-yellow dextrins called British gums. They differ in properties and applications from dextrins from acid hydrolysis. Dextrins are also available by UV irradiation and ionizing radiation, as well as other types of physical action on starch (Tomasik and Zaranyika, 1995). Dextrins are commonly used as food thickeners, plasticizers, and adhesives. Depolymerization of polysaccharides to formaldehyde seems to be particularly promising to the chemical industry as a versatile, renewable source of various chemicals and a key process for utilization of polysaccharides in the 21st century. Reduction of formaldehyde delivers methanol, one of the most important chemical reagents. 5.4.2.2 Chemical Modification of Polysaccharides without Attempted Depolymerization Polysaccharides offer practically the same kind of reactivity as monosaccharides, except the reactions on the anomeric carbon atom, because in a waste majority of polysaccharides the hydroxyl groups at this atom take part in the polysaccharide chain formation via the glycosidic bonds. In the long chains of polysaccharide, only the terminal saccharide units carry free hydroxyl groups at the anomeric carbon atom. However, even such minute modification in this position can be reflected by changes in the rheological properties of polysaccharide solutions, pastes, and gels. There are some polysaccharides naturally containing additional functional groups, other than the hydroxyl groups. Thus, chitin contains acetamido groups. Alginates, many plant gums (Arabic, gatti, karaya, tragacanth, and xanthan, the latter is semisynthetic), pectins, some galactans, and xylans contain the carboxylic groups.
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Heparin, furcellaran, and carrageenans carry sulfate functions. These groups can be utilized in chemical modifications of those polysaccharides. Limitations in the possibility of chemical modifications of starch result from steric hindrances of the reaction sites, solubility, viscosity of the reaction medium, and susceptibility to side reactions. Furthermore, depolymerization almost always accompanies the intended modification. As a rule, polysaccharides are soluble, although frequently only sparingly, in water and dimethyl sulfoxide. Polysaccharides solubilize on xanthation, that is, on reaction with CS2 in an alkaline medium, to form syrups of xanthates. On acidification, the polysaccharides could be recovered together with CS2. This procedure was utilized for several decades for production of artificial silk from cellulose. Polysaccharides undergo hydrolysis; reduction to alcohols, oxidation to aldehydes, ketones, and carboxylic acids; esterification with inorganic (sulfuric, phosphoric, nitric, boric, and sillylic) and organic acids; etherification; acetylation with aldehydes; halogenation with the same reagents as mono- and disaccharides; ammination (usually via halogenated polysaccharides); carbamoylation with acylamides or isocyanates; and metallation. Only some of the large number of potential modifications achieved approval under the food laws of particular countries. Thus far, only starch, cellulose, and pectin are chemically modified for nutritional purposes and can be used in foodstuffs. If biodegradable materials are also considered, modification of other polysaccharides can be taken into account. Practical modification of pectins is limited to changes in the degree of their methylation. In this manner, the strength of their jellies can be controlled. Modification of cellulose for use in the food industry is limited to its esterification. Mainly cellulose acetate is produced. It is used for special membranes for treating water and fruit juices. Among ethers, methyl- and methylhydroxypropyl celluloses deserve particular attention. They are available by methylation with common methylating agents and propylene oxide, respectively. Carboxymethyl cellulose (CMC) is the product of etherification of cellulose with chloroacetic acid; it usually has a degree of substitution from 0.3 to 0.9. All 2-, 3-, and 6-hydroxyl groups do react. Modified and derivatized cellulose have found their application in the food industry as nondigestible components of low-calorie meals. CMC is a texturizing agent and edible adhesive. The chemical modification of starch for nutritional purposes involves oxidation, but only with a limited number of oxidants; esterification with a limited number of reagents; etherification; and complex formation. Metal derivatives might have some significance as carriers of bioelements and therapeutic agents, primarily inulin substitutes (Tomasik et al., 2001). By forming Werner-type complexes with metal ions (Ciesielski et al., 2003; Ciesielski and Tomasik, 2004), the polysaccharides can influence administration, excretion, and functioning of metal ions in living organisms. Oxidation of starch to aldaric and uronic acid-type carboxylic starches for practical purposes should leave no more than one carboxylic group per each 25th glucose unit. The oxidation should be carried out with a possible high depolymerization degree and the formation of the smallest possible number of terminal anomeric carbon atoms. Gels from oxidized starches have low viscosity and good transparency. Such oxidation is provided by sodium hypochlorite. This oxidant only
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randomly oxidizes the 6-hydroxymethyl groups of starch. The latter groups are readily oxidized with nitrogen oxides. Metal ion-catalyzed air oxidation of starch results in simultaneous formation of carboxyl and carbonyl starches. The most useful esters of starch are phosphates, commonly used as gelating agents, acetates, and adipates—the film-forming materials. Only these preparations are utilized in foods, which have a low degree (0.2 to 0.0001) of esterification. Higher derivatized starches are used as fat substitutes and emulsifiers (Wang, 2004). The most important and widely used starch ethers are carboxymethyl, hydroxyethyl, and hydroxypropyl starches. Some functional groups introduced into starch by esterification or etherification can dissociate. For instance, all starch sulfates, phosphates, and carboxylates dissociate in aqueous solution, leaving a negative charge on starch. They are called anionic starches. Such products are capable of forming complexes with metal ions and with proteins (Schmitt et al., 1998). They may be utilized as biodegradable plastics and meat substitutes. If the starch was etherified with a reagent introducing a tetralkylammonium salt function, the dissociation develops a positive charge on the molecule and such starch becomes cationic. Such starches are used in the manufacture of paper and as detergents. Polysaccharides are important complexing agents for inorganic and organic gases, liquids, and solids. Usually surface sorption is involved, but in case of starch, inclusion complexes inside the amylose helix and eventually short helices of amylopectin, and capillary complexes involving capillaries between starch granules are formed. All of them exist in a natural, native form; they can also be formed in several common operations of food processing, such as dough formation, foam beating, and scrambling egg yolk with sugar. Formation of inclusion complexes of starch becomes a more common method of protecting some volatile, as well air-sensitive, food components (microencapsulation). Such complexes are essential for food texturization and overall stabilization. 5.4.2.3 Retrograded, Cross-Linked, and Graft Polysaccharides Retrogradation is a very common reaction of gels of starch polysaccharides. It leads to enhanced molecular-weight systems. Amylose gels retrograde within hours, whereas retrogradation of amylopectin takes days and even weeks. This process is manifested by dendrite formation in the gel and in bread by bread staling and water expulsion. This phenomenon is due to the orientation of chains of polysaccharides with respect to one another to aggregate with involvement of intermolecular hydrogen bonds. The retrogradation affinity depends on the starch variety and decreases in the following order: potato > corn > wheat > waxy corn starch. Evidently, the retrogradation rate and nature of the formed amylose crystals depend on the starch source, amylose-to-amylopectin ratio, and storage temperature. Low temperatures around the freezing point and polar gel additives favor retrogradation. Retrograded starch is utilized as a component of low-calorie foods. Polysaccharide cross-linking frequently occurs when it is acetylated, esterified, or etherified with corresponding bi- and polyfunctional reagents, such as POCl3, polyphosphates, anhydrides, aldehydes, carboxylic acids, and carboxyamides. Grafting is most
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commonly performed with vinyl monomers. Grafting competes with homopolymerization of vinyl monomers. The latter can be suppressed by adjusting the suitable catalyst and reaction mechanism. Usually cross-linked and grafted polysaccharides have enhanced water-binding capacity, lower aqueous solubility, and shear force stability.
5.4.3 ENZYMATIC CONVERSIONS
OF
CARBOHYDRATES
With few exceptions, enzymatic processes cause degradation of carbohydrates. Enzymes are used in the form of pure or semipure preparations or together with their producers, that is, microorganisms. Currently, semisynthetic enzymes are also in use. Alcoholic fermentation is the most common method of utilization of monosaccharides, sucrose, and some polysaccharides, such as starch. Hydrolysis of polysaccharides with alpha-amylase, so-called alpha-amylolysis, is the common way of hydrolysis of starch to maltodextrins. Recently, it has been shown that such reaction can be stimulated by illumination of the enzyme with linearly polarized light. The amylolysis can be accelerated by several orders (Fiedorowicz and Khachatryan, 2003). Lactic acid fermentation is another important enzymatic process. Lactic acid bacteria metabolize mono- and disaccharides into lactic acid. This acid has a chiral center; thus either D(–), L(+), or racemic products can be formed. In the human organism, only the L(+) enantiomer is metabolized, whereas the D(–) enantiomer is concentrated in the blood and excreted with urine. Among lactic acid bacteria, only Streptococcus shows specificity in the formation of particular enantiomers, and only the L(+) enantiomer is produced. Enzymatic reduction of glucose-6-phosphate (Structure 5.85) into inositol-1-phosphate with cyclase and reduced NAD coenzyme, followed by hydrolysis with phosphatase, presents another nondegrading enzymatic process proceeding on hexoses. Inositol (Structure 5.86) resulting in this manner from its phosphate, plays a role in the growth factor of microbes. Its hexaphosphate, phytin, resides in the aleurone layer of wheat grains. There are also known bacteria that polymerize mono- and oligosaccharides. Leuconostoc mesenteroides polymerizes sucrose into dextrin—an almost linear polymer of 400 or more α-D-glucose units. Dextran is also generated in frozen sugar beets. This causes difficulties in sugar manufacturing if the beets have to be stored at low temperature. Dextran serves as a blood substitute and chromatographic gel (Sephadex). Other polysaccharides synthesized by bacteria are levan, a polymer of β-D-fructose, pullulan, a polymer of α-D-glucose, and xanthan gum, a polymer of β-D-glucose and α- and β-D-mannoses. CH2
O
OH
PO3H2
O OH
O OH
OH OH
HO
HO OH
OH 85
STRUCTURES 5.85–5.86
86
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Amylo-1,6- -gluconase (Dextrinase)
-
-
Cyclodextrin
Bacillus macerans
Glucoamylase
-Glucosidase
-Amylase
Glucose Glucose
Maltose
Glucose isomerase Fructose
-Amylase Pullulanase and glucogenase
Dextrin
Dextrin
FIGURE 5.4 Enzymatic transformations of starch.
The enzymatic oxidation of sucrose with glucose oxidase to D-glucose-δlactone consumes oxygen dissolved in beer and juices. In this manner the rate of undesirable processes caused by oxidation, such as color and taste changes, is decreased. All essential enzymatic polysaccharide transformations deal with degradation (Figure 5.4). In the case of cellulose, this degradation leads to glucose. Endoglucanase and cellobiohydrolase attack the amorphous regions of the compact structure of cellulose, producing D-glucose and cellobiose, respectively. Starch, amylose, and amylopectin are not necessarily as deeply degraded. There are several amylolytic enzymes capable of starch degradation. They provide high specificity of their action. Synthesis of cyclodextrins (cycloglucans, Schardinger dextrins) presents a special case. Slightly hydrolyzed starch is transformed into cyclic products composed of six, seven, and eight α-D-glucose units, α−, β−,and γ-cyclodextrins. The yield of cyclodextrins declines with the number of glucose units in cycles. Although higher-membered cyclodextrins are also formed in the reaction mixtures, their minute yield seriously limits their potential applications. Cyclodextrins are sometimes cross-linked into cyclodextrin resins with interesting inclusion properties. There are several other known enzymatic conversions of saccharides, oligosaccharides, and polysaccharides (Bielecki, 2004).
5.4.4 CEREAL
AND
TUBER STARCHES
All botanical varieties of starches can be primarily classified into tuber and cereal starches. Only the true sago starch is isolated from the sago palm trunk (Indonesia).
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Differences in size and shape of the granules, the amylose-to-amylopectin ratio, and protein (7 to 13% content), lipid (1.5 to 6% content), other carbohydrate (5 to 23% content), and mineral (1 to 3% content), do not necessarily depend on whether a given starch belongs to one of the above two classes. Potato starch, the tuber starch, has the largest granules (up to 150 µm) and as the sole starch, it has amylopectin esterified with phosphoric acid (Seideman, 1966; Jane et al., 1994). However, starch granules of wild yam (Diascorea dumetorum), which has one of the finest granules ever seen, also originates from tubers (Nkala et al., 1994). Generally, cereal starches are richer in lipids (e.g,. cornstarch), and tuber starches are richer in proteins, although the oat starch, a cereal starch, is one of the richest in lipids and proteins (see Table 5.2). Polysaccharides, lipids, and proteins usually reside in granules in native complexes. These complexes are stronger in cereal starches. Thus, potato starch can readily be isolated relatively free of proteins, whereas the complete defatting of cornstarch could not be done. It might be due to the structure of such complexes. Residual lipids in cornstarch reside inside of the amylose inclusion complex, and protein molecules are too large to be included in the amylose helix. The main difference between tuber and cereal starches comes from their crystallographic pattern: A for tuber and B for cereal starches. These two patterns result from different mutual orientations of amylose helices inside the granule, for example, single (A-type) and double (B-type) (Figure 5.3). These differences determine several essential properties of both classes of starches: swelling, gelation, and course of pasting, and affinity to various physical, physicochemical, and chemical modifications. There are also differences in the taste, digestibility, and nutritive value of particular starches. The most common sources of starch in various regions of the world are potato, maize, cassava (manioc, tapioca, yucca), and rice. Recently, interest in wheat starch has considerably increased. The popularity of a given starch and starchy plants do not go together. For instance, in several regions of the world rye is commonly used, but isolation of starch from it is difficult due to the mucus present in that grain, which obstructs the isolation. The properties of that starch do not justify the higher costs of its isolation.
5.5 FUNCTIONAL PROPERTIES OF CARBOHYDRATES 5.5.1 TASTE Saccharides are usually associated with sweet taste, although some among them are bitter and nonsweet saccharides (Table 5.3). Except for sucrose, the sweetness decreases with the number of monosaccharide units going toward oligo- and polysaccharides, because only one monosaccharide unit interacts with the mucoprotein of the tongue receptor. The quantum-mechanical treatment of sweetness, in terms of interaction of sweeteners with receptors, was recently given by Pietrzycki (2004). Because a number of powerful synthetic and natural nonsaccharide sweeteners are available on the market, apart from reduction to saccharide alcohols, other derivatizations of saccharides, even if they increase their sweetness (Table 5.3), have no practical significance.
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The following carbohydrate sweeteners are in common use (their relative sweetness [RS] with respect to a 10% aqueous solution of sucrose is given in Table 5.3). D-glucose: because of fast resorption, it is a source of immediately available energy. It is used in injections and infusion fluids for children and patients in their recovery period. Its metabolization requires insulin, and it causes tooth cavitation. D-fructose: the most readily water-soluble sugar. It does not crystallize from stored juices. Because of its hygroscopicity, it retains moisture in sugarpreserved food and intensifies its flavor and aroma. The metabolism of Dfructose delivers less energy than sucrose. This saccharide neither causes nor accelerates tooth cavitation. It accelerates ethanol metabolism. In the organism, D-fructose metabolizes into glycogen, so-called animal starch, which is the energy reservoir stored in the liver. Lactose: a sparingly water-soluble (20% at room temperature) sugar present in mammalian milk (4.8 to 5.1%). It is utilized as a carrier of other sweeteners. It improves flavor, produces a good image of food processed in microwave ovens, and improves the taste of dairy products. Sucrose: the most common sweetener used for its pleasant taste. It is widely used as a preservative of marmalades, syrups, and jams. Osmotic phenomena are involved. Due to the competition of microorganisms and preserved foodstuffs for water molecules, the microorganism tissues undergo plasmolysis. Aqueous solutions containing 30% sucrose do not ferment, and 60% solutions are resistant to all bacteria but Zygosaccharomyces. Maltose: a slightly hygroscopic disaccharide of mild and pure sweet impression. Its solutions have low viscosity. Its color is stable regardless of temperature. Starch syrups: these result from starch saccharification. The saccharification can be completed in various stages. The first sweet product, maltotetraose syrup (RS = 0.25), is viscous. As the saccharification proceeds, the viscosity of syrups declines and their RS increases. Syrups are water-soluble, do not retrograde, and are readily digested. Glucose syrup, the final product of saccharification, may be converted by isomerization into fructose syrups (Table 5.4) or hydrogenated into D-sorbitol. Apart from the sweetness and low energetic value (17.5 kJ/g), the texturizing and filling properties of syrups are utilized in practice. Malt extract from barley malt obtained by aqueous extraction: contains 4 to 5% sucrose; spare amounts of D-glucose, D-fructose, and maltose; proteins; and mineral salts. Maple syrup and maple sugar from juice of Acer saccharium maple trees: contains 98% saccharides, 80 to 98% of which is sucrose. Sugar alcohols (D-sorbitol, D-xylitol, and D-mannitol): perfectly water-soluble, soluble in alcohol, and more stable at low and high pH values than saccharides. Their sweet taste lasts for a prolonged time and is accompanied by a cool impression. They metabolize without insulin. The energetic values of xylitol, sorbitol, and mannitol are 17, 17, and 8.5 kJ/g, respectively.
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Therefore, they can be used as sweeteners for diabetics and consumers with obesity. Because of hygroscopicity, they are used as food humectants. Compounded sweeteners, blends of various sweeteners: the composition of such blends depends on the purpose for which they are designed. Usually, they are blends of various saccharides, but sorbitol, sugar syrups, and even malic acid are also compounded. Honey: this natural product has a composition depending on the harvest time, geographical region, and origin and kind of flowers from which the nectar was collected. Even the variety of insects is a factor. Fructose, glucose, and maltose constitute approximately 90% the total sugar content. There is also a rich variety of free amino acids and other organic acids, minerals, pigments, waxes, enzymes, and pollen. The latter may create allergic reactions. Honey may contain toxic components from poisonous plants, although there are several poisonous plants that give nonpoisonous honey. In some countries, mainly in Eastern Europe, aqueous solutions of honey are fermented into honey-flavored wine (mead). Fermentation involves 1 volume of boiled honey with 0.5 to 4 volumes of water. The fermentation of concentrated solutions provides a soft beverage with up to 18% alcohol and dry product resulting from the fermentation of diluted honey solutions.
5.5.2 COLORANTS Sugars are utilized for generation of caramel, a brown colorant for food (Tomasik et al. 1989). For this purpose, sugar is burned (caramelized). Various additives (caustic soda, caustic sulfite, ammonia, and their combinations) catalyze this process. In laboratory tests some proteogenic amino acids and their sodium and magnesium salts proved to be suitable catalysts (Sikora and Tomasik, 1994). Catalysts accelerate the process and decrease caramelization temperature, usually to the region between 130 and 200°C providing, simultaneously, their good tinctorial strength. Products prepared by noncatalyzed burning sugars at 200 to 240°C have poor tinctorial strength and serve as food flavoring. There is a concern about harm from the free radical character of caramels. However, they were proven (Barabasz et al., 1990) to be nonmutagenic. Thermal processing of saccharide- and polysaccharide-containing foodstuffs results in development of
TABLE 5.4 Saccharide Content (%) in Various Starch Syrups Syrups Glucose conversion Saccharide Glucose Fructose Maltose Higher saccharides
Low
High
Very high
Maltose
Fructose
15 — 11 48
43 — 20 13
92 — 4 2
10 — 40 28
7–52 42–90 4 3–6
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brown color; it originates from caramelization and, in the case of the polysaccharides, dextrinization. Brown-colored dextrins, even if they contain free radicals, are nonmutagenic because such free radicals are unusually stable (Ciesielski and Tomasik, 1996). Depending on the catalyst used, caramels differ in their isoelectric point. If colored matter does not match the isoelectric point of the caramel, micelles of the caramel irreversibly discharge and the caramel separates. Four types and several classes of caramels with widely different properties are manufactured. This variety provides a selection of a proper colorant to all types of foodstuffs.
5.5.3 FLAVOR
AND
AROMA
Burning of sugar in noncatalyzed processes results in the formation of particularly high amounts of furan-2-aldehyde and its derivatives. They constitute the flavor and aroma typical of caramel. Many foodstuffs (meat, fish, bakery products, potato, cocoa, coffee, and tobacco) on thermal treatment (baking, roasting, frying) develop specific aromas. They are volatile derivatives of pyrazine, pyrrole, and pyridine formed on thermal reactions of saccharides and proteins, nucleotides, and amino acids. Saccharides and polysaccharides—starch and cellulose (Bączkowicz et al., 1991), pectins (Sikora et al., 1998), and hemicelluloses (Tomasik and Zawadzki, 1998)— heated with amino acids develop scents specific to polysaccharides, amino acids, and reaction conditions. Thus, supplementation of saccharides and polysaccharides with amino acids and proteins, as well as supplementation of protein-containing products with saccharides, can be useful in generation, modification, and enrichment of flavor and aroma of foodstuffs and tobacco. Scents of plants are developed on the thermal reaction of saccharides with α−hydroxy acids (Sikora et al., 1997).
5.5.4 TEXTURE Concentrated aqueous solutions of carbohydrates form viscous liquids. That property is most commonly utilized in practice for texturizing foodstuffs. Intermolecular inter. actions between the same (Mazurkiewicz and Nowotny-Rożańska, 1998) and different saccharides (Mazurkiewicz et al., 1993; Obanni and BeMiller, 1997; Lii et al., 2002a; Gibiński et al., 2005) and changes in water activity (Mazurkiewicz et al., 2006) are involved. Blending of various saccharides and polysaccharides can result in the formation of numerous edible glues and adhesives. Such interactions are commonly utilized in texturization of puddings, jellies, and foams. Some oligosaccharides and the majority of polysaccharides form hydrocolloids, which build up their own macrostructure. They give an impression of jelly formation, thickening, smoothness, stabilization against temperature and mechanical shock, aging, and resistance on sterilization and pasteurization. Plant gums, pectins, and alginates are particularly willingly utilized for this purpose (Lai and Lii, 2004; Ramsden, 2004). Such properties can be controlled by the addition of salts because various metal ions form Wernertype complexes with saccharides and polysaccharide ligands. Formation of the calcium ion–sucrose complex, commonly utilized in sucrose manufacture, illustrates that phenomenon well. The effect of the metal ions in texturization is particularly visible in the case of anionic polysaccharides (potato amylopectin, pectin, alginates, carrageenans, furcellaran, xanthan gum, and carboxylic starches from starch oxidation).
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The texturizing effect of a given saccharide or polysaccharide and its various blends is developed as a function of the time necessary for the formation of a gel network (a physical cross-linking). The pH and temperature may also be essential factors. If protons (pH <7) or hydroxyl anions (pH >7) and temperature do not evoke any structural changes in the interacting species, the texturizing effect is reversible in pH and temperature. If retrogradation does not take place, the texturizing effect is also reversible in time. Saccharides, oligosaccharides, and polysaccharides also form complexes with proteins and lipids. Such complexes contribute to the texture of foodstuffs. Apart from combinations of natural saccharides, oligosaccharides, and polysaccharides, chemically modified polysaccharides are also utilized for texturization. Frequently, phosphorylated starches are used as gelling agents. Cross-linked starches are also important in that respect. The degree of cross-linking is essential. It should not exceed 0.2. Among cross-linked starches, those esterified with phosphoric acid are particularly favored. They are available by reacting starch with meta- and orthophosphates as well as POCl3 and PCl5. At the same degree of substitution, phosphorylated potato starch is superior and phosphorylated cornstarch is the poorest. A starch sulfate ester is used as a thickener and emulsion stabilizer. It is a typical anionic starch used as a component of anionic starch–protein complexes constituting meat substitutes (Tolstoguzov, 1991, 1995). Other anionic starches, as well as pectins, alginic acid, carrageenans, furcellaran, heparin, xanthan gum, and carboxymethyl cellulose are also used in food texturization (Clark and Ross-Murphy, 1987; Dejewska et al., 1995; Grega et al., 2003; Schmitt et al., 1998; Lii et al., 2002c, 2003c, 2003d; Najgebauer et al., 2003, 2004; Zaleska et al., 1999, 2000, 2001a, b, 2002a, b). Among many available modified polysaccharides, application of only a few of them is legal under the food laws of certain countries. Some restrictions are put on the method of their manufacture and the purity of such products. The replacement of saccharide sweeteners (first of all, sucrose) in food with various natural and synthetic sweeteners of very high RS (currently, mainly saccharin, aspartame, and cyclamates) is a task. It is also demanded by consumers looking for low-calorie foods. Diabetics are also looking for food free of insulin-requiring saccharides and polysaccharides. Following such demands, problems are encountered in providing the anticipated texture of sweet products manufactured without saccharides (Mazurkiewicz et al., 2001).
5.5.5 ENCAPSULATION Various foodstuffs lose their original, desirable flavor, aroma, taste, and color on processing. It is a common result of evaporation of volatile compounds or decomposition of certain food components under the influence of oxygen or light. In this manner the quality of foodstuffs decreases. In order to avoid such effects, volatile and unstable products are either protected in processed sources or, after processing, foodstuffs are supplemented by fragrances, colorants, and other components. Such goals are met by encapsulation and supplementation of microcapsule enclosed additives. Saccharides are suitable for making such microcapsules. Compression of additives (guest molecules) with a saccharide forming the matrix of the microcapsule
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(the host molecule) is a common practice. It is beneficial if there are some otherthan-mechanical interactions between the guest and host that decrease the rate of evaporation or reaction of the guest from the microcapsule. Granular starch can encapsulate guests in capillaries between granules; gelatinized starch, amylose, and amylopectin can trap certain molecules inside helices generated in contact with the guest molecules. Coacervation or coprecipitation of host and guest, and suspension of the guest molecule in polysaccharide gels, followed by drying, is another common procedure. Microcapsules can be made of preswelled granular starches. Potato starch is superior for this purpose for its granular size and easy swelling (Lii et al., 2001b; Korus et al., 2003). Lipids can be encapsulated in starches through short microwave heating with granular starches (Kapuśniak and Tomasik, 2005). α−, β−,and γ−cyclodextrins are the most effective compounds for microencapsulation of food components (Szejtli, 1984, 2004). Cyclodextrins take the form of toruses with cavities of 0.57, 0.78, and 0.95 nm, respectively. Their height is 0.78 nm. The upper and bottom edges of the toruses carry secondary and primary hydroxyl groups, respectively. All hydroxyl groups reside on the external surface of the toruses making cyclodextrins hydrophilic. Simultaneously, their cavity interior is hydrophobic. Cyclodextrins are water-soluble hosts for hydrophobic guests. The formation of inclusion complexes is controlled by the dimensional compatibility of the guest and host cavity. Commercially available dextrins are, in fact, inclusion complexes of cyclodextrins with two water molecules closing the entrance to the cavity. The formation of cyclodextrin inclusion complexes is reversible and, therefore, is governed by concentration of the guests competing for a place inside the cavity.
5.5.6 POLYSACCHARIDE-CONTAINING BIODEGRADABLE MATERIALS There is a growing concern about fully biodegradable plastics—packing and wrapping foils, containers, equipment of fast-food restaurants, disposable bags, and superabsorbents. Currently, several products made of polyethylene modified into biodegradable material are in use throughout the world. Biodegradability of such materials is afforded by admixture of 6 to 15 natural components, such as starch, cellulose, wood, or proteins into polyethylene. Polyurethane foams used as thermal insulators and packing materials contain up to 20% starch. The level of starch in copolymers of ethylene with vinyl chloride, styrene, or acrylic acid may reach 50%. Of course, the effect of biodegradation of such materials has more aesthetic significance than ecological. Although degradation of the finely pulverized synthetic portion of such materials is accelerated, it still takes several decades for depolymerization to come to its end. Apparently, the simplest biodegradable plastics could be prepared of starch solely by its compression up to 106 kPa, provided starch was moisturized up to its natural water-binding capacity (~20w-%) (Kudła and Tomasik, 1991). Following the idea of full biodegradability of materials, attention has been paid to the compositions of plain carbohydrates with either unmodified or modified proteins and of modified carbohydrates with unmodified or modified proteins. Such
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compositions are processed to generate carbohydrate–protein complexes. The thermodynamic and electrical compatibilities of components should be reached in order to afford superior functional properties of the materials. Because of the chemical nature of proteins (cationic character), carbohydrates should be anionic, that is, on dissociation, the negative charge should be left on the polysaccharide moiety. The COOH, PO3H2, and SO3H groups provide such properties. Whenever modification of a carbohydrate is required to make it anionic, the degree of derivatization should not exceed 0.1. It should neither increase the hydrophilicity of the product nor, if possible, decrease its molecular weight.
REFERENCES Angyal, S.Y., Complexes of metal cations with carbohydrates in solution, Adv. Carbohydr. Chem. Biochem., 47, 1, 1989. Bączkowicz, M. et al. Reactions of some polysaccharides with biogenic amino acids, Starch/Staerke, 43, 294, 1991. Ball, S.G., van der Wal, M.H.B.J., and Visser, R.G.F., Progress in understanding the biosynthesis of amylose, Trend Plant Sci., 3, 462, 1998. Barabasz, W. et al. On mutagenicity of caramels, Starch/Staerke, 42, 69, 1990. Bielecki, S., Enzymatic conversions of carbohydrates, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 10. Boruch, M. and Nebesny, E., Die Wirkung von Glucose isomerase auf Oligosacharide in Staerkehydrolysaten, Starch/Staerke, 31, 345, 1979. Ciesielski, W. and Tomasik, P., Starch radicals. Part I, Carbohydr. Polym., 31, 205, 1996. Ciesielski, W. and Tomasik, P., Starch radicals, Part III, Z. Lebensm. Untersuh. Forsch., A207, 292, 1998. Ciesielski, W. and Tomasik, P., Metal complexes of amylose and amylopectins and their thermolysis, J. Inorg. Biochem, 98, 2039, 2004. Ciesielski, W., Tomasik, P., and Bączkowicz, M., Starch radicals, Part IV, Z. Lebensm, Untesuh. Forsch., A207, 299, 1998. Ciesielski, W. et al. Interaction of starch with metal ions from transition groups, Carbohydr. Polym., 51, 47, 2003. Clark, A.H. and Ross-Murphy, S.B., Structural and mechanical properties of biopolymer gels, Adv. Polym. Sci., 53, 57, 1987. Davidek, J. and Davidek, T., Chemistry of the Maillard reactions in foods, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 18. Dejewska, A., et al. Electrochemical formation of polysaccharide-protein-water ternary complexes. Part I. Apple pectin–albumin–water complexes, Starch/Stearke, 47, 219, 1995. Einbu, A. and Vaarum, K.M., Structure and property relationships in chitosan, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 14. Erlander, S., Biosynthesis of starch, Żywn. Technol. Jakość, Suppl. 4, 112, 1998. Fiedorowicz, M. and Khachatryan, G., Effect of illumination with visible polarized and nonpolarized light on alpha-amylolysis of starches of different botanical origin, J. Agric. Food Chem., 51, 7815, 2003. Fiedorowicz, M., Tomasik, P., and Lii, C.Y., Depolymerization of starch by illumination with polarized light. Carbohydr Polym., 45, 75, 2001.
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Fiedorowicz, M., et al. Novel dextrins as potential prebiotics. J. Food Agric. Environ., 1(3 & 4), 54, 2003. Gallant, D.J., Bouchet, B., and Baldwin, P.M., Microscopy of starch: evidence of a new level of granule organisation, Carbohydr. Polym., 32, 177, 1997. Gibiński, M. et al. Thickening of sweet and sour sauces with various polysaccharide combinations, J. Food Eng., submitted, 2005. Grega, T., et al. Products of co-precipitation of potato starch with casein from milk, J. Polym. Environ., 11, 75, 2003. Imberty, A., Buleon, A., and Tran, V., Recent advances in knowledge of starch structure, Starch/Staerke, 43, 375, 1991. Jane, J. et al. Anthology of starch granule morphology by scanning electron microscopy, Starch/Staerke, 46, 121, 1994. Kapuśniak, J. and Marczak, M., Starch modified with hydroxy acids as a potential source of energy and matter for Lactobacillus bacteria, J. Food Agric. Environ., 3, 125, 2005. Kapuśniak, J. and Tomasik, P., Microencapsulation of lipids in granular starches, J. Microencaps., submitted 2005. Korus, J., Tomasik, P., and Lii, C.Y., Microcapsules from starch granules, J. Microencaps., 20, 47, 2003. Kudła, E. and Tomasik, P., Effect of high pressure on starch matrix, Starch/Staerke, 44, 167, 1991. Lai, V.M.-F. and Lii, C.Y., Role of saccharides in texturization and functional properties of foodstuffs, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 11. Lii, C.Y. et al. Behavior of granular starches in air, low-pressure glow plasma. Carbohydr. Polym., 49, 499 2002a. Lii, C.Y. et al. Polysaccharide–polysaccharide interactions in pastes, Pol. J. Food Nutr. Sci., 11/4, 29, 2002b. Lii, C.Y. et al. CMC-gelatin complexes. Carbohydr. Polym., 50, 19, 2002c. Lii, C.Y. et al. Granular starches as dietary fiber and natural microcapsules, Int. J. Food. Sci Technol., 38, 677, 2003a. Lii, C.Y. et al. Effect of corona discharges on granular starches. J. Food. Agric. Environ., 1, 143, 2003b. Lii, C.Y., Liaw, S.C., and Tomasik, P., Xanthan gum–ovoalbumin complexes, Pol. J. Food Nutr. Sci., 12(3), 25, 2003c. Lii, C.Y. et al. Carrageenan–gelatin complexes, J. Polym. Environ., 11, 115, 2003d. Lii, C.Y. et al. Electrosynthesis of κ-carrageenan–ovalbumin complexes. Int. J. Food Sci. Technol., 8, 787, 2003e. MacDougall, A.J. and Ring, S.G., Pectic polysaccharides, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 12. . Mazurkiewicz, J. and Nowotny-Rożańska, M., Viscosity of aqueous solutions of saccharides, Pol. J. Food Nutr. Sci., 7/2, 171, 1998. Mazurkiewicz, J., Rębilas, K., and Tomasik, P., Aspartame as texturizing agent for foodstuffs, Z. Lebensm. Unters. Forsch., A212, 369, 2001. Mazurkiewicz, J., Rębilas, K., and Tomasik, P., Dextrans—low-molecular sweetener interactions in aqueous solutions, Food Hydrocol., 20, 21, 2006. Mazurkiewicz, J., Zapotny, J., and Zaleska, H., Studies on carbohydrate based glues and thickeners for foodstuffs, Part I. Glucose-sucrose-apple pectin ternary systems, Starch/Staerke, 45, 175, 1993. Najgebauer, D. et al. Polymeric complexes from casein and starch phosphate: characteristics and enzyme susceptibility, J. Polym. Environ., 12, 17, 2003.
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Najgebauer, D. et al. Polymeric complexes of cornstarch and waxy cornstarch phosphates with milk casein and their performance as biodegradable materials, Molecules, 9, 550, 2004. Nkala, B. et al. Starch from wild yam from Zimbabwe, Starch/Staerke, 46, 85, 1994. Obanni, M. and BeMiller, J.N., Preparation of some starch blends, Cereal Chem., 74, 431, 1997. Pietrzycki, W., Saccharide sweeteners and the theory of sweetness, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 5. Praznik, W., Huber, A., and Cieślik, E., Fructans: occurrence and applications in food, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 13. Ramsden, L., Plant and algal gums and mucilages, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 15. Ruck, H., The new organosolv pulps: will they outrival starch as an industrial raw material? Żywn. Technol. Jakość, Suppl. 2, 138, 1996. Rybak-Chmielewska, H., Honey, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 6. Seidemann, J., Die Staerkeatlas, Paul Parey, Berlin, 1966. Siemion, I.Z., Biosterochemistry, PWN, Warsaw, 1985, chap. 1 (in Polish). Sikora, M. and Tomasik, P., Caramelization of starch syrups in the presence of amino acids and their metal salts as the catalysts, Starch/Stearke, 46, 150, 1994. Sikora, M., Tomasik, P., and Pielichowski, K., Reaction of starch with amino and hydroxy acids in the field of microwaves, Pol. J. Food Sci. Nutr., 6/2, 23, 1997. Sikora, M., Tomasik, P., and Pielichowski, K., Thermolysis of pectins with amino acids, Pol. J. Food Nutr. Sci., 7/3, 391, 1998. Schmitt, C. et al. Structure and technofunctional properties of protein-polysaccharide complexes. A review, Crit. Rev. Food Sci. Nutr., 38, 689, 1998. Starzyk, F., Lii, C.Y., and Tomasik, P., Light absorption, transmission and scattering in potato starch granule, Pol. J. Food Nutr. Sci., 10/4, 27, 2001. Szejtli, J., Cyclodextrin Inclusion Complexes, Akademiai Kiado, Budapest, 1984. Szejtli, J., Cyclodextrins, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 17. Szymońska, J., Krok, F., and Tomasik, P., Deep freezing of potato starch, Int. J. Biol. Macromol., 27, 307, 2000. Tolstoguzov, V.B., Functional properties of food protein and role of protein–polysaccharide interaction, Food Hydrocoll., 4, 429, 1991. Tolstoguzov, V.B., Some physico-chemical aspects of protein processing foods. Multicomponent gels, Food Hydrocoll., 9, 317, 1995. Tomasik, P. and Schilling, C.H., Complexes of starch with inorganic guests, Adv. Carbohydr. Chem. Biochem., 53, 263, 1998a. Tomasik, P. and Schilling, C.H., Complexes of starch with organic guests, Adv. Carbohydr. Chem. Biochem., 53, 346, 1998b. Tomasik, P. and Schilling, C.H., Chemical modifications of starch, Adv. Carbohydr. Chem. Biochem., 59, 176, 2004. Tomasik, P., Jane, J., and Wang, Y.J., Starch sugar complexes, Starch/Staerke, 47, 185, 1995. Tomasik, P. et al. Potato starch derivatives with some chemically bound bioelements, Acta Pol. Pharm. Drug Res., 58, 447, 2001. Tomasik, P. and Zaranyika, M.F., Nonconventional methods of modification of starch, Adv. Carbohydr. Chem. Biochem., 51, 243, 1995.
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Tomasik, P. and Zawadzki, W., Reaction of plant material with biogenic amino acids, Pol. J. Food Nutr. Sci., 7/1, 29, 1998. Tomasik, P., Pałasiński, M., and Wiejak, S., The thermal decomposition of carbohydrates. Part I., The decomposition of mono-, di-, and oligo-saccharides. Adv. Carbohydr. Chem. Biochem., 47, 203, 1989. Wang, Y.J., Saccharides: modifications and applications, Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, 2004, chap. 3 Zaleska, H. et al. Electrosynthesis of polysaccharide–protein complexes. Part II. Apple pectincasein complexes, Nahrung., 43, 278, 1999. Zaleska H., Ring, S., and Tomasik, P., Apple pectin complexes with whey protein, Food Hydrocoll., 14, 377, 2000. Zaleska, H., Ring, S., and Tomasik, P., Complexes of potato starch with casein, Int. J. Food Chem. Technol., 36, 509, 2001a. Zaleska, H., Ring, S., and Tomasik, P., Electrosynthesis of potato starch–whey protein isolate complexes, Carbohydr. Polym., 45, 89, 2001b. Zaleska, H., Tomasik, P., and Lii, C.Y.., Electrosynthesis of CMC–casein complexes, Food. Hydrocoll., 16, 215, 2002a. Zaleska, H., Tomasik, P., and Lii, C.Y., Formation of CMC–ovoalbumin complexes by electrosynthesis. J. Food Eng., 53, 249, 2002b.
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6
The Role of Proteins in Food Zdzisław E. Sikorski
CONTENTS 6.1
6.2
6.3
6.4
Chemical Structure....................................................................................... 130 6.1.1 Introduction ...................................................................................... 130 6.1.2 Amino Acid Composition and Sequence ........................................ 130 6.1.3 Hydrophobicity................................................................................. 133 6.1.3.1 Average Hydrophobicity................................................... 133 6.1.3.2 Surface Hydrophobicity.................................................... 133 Conformation ............................................................................................... 134 6.2.1 The Native State............................................................................... 134 6.2.2 Denaturation ..................................................................................... 136 Functional Properties ................................................................................... 138 6.3.1 Introduction ...................................................................................... 138 6.3.2 Solubility .......................................................................................... 139 6.3.2.1 Effect of the Protein Structure and Solvent ..................... 139 6.3.2.2 Effect of pH and Ions....................................................... 140 6.3.2.3 Importance in Food Processing........................................ 141 6.3.3 Water-Holding Capacity................................................................... 141 6.3.4 Gelling and Film Formation ............................................................ 141 6.3.4.1 The Gel Structure ............................................................. 141 6.3.4.2 Interactions of Components.............................................. 142 6.3.4.3 Binding Forces and Process Factors ................................ 143 6.3.4.4 Importance in Food Processing........................................ 144 6.3.5 Emulsifying Properties..................................................................... 145 6.3.5.1 The Principle..................................................................... 145 6.3.5.2 Factors Affecting Emulsifying ......................................... 146 6.3.5.3 Determination of Emulsifying Properties ........................ 146 6.3.6 Foaming Properties .......................................................................... 147 Proteins as Important Components in Foods .............................................. 148 6.4.1 Muscle Proteins................................................................................ 148 6.4.2 Milk Proteins.................................................................................... 149 6.4.3 Egg Proteins ..................................................................................... 151 6.4.4 Legume Proteins .............................................................................. 152 6.4.5 Cereal Proteins ................................................................................. 152 6.4.6 Mycoprotein ..................................................................................... 153 129
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6.5
Effects of Heating ........................................................................................ 153 6.5.1 Introduction ...................................................................................... 153 6.5.2 Rheological Changes ....................................................................... 154 6.5.3 Changes in Color ............................................................................. 156 6.5.4 Development of Volatile Compounds.............................................. 156 6.5.5 Reactions at Alkaline pH ................................................................. 156 6.6 Oxidation...................................................................................................... 158 6.7 Enzyme-Catalyzed Reactions ...................................................................... 160 6.7.1 Introduction ...................................................................................... 160 6.7.2 The Plastein Reaction ...................................................................... 160 6.7.3 Transglutaminase Catalyzed Reactions ........................................... 162 6.7.4 Proteolytic Changes in Milk Proteins.............................................. 164 6.7.5 Role of Enzymes in Muscle Foods ................................................. 164 6.7.6 Other Enzymatic Changes in Food Proteins ................................... 167 6.8 Chemical Modifications ............................................................................... 167 6.8.1 Introduction ...................................................................................... 167 6.8.2 Alkylation......................................................................................... 168 6.8.3 Acylation .......................................................................................... 169 6.8.4 N-Nitrosation.................................................................................... 170 6.8.5 Reactions with Phosphates .............................................................. 171 References.............................................................................................................. 172
6.1 CHEMICAL STRUCTURE 6.1.1 INTRODUCTION Proteins are linear condensation products of various α-L-amino acids (a.a.), which differ in molecular weight, charge, and polar character (Table 6.1), bound by transpeptide linkages. They differ also in the number and distribution of various a.a. residues in the molecule. The chemical properties and size of the side chain, as well as the sequence of the a.a., affect the conformation of the molecule, that is the secondary structure containing helical regions, β-pleated sheets and β-turns, the tertiary structure or the spatial arrangement of the chain, and the quaternary structure or the assembly of several polypeptide chains. The conformation affects the biological activity, nutritional value, and functional role of proteins as food components.
6.1.2 AMINO ACID COMPOSITION
AND
SEQUENCE
The proportion of each of the various a.a. residues, calculated as a percentage of the total number of residues, ranges in most proteins from 0 to about 30%. In extreme cases it may even reach 50%. Among the 225 residues of the molecule of phosvitin in egg yolk, there are 122 Ser, most of them phosphorylated, SerP. The typical sequences of phosvitin are . . .Asp-(SerP)6-Arg-Asp. . . and . . .His-Arg--(SerP)6-ArgHis-Lys. . . . In collagens, the content of Gly, Pro, and Ala is 328, 118, and 104 residues/1000 residues, respectively. Grain prolamines are very rich in Glu (up to
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TABLE 6.1 Selected Properties of Proteinogenic Amino Acids
Amino acid
Abbreviation
pKa1
pKa2
Glycine Alanine Valine Leucine Isoleucine Proline Phenylalanine Tyrosine Tryptophan Serine Threonine Cysteine Methionine Asparagine Glutamine Aspartic acid Glutamic acid Lysine Arginine Histidine
Gly Ala Val Leu Ile Pro Phe Tyr Trp Ser Thr Cys Met Asn Gln Asp Glu Lys Arg His
2.34 2.34 2.32 2.36 3.26 1.99 1.83 2.20 2.38 2.21 2.15 1.71 2.28 2.02 2.17 1.88 2.19 2.20 2.18 1.80
9.60 9.69 9.62 9.60 9.68 10.60 9.13 9.11 9.39 9.15 9.12 8.35 9.21 8.80 9.13 3.65 4.25 8.90 9.09 5.99
pKR
10.07 13.60 13.60 10.28
3.65 4.24 10.56 12.48 6.00
Isoelectric point pI
Side chain hydrophobicity (ethanol→water) kJ/mol
6.0 6.0 6.0 6.0 6.0 6.3 5.5 5.7 5.9 5.7 5.6 5.0 5.7 5.4 5.7 2.8 3.2 9.6 10.8 7.5
0.0 3.1 7.0 10.1 12.4 10.8 11.1 12.0 12.5 0.2 1.8 4.2 5.4 0.04 0.4 2.2 2.3 6.2 3.1 2.1
Note: pKa1, pKa2, and pKR are the negative logarithms of the dissociation constants of the acidic, basic, and R groups of a.a. in aqueous solution
55%) and Pro (up to 30%). Paramyosin, abundant in the muscles of marine invertebrates, is rich in Glu (20 to 24%), Asp (12%), Arg (12%), and Lys (9%). Most food proteins, however, do not differ very much in their a.a. composition. Generally, the content of acidic residues is the highest, and that of His, Try, and sulfur containing a.a. is the lowest. However, the number of residues capable of accepting a positive charge is often higher, especially in plant proteins because about 50% of the sidechain carboxyl groups are amidated. The composition affects the value of food proteins as the source of essential a.a. Most proteins of meat and fish muscles have very high biological value, while cereal proteins are generally poor in Lys. Several major grains are deficient also in Thr, Leu, Met, Val, and Trp. In most collagens there are no Cys and Trp residues. Several milk proteins, as well as proteins of other origin, contain short a.a. sequences corresponding to different peptides known for their biological activity. The sequence of the a.a. residues in the polypeptide chains is critical for the behavior of the proteins in food systems. The antifreeze fish serum glycoproteins, which contain several a.a. sequences Thr–X2–Y–X7, where X is predominantly Ala
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and Y a polar residue, have the ability to interact with ice crystals. The molecule of β-casein has a polar N-terminal region (residues 1–43) with a charge of –16, and an apolar fragment, containing 34 of the total number of Pro residues. This sequence favors the temperature-, concentration-, and pH-dependent associations into threadlike polymers, stabilized mainly by hydrophobic adherences. Lysosyme, a basic protein of egg white and other organisms, containing four –S–S– cross-links in a single polypeptide, retains its enzymatic activity in acidic solution even after heating to 100°C. The Bowman-Birk trypsin inhibitor consists of 71 a.a. residues in one polypeptide chain with loops due to seven –S–S– bonds, and is characteristic for its high thermal stability. The bovine serum albumin has one SH group and 17 intramolecular –S–S– bridges per molecule. Many a.a. residues undergo posttranslational enzymatic amidation, hydroxylation, oxidation, esterification, glycosylation, methylation, or cross-linking. Some segments of the original polypeptide chains may be removed (Figure 6.1). Modified residues present in a given protein can be used for analytical purposes, such as hydroxyproline (ProOH), which is characteristic for collagens.
Polyrybosome
Hydroxylases
Procollagen
Glycosyltransferases Endopeptidases
Tropocollagen
Collagen fibers
FIGURE 6.1 Posttranslational modifications in collagen (From Sikorski, Z.E., Chemical and Functional Properties of Food Components, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 1997. With permission.)
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Posttranslational modifications may result in covalent attachment of various groups to the proteins. They may change the ionic character of the molecule, for example the phosphoric acid residues or some saccharides. The residues involved in phosphorylation and binding of saccharide moieties are Ser, Thr, LysOH, ProOH, His, Arg, and Lys. Among the highly phosphorylated proteins is αS-casein. In the central region of αS1-casein, SerP occurs in sequences . . .SerP-Ala-Glu. . ., . . .SerPVal-Glu. . ., . . .SerP-Glu-SerP. . ., and . . .SerP-Ile-SerP-SerP-SerP-Glu. . . . Such distribution favors oligomer formation due to hydrophobic interactions of the apolar fragments of the molecules, while the charged sequences are exposed to the solvent. High saccharide content is characteristic for the allergenic glycoproteins of soybeans (up to about 40%), several egg white proteins (up to 30%), albumins of cereal grains (up to 15%), whey immunoglobulin (up to 12%), and collagens of marine invertebrates (up to 10%). In κ-casein there is a hydrophobic N-terminal part (residues 1 to 105) and a hydrophilic macropeptide (106 to 169), or a glycomacropeptide, with a saccharide moiety (0.5%) composed of N-acetylneuraminic acid, D-galactose, Nacetylgalactosamine, and D-mannose residues.
6.1.3 HYDROPHOBICITY 6.1.3.1 Average Hydrophobicity The nonpolar character of an a.a. can be expressed by hydrophobicity, that is, change of the free energy Fta accompanying the transfer of the a.a. from a less polar solvent to water. Exposure of an a.a. with a large hydrocarbon side chain to the aqueous phase results in a corresponding decrease in entropy due to structuring of water around the chain. The hydrophobicity of the side chain of an a.a. is: Ftr = Fta – FtGly where FtGly is the hydrophobicity of Gly The average hydrophobicity Ftav of a protein can be estimated as Ftav = ΣFta/n where n is the number of a.a. residues in the protein molecule. It is not possible to predict the conformation and behavior of a protein in solution on the basis of Ftav. However, proteins of high Ftav yield bitter hydrolysates. 6.1.3.2 Surface Hydrophobicity The interior of the native molecule of a globular protein contains most of the hydrophobic a.a. residues. However, some of them form hydrophobic clefts or occur on the surface as patches of various sizes. Phe, Tyr, and Try in food proteins can be monitored by measuring the intrinsic fluorescence. They absorb ultraviolet radiation and emit fluorescence in the following order:
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Phe Tyr Try
260 nm 275 nm 283 nm
283 nm 303 nm 343 nm
The intensity of fluorescence and the wavelength of maximum intensity depend upon the polarity of the environment. Thus the Try residue located in a nonpolar region emits fluorescence at 330 to 332 nm, while at complete exposure to water, the emission wavelength is 350 to 353 nm. Furthermore, electron withdrawing groups, like carboxyl, azo, and nitro groups, as well as different salt ions, have a quenching effect on fluorescence. Measurements of intrinsic fluorescence and of fluorescence quenching have not found, however, wide application in hydrophobicity determinations, as they are restricted to the effect of aromatic a.a. residues. The simplest and most commonly used are hydrophobic probes based on the phenomenon that the quantum yield of fluorescence of the compounds containing some conjugated double-bond systems is in a nonpolar environment about 100 times higher than in water. Thus hydrophobic groups can be monitored by aromatic or aliphatic probes and fluorescence measurements. Most often used is 1-anilinonaphthalene-8-sulfonate (ANS) (Formula 6.1) and cis-parinaric acid (CPA) (Formula 6.2). Also the binding of triacylglycerols or sodium dodecylsulphate may be determined.
H N
-
SO 3
CH3 CH2 (CH = CH)4 (CH2 )7 COOH
(6.1)
(6.2)
6.2 CONFORMATION 6.2.1 THE NATIVE STATE Proteins in a natural environment fold spontaneously from an extended form L, to the native conformation N, which is affected by the primary structure: L↔N This is accompanied by a decrease in free energy: –RTlnK = ∆G = ∆H – T∆S where R = gas constant, T = temperature, H = enthalpy, S = entropy, and K = equilibrium constant (K = [N]/[L]).
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The conformation of proteins in solutions is affected by hydrogen bonds and hydrophobic effects. The hydrogen bonds between water and hydrophilic residues lead to enthalpy changes, while the effects of nonpolar groups in the aqueous environment bring about changes in entropy. This is reflected in the total free energy change ∆Gt ∆Gt = ∆Hp + ∆Hw – T∆Sp – T∆Sw where the subscripts p and w refer to protein and water, respectively. Various forces stabilize the native conformation. The dipole–dipole interactions, depolarization, and dispersion forces are significant only at very close distance r of the atoms because the energy of interactions decreases with r–6. The hydrogen bonds, abundant in proteins, differ in energy from approximately 2 to about 12 kJ/Mol, depending on the properties and positioning of the groups involved. The strength of the H-bonds does not depend significantly on temperature, but increases with pressure. The energy of the ionic bonds is affected by the dielectric constant and may reach in the hydrophobic core of a globular protein about 21 kJ/Mole between the ionized residues of Asp and Lys. The energy of hydrophobic interactions increases with temperature and decreases with increasing pressure. Covalent bonds other than those in the polypeptide chain, although of highest energy, are generally very limited in number. However, some proteins rich in such bonds may have high thermal stability, for example, mature collagens containing different cross-links generated in reactions of the oxidized ε-NH2 group of Lys and LysOH Prot
NH2
lysyl oxidase
Prot
C
H O H
O Prot
C
H O
+
H O
O
C C
CH2 Prot
Prot
CH
CH
H2O Prot
H C
Prot
CH
C
Prot
OH N NH
CH2 Prot Prot N
O
CH Prot
CH
CH
Prot
Prot NH2
Prot
N
REACTION 6.1
CH
CH
Prot
N CH2
N
H C
Prot
CH2 N
Prot
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and in the Maillard reaction of the saccharide moieties of the molecules, as well as proteins containing many –S–S– bridges, for example, several proteinase inhibitors. A very significant effect on the properties of proteins is exerted by their quaternary structures and micellar associations. Soybean glycinin, composed of 6 basic and 6 acidic subunits has a structure of two superimposed rings. In each ring the 3 acidic and 3 basic subunits are arranged alternatively. Thus ionic interactions are possible both within each ring and between the rings. Because the conformation of the oligomer is buttressed by noncovalent forces, the addition of urea and changes in pH and ionic strength lead to dissociation of the protein into subunits. In unheated milk the caseins are present as a colloidal dispersion of particles containing about 6 to 7% calcium phosphate, known as micelles, and as smaller particles without calcium phosphate. These forms are in equilibrium, which is affected by temperature, pH, and the concentration of Ca2+. In fresh milk about 80 to 90% of the mass of caseins is in the micellar form. The micelles are porous and hydrated, have a diameter ranging from a few to about 600 nm, and a weight average molecular mass of 600 MDa. Numerous investigations have resulted in various models of the structure of casein micelles. According to one group of models, the micelles are formed from several hundred subunits called submicelles differing in composition and size. In these models the subunits are either linked by calcium phosphate or the calcium phosphate is located as discrete packages within the submicelles. According to the nanocluster model there are no subunits, but the polypeptide chains form a matrix in which calcium phosphate nanoclusterlike particles are embedded (Figure 6.2). The outer parts of the micelles are occupied by hydrophilic polypeptide chains, including the macropeptide of κ-casein and Ca2+sensitive peptides, and form a hairy layer (Holt and Rogiński, 2001)
6.2.2 DENATURATION The native conformation of proteins is generally stabilized by a small amount of energy. The net thermodynamic stability of the native structure of many proteins is as low as about 40 to 80 kJmole–1. The unfolding enthalpy of metmyoglobin and lysozyme is about 285 and 368 kJ mole–1, respectively. Therefore ionizing radiation, shift in pH, change in temperature or concentration of various ions, or addition of detergents or solvents, may cause dissociation of the oligomers into subunits, unfolding of the tertiary structure, and uncoiling of the secondary structure (Figure 6.3). These changes are known as denaturation. Exposure of the a.a. residues originally buried in the interior of the molecule changes the pI, surface hydrophobicity, and the biochemical properties of proteins. Denaturation may be reversible, depending on the degree of deconformation and environmental factors. This may affect the results of assays of enzyme activity used as a measure of, for example, the severity of heat processing in food operations. For monitoring milk pasteurization, the determination of γ-glutamyltransferase can be used because the enzyme undergoes complete inactivation after 16 s at 77°C, and no reactivation has been evidenced (Zehetner et al., 1995).
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FIGURE 6.2 The calcium phosphate nanocluster model of a casein micelle. Substructure arises from the calcium phosphate nanocluster-like particles in the micelles (dark spheres). There is a smooth transition from the core to the diffuse outer hairy layer that confers steric stability on the micelle. (Courtesy Holt, C. and Rogiński, H., Chemical and Functional Properties of Food Proteins, Sikorski, Z. E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 2001.) (a)
(b)
(c)
ss
M2+
ss
ss ss
FIGURE 6.3 Protein denaturation (a) native molecule, (b) molecule in a changed conformation with ruptured disulfide bridges and ionic bonds, (c) denatured molecule with randomly extended polypeptide chains. (From Sikorski, Z.E. Chemical and Functional Properties of Food Components, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 1997. With permission.)
Generally food processing causes irreversible denaturation followed by reactions of the thermally denatured proteins with other components in the system. This second step may lead to loss in food quality, but the denaturation may have beneficial or detrimental effects in foods. The main effects comprise changes in pI, hydration, solubility, viscosity of solutions, biological activity, and reactivity of a.a. residues.
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6.3 FUNCTIONAL PROPERTIES 6.3.1 INTRODUCTION The functional properties important for the food processor are attributes, which at the proper concentration of the respective components or additives and at appropriate conditions, provide for the desirable characteristics of the product. These properties of proteins are displayed in interactions with the surrounding solvent, ions, other proteins, saccharides, lipids, and numerous other components, as well as in surface phenomena. The most important properties in food processing can be roughly grouped as seen in Table 6.2. They affect the appearance, color, juiciness, mouth feel, and texture of a large variety of foods, as well as cutting, mincing, mixing, formation of dough, fibers, foils, and bubbles, shaping, and transporting of food materials. The term functional with respect to food has, since the mid-1980s, had one more meaning. It has been used to describe foods or food ingredients that provide for additional health benefits to consumers beyond those of satisfying the basic nutritional requirements, that is, they aid specific bodily functions. Such functional components have been found predominantly in foods of plant origin, especially oat products as a source of β-glucans; soybean, containing various protease inhibitors, phytosterols, saponins, phenolic acids, phytic acid, and isoflavones; flaxseed rich in α-linolenic acid and lignan precursors; garlic, providing diallyl sulfide; broccoli and other cruciferous vegetables, which are rich sources of glucosinolates; citrus fruits containing folate, fiber, and limonoids; tea, particularly green tea; and red wine and grapes with their polyphenolic constituents. Foods of animal origin also contain functional constituents. Fish, especially marine fish, are rich in n-3 fatty acids; dairy products are one of the best sources of Ca, and the fermented commodities supply large beneficial microbial populations; beef contains conjugated linoleic acid, and in many proteins there are sequences of a.a., which after digestion yield peptides known for their various biological activities. In a large number of experiments, the following beneficial health effects of functional foods have been found: cancer chemopreventive, antihypertensive, cholesterol-lowering and reducing the risk of coronary heart disease, increasing cellular antioxidant defense, contributing to maintenance of a healthy immune function, preventing osteoporosis, antibiotics, and improving intestinal microbial balance.
TABLE 6.2 Functional Properties of Proteins Displayed in Interactions with Different Food Constituents Interactions with Water
Water and proteins
Lipids or gases
Wet ability Swelling Rehydration Water holding Solubility
Viscosity inducing Gelling Fiber forming Dough forming Membrane forming
Emulsifying ability Emulsion stabilization Foaming ability Foam stabilization
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The functionalities can be modified by using enzymatic and chemical processes that change the structure of the proteins. They depend also on the pH, ionic strength, and temperature in the food system. By better understanding of the tertiary structure of many food proteins, it should also be possible to modify their functionality using genetic engineering. To evaluate the functional properties of some proteins in different systems, the quantitative structure–activity relationship approach may be applied (Nakai and Li-Chan, 1988).
6.3.2 SOLUBILITY 6.3.2.1 Effect of the Protein Structure and Solvent The solubility or extractability of proteins is often defined in food chemistry as the percent of the total quantity of protein contained in the food material that can be extracted by water or a suitable solvent in specified conditions. It depends on the properties of the protein and of the solvent, pH, concentration and charge of other ions, ratio of sample weight to solvent volume, particle size of the sample, duration of extraction, and on temperature. Generally, proteins rich in ionizable residues, of low surface hydrophobicity, are soluble in water or dilute salt solutions, for example, the proteins found in egg white. Proteins abundant in hydrophobic groups readily dissolve in organic solvents. The classification of cereal proteins into albumins, globulins, prolamines, and glutelins soluble in water, dilute salt solutions, 60 to 80% aliphatic alcohols, and 0.2% NaOH, respectively, may also be used for characterization of other proteins. Stabilization by cross-linking is of crucial importance, for example, the solubility of collagen from different connective tissues depends on the type and age of the tissue. Young tropocollagen can be solubilized in a neutral or slightly alkaline NaCl solution, tropocollagen containing intramolecular covalent bonds are soluble in citric acid solution at pH 3, while mature collagen with covalent intermolecular cross-links is not soluble in cold, dilute acids and buffers. It can, however, be partially solubilized in a highly comminuted state or after several hours of treatment in alkaline media. Differences in solubility are essential for various procedures of isolation of individual proteins and groups of proteins from foods (Kristinsson, 2001). Denaturation may decrease solubility, for example, the fish protein concentrate produced by extraction of minced fish with a boiling, azeotropic solution of isopropanol, is scarcely soluble in water. In organic solvents, due to their low dielectric constant, the energy of interactions between charged a.a. residues is higher than in water. This may favor unfolding of the molecules and exposing of the hydrophobic residues, which cannot be counterbalanced by entropy forces. Thermal denaturation followed by aggregation due to interactions of the surface-exposed reactive groups leads generally to loss in extractability. On the other hand, if heating brings about deconformation of the quaternary and tertiary structures it may increase the solubility, for example, in collagen. Adding antioxidants to defatted soy flour prior to alkaline extraction enhances the solubility of the protein isolate in proportion to the decrease in oxidation of thiol groups (Boatright and Hettiarachchy, 1995).
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6.3.2.2 Effect of pH and Ions In water solutions, the solubility of proteins has a minimum at the pI (Figure 6.4). At such pH there is no electrostatic repulsion between the molecules, hence the hydration layer alone cannot prevent aggregation. Although the attraction of water dipoles by ionized groups of opposite charges in a.a. residues largely offsets the electrostatic binding between the ions; the net balance of the attraction and change in solvent entropy favors salt-bridge formation. At pH values below or above pI of the protein the solubility increases due to repelling of the positive or negative ions, as well as due to increased interaction of the charged polypeptide chains with water dipoles. The pI of a protein may shift slightly with a changing concentration of salts in the solution. The effect of ions on the solubility of a protein depends on their ionic strength µ, and their effect on the surface tension of the solvent, as well as on the dipole moment and the decrease in the molecular surface area of the protein upon aggregation. Various ions, depending on their size and charge, favor or lower the solubility of proteins. In the low range of concentration, that is, µ = 0.5 to 1.0, the solubility increases with the concentration of neutral salts. This is known as salting in. The ions have a screening effect on the charged protein molecules. Being surrounded by water dipoles they add to the hydration layer, which favors solubilization of the macromolecules. At higher concentration the effect depends mainly on the ability of the salts to affect the water structures. Salts containing polyvalent anions at appropriate concentrations precipitate protein from solutions; this is known as salting out. Most widely used for this purpose is (NH4)2SO4 or Na2SO4. Various proteins precipitate from solution at different percentages
FIGURE 6.4 The effect of pH on the solubility of various proteins. (From Sikorski, Z.E. (Ed.), Chemical and Functional Properties of Food Components, Second Edition, CRC Press, Boca Raton, FL, 2002. With permission.)
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of saturation of the salts. Thus salting out is used for protein fractionation because by changing the concentration of the salt, various fractions of the mixture of proteins present in the original solution can be precipitated. 6.3.2.3 Importance in Food Processing The solubility versus pH curve can be used for selecting parameters for extraction of proteins from different sources. Adding a required amount of salt to meats during cutting and mixing in a silent cutter is a prerequisite for extracting myofibrillar proteins from the tissue structures, and for forming a sausage batter of adequate quality. CaCl2 is used to precipitate the whey proteins, while CaSO4 coagulates soy proteins in tofu manufacturing in soybean processing. Solubility also contributes to gelling and emulsifying. It may also be required for efficient use of various protein isolates as functional food additives in products differing in pH and salt content. The loss in solubility due to abusive treatment is often indicative of protein denaturation and subsequent cross-linking. Therefore, solubility data, if used to characterize commercial protein products, should be determined using standardized procedures (Kołakowski, 2001).
6.3.3 WATER-HOLDING CAPACITY The ability of many foods to retain water is affected by the involvement of proteins in different structures. In meat and fish tissues, the state of water depends on interactions of water structures with proteins and other solutes. Furthermore, because of the fibrous nature and compartmentalization of the muscle, water is also held in the meat by physical entrapment. Alterations in the spatial arrangement of the proteins and in the integrity of tissue structures caused by biochemical and processing factors are responsible for shrinking or swelling of the material, and thus for retention or exudation of water. Classical investigations on the effect of pH, divalent cations, postmortem changes, freezing and thawing, heating, salting, polyphosphates, and citrates on the water-holding capacity (WHC) of meat were made by Hamm (1960). WHC has a large impact on the texture and juiciness of meat and fish products. A decrease in WHC brings about excessive cooking loss and thawing drip. Changes in WHC may also be used for evaluating the effect of processing on the structure of proteins and on the quality of muscle foods. To be used as a quality index, WHC should be determined using standardized procedures. They are based either on measurements of loss of water from the original sample due to centrifugation, pressing, or capillary force, or on measuring the quantity of liquid separated under the action of a force from a sample with added water or aqueous solution.
6.3.4 GELLING
AND
FILM FORMATION
6.3.4.1 The Gel Structure A gel consists of a three-dimensional lattice of large molecules or aggregates, capable of immobilizing solvent, solutes, and filling material. Food gels may be formed by proteins and polysaccharides, which may participate in gel formation in the form of
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solutions, dispersions, micelles, or even in disrupted tissue structures, as in meat and fish products. Generally gelation is a two-step phenomenon (Damodaran, 1989). The first step usually involves dissociation of the quaternary structure of the protein, followed by unfolding. In several proteins, heating to about 40°C is sufficient. Some fish protein sols turn slowly into gels even at 4°C. Preheating at 25 to 40°C, called ashi or setting, is applied prior to cooking in manufacturing gelled, elastic fish–meat products. During setting, the endogenous transglutaminase catalyzes the formation of crosslinks between myosin heavy chains. In ovalbumin solutions, gelling starts at 61 to 70°C. In the second step, at higher temperature, the unfolded molecules rearrange and interact, initially usually with their hydrophobic fragments, forming the lattice. Ovalbumin gels increase in firmness when heated up to about 85°C. Subsequent cooling generally stabilizes the gel structure. If the rate of the structuring stage is lower than that of denaturation, the unfolded molecules can rearrange and form an ordered lattice of a heat-reversible, translucent gel. Too rapid interactions in the denatured state lead to an irreversible coagulum due to random associations with insoluble, large aggregates. In the gel network there are zones where the polymers interact, and large segments where the macromolecules are randomly extended. The lattice is responsible for the elasticity and the textural strength of the product. In multicomponent gels all constituents may form separate or coupled networks, or one component, not involved in network formation, may indirectly affect the gelling by steric exclusion of the active molecules. Such exclusion increases the concentration of the active component in the volume of the solution where the gel is formed. In composite gels made from minced squid meat at 1.5% NaCl, the added carrageenan and egg white form separate networks, which support the structure, made of squid proteins, while added starch fills the lattice, swells, and retains water (Gomez-Guillen et al., 1996). K-carageenan added to pork batter formulation increases the hydration and thermal stability of the gels (Pietrasik et al., 2005). Proteins and polysaccharides that have opposite net charge, when in mixed solutions, may form different soluble and insoluble complexes held by ionic bonds. Lipid-filled milk protein gels containing small fat globules with a narrow particle size distribution have smooth texture and high shear modulus. The formation of a three-dimensional network of partially unfolded molecules is also crucial for preparing proteinaceous films. These films are usually made from a protein solution at pH values far from the pI. The process comprises controlled denaturation of the molecules due to heating or shear, addition of plasticizers, degassing, casting or extruding through a nozzle, and drying to evaporate the solvent. 6.3.4.2 Interactions of Components The structure of gels depends upon the components and the process parameters. Proteins containing over 30% hydrophobic residues form coagulum-type gels, such as hemoglobin and egg-white albumin. The gelling-type proteins contain less hydrophobic residues and are represented by some soybean proteins, ovomucoid, and gelatin.
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The interactions of different macromolecules may decrease the gel strength, may have no influence on the rheological properties of the gel, or may have a synergistic effect. Casein micelles in a whey protein matrix may enhance or decrease gelling, depending on pH. Heat coagulation of sarcoplasmic proteins impairs the gelation of actomyosin in gels made from the meat of pelagic fish. In minced heated fish products the proteinase catalyzed softening known as modori may be decreased by adding protease inhibitors from potato, bovine plasma, porcine plasma, or egg white. Also, inhibitors from various legume seeds are effective against fish muscle proteinases (Benjakul et al., 2001; Matsumoto and Noguchi, 1992). The impact of other factors may be controlled by applying optimum processing parameters. 6.3.4.3 Binding Forces and Process Factors The hydrophobic interactions prevail at higher temperatures and probably initiate the gel lattice formation, while hydrogen bonds increase the stability of the cooled system. The electrostatic interactions depend upon pH, charge of the molecules, ionic strength, and divalent ions. Intermolecular –S–S– bridges, as well as covalent bonds formed due to the activity of transglutaminases, may also add to the gel formation. In gelled fish products, –S–S– bonding occurs during cooking at about 80°C (Hossain et al., 2001). Gels stabilized mainly at low temperature by hydrogen bonding are heat-reversible; that is, they melt due to heating and can be set again by cooling. Gels stabilized by hydrophobic interactions and covalent bonds are heat stable. Depending on the properties and concentration of the protein, ionic strength, and pH, even a coagulum-type gel such as that of ovalbumin can be melted by repeated heating and set again when cooled (Shimizu et al., 1991). Heat-induced gels may melt under increased pressure at room temperature, while cold-set gels of gelatin are resistant to such conditions (Doi et al., 1991). Optimum ionic strength and concentration of Ca2+ are required for producing well-hydrated, heat-set gels from whey proteins. There is generally a pH range at which the gel strength in the given system is the highest. It depends on the nature of the polymers participating in cross-linking, and increases with protein concentration. At the pI of the proteins, due to lack of electrostatic repulsion, the rate of aggregation is usually high, leading to less ordered, less expanded, and less hydrated gels. In heat-induced gels made of minced, waterwashed chicken breast muscle at pH 6.4 and low NaCl concentration, the myofibrils, insoluble at such conditions, form local networks of aggregates with large voids between them. Increasing pH to 7.0 results in a gel with an evenly distributed network of myofibrils and an additional network of fine strands, smaller intramyofibrillar spaces, increased stress and strain values, and higher water-holding capacity (Feng and Hultin, 2001). The ovalbumin gel has optimum rheological properties at pH 9, while at pH <6 it is brittle and has low elasticity. Transparent ovalbumin gel can be made by heating at pH other than pI at a certain salt concentration. In a two-step procedure, transparent gels can be made from ovalbumin, bovine serum albumin, and lysozyme over a
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broad range of salt concentrations by heating the protein solutions first without salt, and after cooling by repeated heating in the presence of added salt (Tani et al., 1993). The pH range for gelation of whey proteins is 2.5 to 9.5, although near the pI, which is about 5, the gels are opaque, coarse, and may turn into curdlike coagulum. In the neutral to alkaline pH, gels made of fat-free whey protein isolates or purified β-lactoglobulin are translucent, smooth, and elastic. In acid conditions, if high shear force is applied for a short time at denaturation temperature, whey proteins aggregate to microparticles. This leads to well-hydrated gels of a smooth, nonelastic texture, similar to that of a fat emulsion. In a slightly alkaline environment at temperatures above 60°C, insoluble aggregates are formed due to the denaturation of β-lactoglobulin, the major component of whey proteins. The rheological properties of whey protein gels, at different pH, depend also on the concentration of Ca2+. The –S–S– bridges are responsible for the thermal stability of some gels. Such bonds add to the elasticity of heat-set whey protein gels at neutral to alkaline pH, but not in acidic conditions when the thiol has low reactivity (Jost, 1993). Ascorbic acid improves the formation of heat-set gels of ovalbumin and fish proteins by undergoing rapid oxidation to dehydroascorbic acid, which affects polymerization by intermolecular –S–S– bridges. In making edible films from wheat gluten, the exposed SH groups of the protein, heat-denatured in an alkaline solution, form –S–S– cross-links due to air oxidation during drying (Roy et al., 1999). 6.3.4.4 Importance in Food Processing Gelling is important for the quality of comminuted-type, cooked sausages, and for gelled fish products. The gel strength of such commodities is mainly affected by the properties of myosin and the processing conditions. Comminuting of the meat with salt results in unfolding of the myosin microfibrils and increases the surface hydrophobicity. This leads to hydrophobic associations in the lattice structure. Heating to 50 to 80°C favors deconformation of the myosin heads (Figure 6.5) and their interactions. Although myosin has the highest gel-forming ability of all muscle proteins, the whole myofibrillar protein fraction, the sarcoplasmic, and the connective tissue proteins are also capable of gelation. The overall gel strength depends on the concentration and interactions of different proteins. Edible films may be used for their barrier properties to prevent the migration of water, oil, oxygen, and volatile aroma compounds between food and the environment. The coatings prevent oxidative browning of sliced fruits and vegetables due to their antioxidant properties. The oxygen radical scavenging capacity of films made of
FIGURE 6.5 A schematic representation of the myosin molecule. (From Sikorski, Z.E. (Ed.), Chemical and Functional Properties of Food Components, Technomic Publishing Co., Inc., Lancaster, PA, 1997. With permission.)
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commercial concentrated whey protein powder has been found to be higher than that of coatings based on calcium caseinate. Addition of carboxymethyl cellulose to the formulation increases the antioxidant capacity of the products (Le Tien et al., 2001). Films can also find application as enzyme supports and carriers of food ingredients. For edible sachets used for delivering premeasured quantities of ingredients in food processing, the heat-seal ability of the material is important. Antimicrobial agents added to edible coatings used for food protection may affect the mechanical strength and barrier properties of the material (Ko et al., 2001). Different films have unique functional properties best suited to fulfill the needs of specific food applications. The films can be made of proteins, or as a composite material with saccharides or lipids. For these purposes collagen, gelatin, casein, total milk proteins, whey proteins, wheat gluten, corn zein, water-soluble fish proteins, and soy proteins are used. The barrier properties, appearance, tensile strength, thermal stability, and the heat-seal ability of the products depend on the characteristics and proportions of the gelling components, interactions and contents of plasticizers, and conditions of fabrication. Films made of whey protein are transparent, bland, and flexible, have very high oxygen, oil, and aroma barrier properties in an environment of low humidity, and are poor protection against water vapor migration. Some of their characteristics depend on the degree of thermal denaturation of the protein before casting. Increasing the degree of denaturation by heating leads to higher tensile strength and insolubility, and to lower oxygen permeability of the films (Perez-Gago and Krochta, 2001). Water vapor permeability may be decreased by laminating a lipid layer over a protein film or by uniformly dispersing the lipid in the proteinaceous component. The result depends on the properties and amount of added lipid.
6.3.5 EMULSIFYING PROPERTIES 6.3.5.1 The Principle Proteins help to form and stabilize emulsions, such as dispersions of small liquid droplets in the continuous phase of an immiscible liquid. The decrease in the diameter of the droplets due to agitation increases exponentially the interfacial area. The work (W) required for the increase in surface area (∆A) can be decreased by lowering the surface tension (z): W = z∆A due to attachment of proteins to the droplets. The protein film around the lipid globules, with its electrostatic charge and steric hindrance, prevents flocculation, that is, formation of clusters of globules and thus rapid creaming due to the action of gravitational force: V = 2r2g∆P / 9µ where V = velocity of the droplet, g = gravitational force, ∆P = difference in density of both phases, µ = viscosity of the continuous phase, and r = radius of the droplet or cluster of droplets.
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Stable films around the fat globules also prevent the coalescence of the dispersed phase, that is, joining of the fat globules to form a continuous phase. Furthermore, soluble proteins increase the viscosity of the dispersing phase, thus reducing the rate of creaming and coalescence. The efficiency of proteins as emulsifiers depends upon their surface hydrophobicity and charge, steric effects, elasticity or rigidity, and viscosity in solution. Globular proteins, which have stable structures and are very hydrophilic, are good emulsifiers only when unfolded. However, the emulsifying properties do not increase linearly with the hydrophobicity of the protein, as they depend on the hydrophile–lipophile balance (HLB), which is defined as HLB = 20Wh /Wt where Wh = weight of hydrophilic groups, Wt = total weight of the molecule. The emulsifiers with HLB <9 are regarded as hydrophobic, HLB 11 to 20 as hydrophilic, and HLB 8 to 11 as intermediate. There is an effect of protein solubility, as the molecules must migrate to the surface of the fat globules. However, in comminuted sausage batters in the presence of salt, the insoluble proteins also may participate in the formation of fat dispersions. After a few minutes of homogenization, about 90% of initially insoluble meat proteins of the stroma can be found in the emulsion layer (Nakai and Li-Chan, 1988). The quantity of protein required for stabilization of an emulsion increases with the volume of the dispersed phase and with the decrease in diameter of the droplets. The concentration of proteins forming a monomolecular layer at the interface is on the order of 0.1 mg/m2, and the effective concentrations are in the range 0.5 to 20 mg/m2. For a high rate of film formation, the required concentration of protein in the emulsion may be as high as 0.5 to 5%. 6.3.5.2 Factors Affecting Emulsifying The pH of the environment affects the emulsifying properties by changing the solubility and surface hydrophobicity of proteins, as well as the charge of the protective layer around the lipid globules. Ions alter the electrostatic interactions, conformation, and solubility of the proteins. However, in many foods, mainly comminuted meat batters, the concentration of NaCl is considerably high for sensory reasons. Thus small changes in the salt content within the accepted range may have no significant effect on the properties of proteins. Heating to about 40 to 60°C, causing partial unfolding of the protein structure without loss in solubility, may induce gelation of the protective layer, as well as decrease the viscosity of the continuous phase. Therefore moderate heating may improve the emulsifying properties of proteins. 6.3.5.3 Determination of Emulsifying Properties Several procedures are used to determine the efficiency of proteins in emulsifying lipids and the stability, which the proteins impart to the emulsions. The emulsifying capacity is represented by the volume of oil (cm3) that is emulsified in a model system by 1 g of protein when oil is added continuously to a
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stirred aliquot of solution or dispersion of the tested protein. It is determined by measuring the quantity of oil at the point of phase inversion. The latter can be detected by a change in color, viscosity, or electrical resistance of the emulsion, or the power taken by the stirrer engine. The emulsifying capacity decreases with increasing concentration of protein in the aqueous volume. It is affected by the parameters of emulsification depending on the equipment, as well as by the properties of the oil. The emulsion stability is measured as the final volume of the emulsion after centrifuging the initial volume or standing for several hours at specified conditions. It may also be determined as the quantity of oil or cream separated from the emulsion, or the time required for the emulsion to release a specified quantity of oil.
6.3.6 FOAMING PROPERTIES Food foams are dispersions of gas bubbles in a continuous liquid or semisolid phase. Foaming is responsible for the desirable rheological properties of many foods, such as the texture of bread, cakes, whipped cream, ice cream, and beer froth. Thus foam stability may be an important food quality criterion. However, foams are often a nuisance for the food processor, such as in the production of potato starch or sugar, and in the generation of yeast. Residues of antifoaming aids in molasses may drastically reduce the yield in citric acid fermentation. The gas bubbles in food foams are separated by sheets of the continuous phase, composed of two films of proteins adsorbed on the interface between a pair of gas bubbles, with a thin layer of liquid in between. The volume of the gas bubbles may make up 99% of the total foam volume. The contents of protein in foamed products is 0.1 to 10% and of the order of 1 mg/m2 interface. The system is stabilized by lowering the gas–liquid interfacial tension and formation of rupture-resistant, elastic protein film surrounding the bubbles, as well as by the viscosity of the liquid phase. The foams, if not fixed by heat setting of the protein network, may be destabilized by drainage of the liquid from the intersheet space due to gravity, pressure, or evaporation, by diffusion of the gas from the smaller to the larger bubbles, or by coalescence of the bubbles resulting from rupture of the protein films. Factors facilitating the migration of the protein to the interface and formation of the film are important for foaming. The foaming capacity or foaming power— the ability to promote foaming of a system, measured by the increase in volume— is affected mainly by the surface hydrophobicity of the protein. The stability and strength of the foam, measured by the rate of drainage and the resistance to compression, respectively, depend on the flexibility and the rheological properties of the film. Other components of the system, mainly salts, sugars, and lipids, affect the foam formation and stability by either changing the properties of the proteins or the viscosity of the continuous phase. The standard whey protein concentrates, which contain 4 to 7% of residual milk lipids, have significantly inferior foaming properties than lipid-free isolates. Excellent foaming ability is characteristic for the egg-white proteins, especially ovalbumin, ovotransferrin, and ovomucoid. Chilling of the egg white below room temperature or the presence of sugar or lipids decrease the foaming, pH less than 6 increases the foaming capacity, while heating of the dried proteins at 80°C for a few
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days before use increases the foam stability. The foaming of whey protein isolate can be improved by addition of Ca2+ or Mg2+. The ions are effective only immediately after the salts are added. According to Zhu and Damodaran (1994), the ions might cause unfolding and polymerization of the proteins at the interface via ionic linkages. On the other hand, prolonged incubation of the isolate solution with the salts slightly reduces the film-forming ability, possibly by promoting aggregation and micellization of the protein.
6.4 PROTEINS AS IMPORTANT COMPONENTS IN FOODS 6.4.1 MUSCLE PROTEINS The meat of slaughter animals, fish, mollusks, and crustaceans is predominantly used to prepare different dishes, a variety of canned, smoked, marinated, salted, and dried products, as well as a large number of sausage assortments and gels. In these products the functional properties of the muscle proteins are responsible for the desirable sensory attributes. However, an increasing proportion of the raw material, that is less suitable for producing high-quality products by the meat, poultry, and fish industry, is used for manufacturing protein concentrates, preparations, and hydrolysates. These products can be incorporated into various food commodities for nutritional reasons and as functional ingredients. Much research effort has been devoted to work out optimum parameters for producing different protein concentrates from fish and krill. While the products have high nutritional value and many are tasteless and odorless, some, manufactured in denaturing conditions, lack the desired functional properties. A good example is the fish protein concentrate obtained by extraction with a hot, azeotropic solution of isopropanol. On the other hand, a concentrate of myofibrillar proteins known as surimi, produced mainly from fish and to a lesser extent from poultry and meat, is highly functional. Surimi is originally a Japanese product obtained by washing minced fish flesh several times with fresh water. This treatment removes most of the sarcoplasmic proteins, including enzymes and pigments, nonprotein nitrogenous compounds, various odorous substances, other soluble components, and fat. The remaining myofibrillar proteins have higher gel-forming ability than the original protein mixture. Nowadays, fish surimi is produced mainly onboard vessels. The typical commercial surimi is made from Alaska pollock. In order to prevent deterioration of its functional properties during frozen storage, different cryoprotectants, predominantly saccharides, are added prior to freezing (Figure 6.6). Surimi is used mainly for manufacturing traditional Japanese gelled products, obtained by mixing it with salt, grinding, forming, and steaming or broil-cooking (kamaboko) or frying (tempura). Another growing outlet is the production of a variety of fabricated specialties, including molded (shrimp-type) and fiberized (crab-leg type) shellfish analogs. The most important topics in this area in the last two decades have been the suitability of fish of different lean and fatty species, including freshwater fish, as raw material for surimi (Luo et al., 2001); mechanisms of protein changes in frozen stored material
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White Fish
Water
Water
Washing
Wastewater, Scales
Heading Gutting
Heads, Viscera
Washing
Wastewater, Scales
Meat Separating and Mincing
Skin, Fins, and Bones
Leaching
Water 3x
Draining
Water-Soluble Components
Refining
Scraps of Skin, Scales
Dewatering
Cryoprotectants
Surplus Water
Mixing
Surimi
FIGURE 6.6 A flow sheet of the process used for manufacturing surimi.
and selection of optimum cryoprotectants; the effect of different ingredients on the texture and water binding of various gelled products; as well as other factors affecting gelation of surimi (Niwa, 1992; Jiang et al., 1998).
6.4.2 MILK PROTEINS Milk proteins have found various applications in formulated foods, and as meat extenders. Initially only the caseins were used, but the recovery of whey proteins and their fractions is economically also feasible.
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Least soluble is acid casein. Its solubility and foaming properties can be improved by glycosylation of the ε-NH2 groups. Sodium and calcium caseinates prepared by acid precipitation and neutralization are soluble, very heat stable, have high WHC, emulsifying, foaming, and gelling ability. Rennet casein has low solubility in the presence of Ca2+. Precipitates produced by heat denaturation of the whey proteins and coprecipitation with casein by the addition of acid or Ca2+ salts are more soluble and have higher nutritional value than the acid and rennet casein. Different whey protein concentrates and isolates are manufactured by complexing with phosphates or polysaccharides, by gel filtration, ultrafiltration, or heat denaturation. At pH of about 4.2 and 55 to 65°C α-lactalbumin undergoes isoelectric precipitation due to dissociation of the Ca2+ and the hydrophobic interactions (Bramaud et al., 1995). Other minor whey proteins precipitate, while the soluble β-lactoglobulin is separated, concentrated by ultrafiltration, neutralized, and spray-dried. The product has superior WHC and gelling properties in meat products. β-lactoglobulin, however, denatures and forms insoluble aggregates at temperatures above 60°C at pH 8. This is a limitation for the use of whey protein isolates in pasteurized products. The thermal stability of β-lactoglobulin can be improved by controlled hydrolysis. Doucet et al. (2001) have shown that hydrolysis of a whey protein isolate by Alcalase to a degree, when only less than 16% and 4% of β-lactoglobulin and α-lactalbumin, respectively, remained unchanged, induced gel formation. Gelation was preceded by the formation of aggregates. The structure of the gel was stable over a range of temperature from 30 to 65°C. Other, undenatured forms of whey proteins are also used as foam stabilizers and gelling agents; some are soluble under acid conditions. The purified α-lactalbumin fraction is more suitable for infant food formulations than the whole whey protein concentrate because human milk does not contain β-lactoglobulin. Milk proteins have high nutritional value due to the very favorable a.a. composition, and they do not contain significant amounts of antinutritional factors, except for some allergenic activity. Furthermore, several milk proteins have antimicrobial properties. The whey protein lactoferrin has antibacterial activity due to its iron-sequestering ability and the strongly bactericidal N-terminal domain. Lactoperoxidase in the presence of H2O2 in milk oxidizes thiocyanate (SCN–), derived from enzymatic hydrolysis of cyanogenic glycosides from the cow’s feed, to different oxidized forms, which inhibit the growth of bacteria due to oxidation of SH groups, NADH, and NADPH. In the polypeptide chains of various milk proteins are some a.a. sequences that can be liberated as biologically active peptides with some positive effects on the functioning of the human organism. Generally these oligopeptides contain predominantly the residues of hydrophobic a.a., Lys, and Arg. They are very resistant to the digestive peptidases. Especially rich in biologically active peptides are β-lactoglobulin, αlactalbumin, lactoferrin, αs1-casein, β-casein, and κ-casein. The peptides Val-ProPro and Ile-Pro-Pro (known as inhibitors of the angiotensin I-converting enzyme), which decrease the systolic and diastolic blood pressure, have been isolated from milk fermented in the presence of Lactobacillus helveticus. Many other fermented milk products have been found to contain other peptides capable of decreasing blood pressure, including
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Tyr-Pro Ile-Pro-Ala Tyr-Leu-Leu-Phe Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln Various milk proteins contain, in their polypeptide chains, some a.a. sequences that correspond to those of peptides having opioid, immunomodulating, Ca2+ binding, antioxidant, antibacterial, or antiviral activity. Glycomacropeptide, the product of hydrolysis of κ-casein, has the ability to bind some pathogenic bacteria, prevent adhesion of bacteria and viruses, promote the growth of bifidobacteria, exert immunomodulatory activity, and cause gastric hyposecretion. The antihypersensitive, opioid, immunomodulatory, and Ca2+ binding milk peptides, the antiviral properties of various milk components, as well as the antimicrobial activity of lactoperoxidase, lactoferrin, lactoferricins, casein peptides, and peptides from α-lactalbumin have been concisely treated by Holt and Rogiński (2001). A large number of biologically active peptides have also been liberated by different hydrolytic enzymes and acid hydrolysis from other sources, including the proteins of fish muscle, soybeans, corn, wheat, barley, and rice. Angiotensin Iconverting enzyme inhibitory activity was also recently found in hydrolysates of animal by-products—defatted cracklings of pork and chicken feathers (Karamać et al., 2005).
6.4.3 EGG PROTEINS Egg albumen is used in various food formulations because of its foaming properties and heat-gelling ability, while egg yolk serves as an emulsifying agent. The functional properties of egg proteins have been thoroughly treated by Ternes (2001). The strength of egg-white gels can be increased by preheating the dry protein at 80°C before use (Kato et al., 1990a). In the gels formed by preheated egg white, the molecular weight of the aggregates is much smaller than in gels made of nonpreheated proteins. Preheating of dry egg white also confers on the protein lower enthalpy and temperature of denaturation. This results in increased flexibility of the molecules, leading to more cohesive interfacial films, that is, improved surface functional properties. The more cohesive films composed of overlapping polypeptides are more capable of expanding under stress than the films formed from nonpreheated proteins (Kato et al., 1990b). Egg-white and egg-yolk gels can also be prepared without heating by applying high pressure (Hayashi et al., 1989). The albumen and the yolk from fresh eggs form stiff gels after exposure for 30 min to a pressure above 6,000 kg/cm2 and 4,000 kg/cm2, respectively. The pressure-induced gels are more adhesive, more elastic, and more digestible than the boiled egg. Lysozyme, which makes up 3.5% of total egg-white proteins and can be easily separated by ion-exchange techniques, is recognized as a safe, antimicrobial agent
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to be used for food preservation. It is stable up to about 100°C, has maximum activity at pH 5.3 to 6.4, and inhibits several pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum, Yersinia enterocolitica, and Campylobacter jejuni (Kijowski and Leśnierowski, 1999).
6.4.4 LEGUME PROTEINS Soybean proteins are used in a variety of traditional products, such as soymilk or fermented items, defatted flour, grits, concentrates, or isolates. The traditional products are prepared according to procedures known in the Orient involving water extraction, cooking, coagulation of the proteinaceous curd, roasting, and fermentation, often in combination with other components. Different forms of concentrated or isolated protein products are prepared by milling, toasting, extraction of fat and saccharides, and isolation of protein fractions. The antinutritional factors present in soybeans are usually inactivated or removed in processing. The “beany” or “painty” off-flavor in soymilk due to volatiles generated in lipoxygenase-catalyzed reactions is prevented by thermal denaturation of the enzyme before or during grinding with water (Kwok and Niranjan, 1995). The protein isolates may be tailor-made, that is, various fractions are separated in order to have the desirable a.a. composition and functional properties. Grits, flours, and isolates for food applications are also produced from other legumes, mainly peanuts, beans, broad beans, and peas (Lampart-Szczapa, 2001).
6.4.5 CEREAL PROTEINS Cereals are an important source of proteins because the grains contain, depending on the species, from about 7.5 to 14% of N×6.25. They are used in foods mostly as flour—the main raw material for producing breads, noodles, cakes, and other commodities of the baking industry. The biological value of cereal proteins is significantly lower than that of meat proteins because of a deficiency of lysine, and the content of methionine residues in the proteins of barley, maize, oats, rye, and wheat is also too low. The prolamins and glutelins present among the storage proteins of wheat, rye, triticale, barley, and oats may evoke in some persons various symptoms of dietary intolerance. A very serious disease caused by gluten intolerance is celiac disease, known also as celiac sprue. This enteropathy is caused by the gliadin peptides altered by the human tissue transglutaminases. These peptides are recognized as foreign by local intestinal T cells. Such stimulation of the immune response leads to accumulation of lymphocytes, plasma cells, and macrophages in the lamina propria and an increased number of lymphocytes in the surface layer of the epithelium, which results in shortening and flattening of intestinal villi. In patients who do not exclude gluten from their diets, the disease may cause lymphoma of the gastrointestinal tract. The most important role of wheat storage proteins in food processing is that played by gluten in the formation of dough and in the texture of bread crumbs, noodles, and cakes (see Chapter 12). Cereals also contain different enzymes, which catalyze various changes in proteins, saccharides, lipids, and other constituents
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during storage in the grains and flour, as well as during baking of bread and malting. A brief but comprehensive presentation of cereal grain proteins has been presented by Wrigley and Bekes (2001).
6.4.6 MYCOPROTEIN One of the results of research in the second half of the 20th century on single-cell protein for human food and for animal feed was the development of the process of manufacturing mycoprotein. The product has been approved in several European countries for general food use. It has the form of insoluble hyphae, typically 400 to 700 µm × 3 to 5 µm with low frequency of branching. Mixing with proteinaceous binders, flavorings, and colors, followed by forming and heating leads to meat product analogs. Mycoprotein can also be used in extruded commodities and as fat replacement in yogurt and ice cream (Rodger, 2001).
6.5 EFFECTS OF HEATING 6.5.1 INTRODUCTION Heating affects the conformation of proteins, enzyme activity, solubility, and hydration, and leads to thermal and hydrolytic rupturing of peptide bonds, thermal degradation, and derivatization of a.a. residues, cross-linking, oxidation, and formation of sensory active compounds. Most of these reactions are reflected in foods in desirable or detrimental changes in color, flavor, juiciness, rheological properties, enzyme activity, and toxicity. These processes are affected by the temperature and time of heating, pH, oxidizing compounds, antioxidants, radicals, and other reactive constituents, especially reducing saccharides. Some of the undesirable reactions can be minimized by appropriate treatments. Stabilizers, such as polyphosphates and citrate, which bind Ca2+, increase the heat stability of whey proteins at neutral pH. Lactose present in whey in sufficiently high concentrations may protect the proteins from denaturation during spray drying (Jost, 1993). The heat-induced unfolding of a protein usually proceeds in several steps because separate domains of the molecule denature at different temperatures, depending on the forces stabilizing their structure. The molecular transition temperature, that is, the point at which major change in conformation occurs, can be determined by various techniques, mainly by differential scanning calorimetry. The susceptibility of proteins to thermal denaturation depends on their structure, predominantly on the number of cross-links, as well as on simultaneous action of other denaturing agents. Salt bridges, side chain–side chain hydrogen bonds, and a large proportion of residues in α-helical conformation increase the thermal stability of many proteins (Kumar et al., 2000). In some proteins the first changes appear at 35 to 40°C. The peak maximum temperature of transition of myosin in the myofibrils ranges from 43°C in cod muscle to 60°C in the bovine M. semimembranosus (Howell et al., 1991). Sodium chloride decreases the thermal stability of meat proteins by up to about 30%. In the experiments of Fernandez-Martin et al. (2000), pressurization of sausage formulations at 300 MPa at 10°C for 20 min significantly destabilized the
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proteins. However, the thermal stability of proteins in formulations heated under hydrostatic pressurization was higher than that in control samples. The stabilizing effect was related to the temperature of heating. Heating of soluble collagen brings about uncoiling of the superhelix and unfolding and separation of the polypeptide chains from each other, if stable bonds do not bind them. This results in a decrease in viscosity of the solution. The denaturation temperature Td can be measured in the range of rapid change in viscosity. Insoluble collagen fibers shrink due to heat denaturation by up to 75% of their original length. The shrinkage temperature Ts is usually about 20°C higher than Td. It increases with the number of cross-links, and in fish collagen with the contents of PrOH. The denatured collagen turns into gelatin after prolonged boiling in water, due to partial hydrolysis. This phenomenon affects the rheological properties of cooked meats containing much connective tissue. Thermal changes comprise loss in solubility due to aggregation of the proteins, for example, β-lactoglobulin and the immunoglobulins, or increase in solubility resulting from breakdown of superstructures, as in collagen. Heating of many proteins to 105 to 140°C at low water content in conditions resembling those during extrusion cooking leads to increased solubility. This concerns proteins that have an open random coil structure and low number of –S–S– bonds and are not able to form many other covalent cross-links (Mohammed et al., 2000). Further effects of heating include formation of gels, such as those in most types of sausages, kamaboko, meat gels, and several types of cheeses, development of gas-retaining structures as in dough and bread, hydrolytic changes, and alteration of the rate of proteolysis, modification of nutritive value, and inactivation of some allergens.
6.5.2 RHEOLOGICAL CHANGES Proteins are primarily responsible for the texture of meat, poultry, fish, meat and fish gels, cheese, bread, cakes, and many other foods. The texture of muscle foods is affected mainly by the • •
•
Contents and cross-linking of collagen, and the morphological structure of the tissues, e.g., meat, fish, and squid Biochemical state of the muscle, that is, interactions of the proteins of the myofibril, such as those in rigor mortis, cold shortening, thaw rigor, as well as the proteolytic changes during aging of meat Mechanical disintegration of the muscle structure
Abusive treatment during storage, processing, and preparation may lead to toughening and partial loss of the gel-forming ability of frozen stored fish, shrinking and toughening of pasteurized ham, toughening and formation of grainy structure in casein curd, and separation of fat layers in sausages. The thermal changes in meat commence at about 40°C, and some of the meat proteins in solution coagulate at that temperature. Further heating results in shrinkage of collagen (at 50 to 60°C), followed by gelatinization in a moist environment. At about 65°C, hardening of the myofibrils occurs. The final texture of the product
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depends upon hardening of the myofibrillar structure and gelatinization of collagen in the given meat at the particular state of postmortem changes, heated according to a particular time and temperature regime. The rheological changes in meat and fish are reflected in drip loss (20 to 40% of the original weight), and shrinkage. Wheat flour, when mixed with water, forms a dough that retains gas and sets due to heating during the baking of bread. The agent responsible for these properties of wheat flour is gluten, which develops in mixing of the flour with water. Dry gluten contains 80 to 90% proteins, 5 to 10% saccharides, 5 to 10% lipids, and minerals. The gluten proteins are composed of 40 to 50% gliadin, 35 to 40% glutenin, and 3 to 7% soluble proteins. During mixing with water the flour particles, containing starch granules embedded in the protein matrix, are progressively hydrated and form the viscoelastic dough. SH/S–S interchange reactions result in formation of intermolecular –S–S– bridges. Generally the resistance to extension of the dough increases to a peak value. Overmixing usually results in a decrease in resistance and is caused by excessive breaking of the –S–S– bonds, not accompanied by the formation of new intermolecular bridges. This ends in depolymerization of the protein and thus increased solubility and decreased viscosity of the dough. The required mixing time can be controlled, within limits, by changing the pH, adding oxidants, or mixing in an inert atmosphere. The gas-retaining ability depends on the viscosity of the hydrated protein-carbohydrate-lipid system. Rice and corn flours do not decrease the gas diffusion rate as effectively, because the dough prepared from them is not as viscoelastic as dough that contains gluten. The relatively high gas retention of rye-flour dough is due to the viscosity of soluble pentosans. A similar effect can be achieved in gluten-free dough by adding surfactants, particularly natural flour lipids, monoacylglycerols, or xanthan gum (Hoseney and Rogers, 1990). During baking the expansion of the dough stops due to polymerization or cross-linking, as a result of SH/SS interchange reactions. The setting prevents further expansion of the dough and the collapse of the loaf of bread. Very severe heating of foods, at much higher temperature and time than required for sterilization, may lead to the formation of isopeptide cross-links between the free NH2 group of Lys and the carboxylic group of Asp or Glu. O HN
CH
O
C
HN
(CH2)2
CH
C
(CH2)2
COOH
C
heating
+
O
NH NH2 (CH2)4 HN
CH
C
(CH2)4 HN
CH
C O
O - N ( -glutamyl) lysylamide crosslink
REACTION 6.2
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These reactions tighten the structure of the products and may decrease the biological availability of Lys, as well as the digestibility of the proteins.
6.5.3 CHANGES
IN
COLOR
Pig, sheep, beef, and whale contain about 0.2 to 2.4, 4.5 to 5.5, 1 to 20, and 50 mg of myoglobin in 1g of muscle, respectively. The total content of chromoproteins in the red muscles of fish ranges from a few to 20 mg/g, and is about 20 times higher than in the white muscles. The skeletal muscles of whales contain as much as 5 g of MbFe(II)/100 g. Changes in the chromoproteins due to oxidation and reduction, denaturation, curing, and reactions with sulfur-containing compounds lead to desirable or undesirable alterations in color. Beef heated at 58 to 60°C internal temperature is rare, at 66 to 68°C medium rare, at 73 to 75°C medium, and 80 to 82°C well done. Recommended end point temperature in pork is 77°C and in poultry 77 to 82°C. The reactions of myoglobin in cured, heated meat resulting in the formation of heat-stable nitrosylhemochromogen and a nitrite–protein complex proceed according to Killday et al. (1988) as follows: -
OH
OH
NO
NO
Fe3+
Fe2+
Fe2+
N
N
N autoreduction
NH
-
+
NO
NH
NH
Globin
metmyoglobin
heating
reduction
Globin
nitrosyl myoglobin radical
NO 2
NO Fe2+ + NO 2 Globin
Globin (or Globin radical)
nitrosyl myoglobin
nitrosylhemochromogen
REACTION 6.3
In proteinaceous foods rich in saccharides or secondary lipid oxidation products, the Maillard reaction prevails. Various products of this reaction can be determined and used as indicators of thermal changes in proteins (see Chapter 12).
6.5.4 DEVELOPMENT
OF
VOLATILE COMPOUNDS
Severe heating of proteinaceous foods leads not only to generation of flavor compounds due to the Maillard reaction, but also to thermal degradation of Met and Cys residues in proteins, as well as those of different low-molecular-weight compounds. These reactions are discussed in Chapter 11 of this volume.
6.5.5 REACTIONS
AT
ALKALINE PH
Alkaline treatment is used for peeling of fruits and vegetables, for producing protein isolates, for removing nucleic acids from single-cell protein preparations, and for
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inactivating mycotoxins and proteinase inhibitors. Severe changes in reactive a.a. residues take place in protein solutions and in foods at high pH even at temperatures as low as 50°C. They lead to cross-linking and formation of nontypical a.a. residues. Cross-linking is based on β-elimination, mainly in Cys, Ser, SerP, or Thr, followed by a nucleophilic addition of the ε-NH2 of Lys, δ-NH2 of ornithine, SH of Cys residues, or of NH3 to the double bond of dehydroalanine or 3-methyldehydroalanine: O HN
CH
O
O OH-
C
HN
C
CHR
CHR
Y
Y
HN
+ H2O
C
C
+ Y
C
-
CHR dehydroalanine residue
where: R=H, CH3; Y=OH, OPO3H2, SH, SR+, SSR
O HN
C
O
C
HN
CH2
+
NH
NH2
C
(CH2)4
(CH2)4 HN
CH
CH2
CH
HN
CH
C
C O
O
REACTION 6.4
Reactions of Cys at alkaline pH also liberate free sulfur and a sulfide ion: O HN
CH
C
HN
CH2 -
S
OH
S
S
CH2 CH
C
HN
CH
S
C
C
CH2 S
+ H2O
-
S
CH2 C O
HN
CH
O
REACTION 6.5
CH2
-
C
OH
HN
C
C O
+ H2O + S 2 -
-
+ S
CH2 C O
-
CH
S
CH2 C O
CH2 HN
HN
C
CH2
S
HN
O
O
HN
CH
C O
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At a given temperature, the overall rate of reaction depends upon the rate of β-elimination and on the conformation of the protein. This is because the accessibility of the dehydroalanine residue for the nucleophilic attack depends on the spatial arrangement of the reacting groups. The reaction can be inhibited by acylating the nucleophilic groups in proteins or by adding thiol compounds, which compete with a.a. residues for the dehydroalanine double bond.
NH
NH R
S + H2C
+ H+
C C
R
S
CH2 C
H
C
O
O
REACTION 6.6
Alkaline conditions favoring cross-linking in proteins also lead to racemization of a.a.: L-amino acid ↔ D-amino acid That is, after the first step in the reaction sequence, the carboanions recombine with protons to the L and D forms. The rate of racemization is affected mainly by the properties of the residues (it is the lowest in aliphatic a.a.) and on the structure of the protein. Generally the rate of the process is about 10 times higher in proteins than in free a.a. Severe heating at alkaline pH may decrease the digestibility and biological value of proteins that result from cross-linking and racemization. The rate of absorption in the organism of some D-a.a. is lower than that of the corresponding L forms. Not all D-a.a. can be metabolized. Furthermore, some of the modified residues, mainly lysinoalanine, induce pathological kidney changes in experimental animals.
6.6 OXIDATION Oxidation of a.a. residues in proteins is initiated in most proteinaceous foods by radicals and different reactive forms of oxygen—singlet oxygen 1O2, super oxide anion radical O2–, and hydroxyl radical ⋅OH. These reactive oxygen species appear in the living organism as the result of natural oxidative metabolism. In food they are generated by light, ionizing radiation, catalytic action of cations, especially heavy metals, pesticide residues, and the activity of enzymes. Also lipid peroxides and other oxidation products are involved. Polyphenols, present in many foods, are prone to oxidation to quinones by oxygen at neutral and alkaline pH. They can also act as strong oxidizing agents in different products. Also H2O2, if abused as a bactericidal agent, such as in the treatment of storage tanks, packaging materials, or proteinaceous meals, may cause oxidation of proteins. The effect of oxidative changes in proteins depends upon the activity of the oxidizing agent, the presence of sensitizers, such as riboflavin, chlorophyll, and erythrosine, temperature, and the reactivity of the a.a. residues. Several tissues contain various prooxidants, including transition metals and hem pigments, lipooxygenases,
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and peroxidases, as well as endogenous antioxidants, such as glutathione, super oxide dysmutase, and catalase. Abstraction of a hydrogen atom on the α-carbon or in the a.a. side chain of a protein leads to the formation of protein radicals. These may polymerize with other protein or lipid radicals. Different scission products may also be formed. H2S and free sulfur may be generated due to oxidation of the –SH group. The sulfur-containing a.a. are converted to many oxidized compounds, including cysteine sulfenic, sulfinic and sulfonic acids, mono- and disulfoxides, and mono- and disulfones. The a.a. most prone to oxidation in different model systems and in foods are Met, Cys, Trp, Tyr, and His. Oxidation of Trp leads to kynurenine (Friedman and Cuq, 1988):
R HN
N
N
CH2 CH H2N
CH2
CH2
CH
CH H2N
COOH
H2N
COOH
COOH
O2
C
H2N
O
C
HN
CH2
CH2
CH COOH
kynurenine
O
N
CHO CH2
CH H2N
O
O
CH COOH
H2N
H2N
COOH
N-formylkynurenine
REACTION 6.7
and of His to Asp: N NH CH2 H2N
+
CH2
h , O2
H2N COOH
REACTION 6.8
C
O NH2
CH
CH
NH2
COOH
sensitizer
COOH
+ other products
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In food systems the oxidation of proteins is often preceded by lipid oxidation, mainly by the primary lipid peroxide products or the radicals produced due to their breakdown (O’Grady et al., 2001). The products of oxidation affect the flavor of foods, either directly or by reacting with precursors. Their biological value depends generally upon the degree of change due to scission, polymerization, and oxidation. Furthermore, in heavily oxidized foods the essential a.a. may become limiting in the diet. Formation of protein–protein and protein–lipid cross-links decreases the digestibility of proteins.
6.7 ENZYME-CATALYZED REACTIONS 6.7.1 INTRODUCTION Reactions in proteins and other nitrogenous compounds catalyzed by endogenous enzymes are responsible for many desirable and undesirable sensory attributes of foods—color, flavor, and texture. They also contribute to the generation of compounds that are nutritionally beneficial or may have detrimental effects on human health. The use of added enzymes or enzyme sources is also an essential part of many traditional methods of food processing. Because the conditions of enzymatic reactions are much milder than those applied in chemical treatments, various added enzymes are increasingly used to modify the functional properties of food proteins. Among many enzymes involved in modification of proteinaceous foods, the most important are probably various proteinases and peptidases. Proteolysis leads to increased protein solubility and extractability by hydrolyzing large, highly crosslinked insoluble proteins into smaller units, removing some hydrophobic sequences or splitting off nonprotein fragments of the molecules. Proteinase catalyzed reactions can also increase the solubility of proteins by binding of hydrophilic a.a. residues to the polypeptide chains. Some proteins affect the sensory and functional properties of foods by exhibiting enzymatic activity in their natural environment or when added intentionally during processing in the form of pure enzyme preparations, enzyme-rich materials, or starter cultures of microorganisms. The enzymatic reactions involve not only changes in proteins, but also in lipids, saccharides, and a large number of other food constituents. The examples of effects involving protein changes include loss of prime freshness in fish after catch, rigor mortis and tenderization of meat, ripening of salted fish, manufacturing of caviar, protein isolates and hydrolysates, softening of fish gels, formation of casein curd and ripening of cheese, fermentation in soybean processing, chill-proofing of beer, and proteolytic changes in wheat flour. Enzymatic modification of proteins in food systems has been treated exhaustively by Haard (2001) and recently by Kołakowski (2005).
6.7.2 THE PLASTEIN REACTION Enzyme catalysis can be used for transformation of different proteins into polypeptides with molecular mass of up to 3 kDa known as plasteins, which may have a tailormade a.a. composition and functional properties. These products can be synthesized
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at a high concentration of an appropriate endopeptidase (E–OH) as a result of transpeptidation in a mixture of peptides, generally protein hydrolysates, or by formation of peptide bonds with a.a. esters added to the reaction mixture. In the first case, two smaller peptides are formed due to hydrolysis of the intermediary acyloenzyme R–CO–O–E, or in reaction with some other peptide H2N–R2 a plastein of larger molecular weight is produced: R-CO-NH-R1 + E-OH
R-CO-O-E + H2N-R1
H2O R-COOH + E-OH
H2N-R2
R-CO-NH-R2 + E-OH
REACTION 6.9
Addition of a.a. esters leads to incorporation of desirable a.a. residues into the structure of the generated plasteins. The esters are used instead of free a.a. in order to increase the nucleophilic character of the reacting amino group. The protection of the carboxyl group can be removed after synthesis by hydrolysis in alkaline conditions (Figure 6.7). The rate and yield of the plastein reaction depend on the pH in the reaction mixture, the properties of the endopeptidase, hydrophobicity of the attached a.a. residues, and the concentration of the substrates. At optimal temperature and pH Protein
Endopeptidases
Hydrolysis
Exopeptidases, Organic Solvent
Modification of Peptides
Endopeptidases, Amino Acid Esters
Plastein Reaction
Ethanol
Extraction
Plasteins
FIGURE 6.7 Utilization of the plastein reaction.
Undesirable Amino Acids, Impurities
Amino Acids, Small Peptides
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conditions, 50% concentration of the substrates, and enzyme-to-substrate ratio of 1:50, the reaction time is usually several tens of hours. Incubation of a protein hydrolysate, concentrated to 30 to 40%, with ethyl esters of, for example, Lys, Met, or Trp, with an appropriate endopeptidase at pH 4 to 7 at about 37°C leads, after a few days, to the accumulation of peptides of 2 to 3 kDa enriched in the respective a.a. residues. Also plasteins free of Phe residues can be obtained for phenylketonuric patients. By hydrolysis of a protein in the presence of pepsin, which cleaves the peptide bonds close to large a.a. residues, followed by further treatment of the hydrolysate by pronase, which liberates terminal, hydrophobic residues, the phenylalanine residues can be split from the peptide chain. The free phenylalanine can be removed by gel chromatography, while the hydrolysate can be used for preparing plasteins suitable for phenylketonuric patients. The a.a. composition of the plasteins can also be modified by selecting various proteins as the source of hydrolysates. The rate of incorporation of hydrophobic a.a. residues is higher than that of hydrophilic substrates. Therefore, the plastein reaction may serve to decrease the bitterness of the products by positioning the hydrophobic a.a. residues inside the long polypeptide chains of the plasteins. Furthermore, the selective removal of hydrophobic a.a. from the hydrolyzate decreases the intensity of its bitter taste.
6.7.3 TRANSGLUTAMINASE CATALYZED REACTIONS Transglutaminase (TGase), or R-glutaminyl-peptide- γ-glutamyltransferase, has been found in many isoforms in a large number of organisms. It catalyses the transfer of the acyl group of glutamine residues in proteins or peptides on primary amines or a water molecule: O Prot
(CH2)2
C
NH2 + H2N
O
TGase
R
Prot
(CH2)2
Prot
(CH2)2
C
NH
R + NH3
acyl transfer
O Prot
(CH2)2
C
NH2 + H2N
O
TGase (CH2)4
Prot
C
NH
(CH2)4
Prot + NH3
crosslinking
O Prot
(CH2)2
C
NH2 + H2O
O
TGase Prot
(CH2)2
C
O H + NH3
deamidation
REACTION 6.10
TGase performs, in plant and animal organisms, various catalytic, regulatory, signaling, and immunological functions, due to its protein cross-linking and modifying properties. It is also produced as an extracellular enzyme by Streptoverticillium sp., Physarium sp., and other microorganisms. The red sea bream TGase, cloned in Escherichia coli, is an intracellular enzyme. Generally the activity of TGase of plant and microbial origin is independent on Ca2+, while the enzyme present in animal
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tissues is Ca2+ dependent. However, the Ca2+ requirement depends not only on the source of the enzyme, but also on the type of substrate. TGases occur in the form of a monomer, dimer, or tetramer, soluble in the cytosol or bound in mitochondria and lysosomes. Although they have maximum activity at 50°C, they can be effectively used for modification of food proteins in the temperature range of 5 to 20°C. The optimum pH range for the activity of TGase of different origins is 6 to 9.5. The role of TGase in food processing is due to protein crosslinking, incorporation of Lys and amines, as well as deamidation of Gln residues. The effect of the enzyme activity expressed as cross-linking depends on the concentration of NaCl as well as on the properties of the protein substrate (Ashie and Lanier, 2000). The TGase-mediated incorporation of amines into a polypeptide chain is effective in TGase-specific sites, for example, when the Gln residues are located close to Pro, Ser, Thr, or Tyr. Therefore the efficiency of the enzyme treatment depends not only on the total number of Gln residues in the protein molecule, but also on their location in the polypeptide chain (Kędzior, 2004). The yield of the reaction products can be significantly increased by acylation of the Lys residues in the protein. The deamidation activity of TGases of various origins is affected by the enzymes’ substrate specificity (Ohtsuka et al. 2001). Endogenous TGase activity is responsible for the formation of ε(γ-glutamyl)lysine cross-links in dried fish, in frozen-stored surimi (Haard et al., 1994), and in polymerization of myosin heavy chains during setting of surimi in the manufacture of kamaboko (Kumazawa et al., 1995). Commercially produced microbial TGase has found numerous applications in food processing. It can be used for increasing the gel strength of kamaboko made of surimi produced from fish of low gel-forming ability, in manufacturing various meat commodities, and for improving the rheological properties of dairy products. TGase derived from Streptoverticillium can induce gelation of glycinin and legumin at 37°C, and the gels are more rigid and elastic than thermally gelled products (Chanyongvorakul et al., 1995). Cross-linking of wheat proteins, especially of the high-molecular-weight prolamines, may improve the baking properties of wheat flour that had been deteriorated by proteolysis induced by proteases from some grain pathogens. TGase-mediated enrichment in Lys may increase the biological value of wheat proteins, while blocking of some Gln residues may decrease the allergenic properties of the peptides resulting from prolamine digestion. Reactions catalyzed by added TGase can lead to tailor-made protein preparations, such as edible films of defined barrier properties from whey proteins, and to covalent binding of saccharides to plant proteins rich in Gln residues (Colas et al., 1993). Ca2+-independent enzymes, added at optimum concentration to fish skin gelatin increases the melting point, strength, and viscosity of the gels. The activity of TGase during storage of the product might change the rheological properties of the gel. It can, however, be arrested by heating to 90°C just after incubation of the gelatin solution with the enzyme (Gómez-Guillen et al., 2001). Cross-linking of proteins catalyzed by TGase does not impair the nutritional value of the products. Kączkowski (2005) recently reviewed the biological role and the possibilities of application of TGase as a proteinmodifying factor in food processing.
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6.7.4 PROTEOLYTIC CHANGES
IN
MILK PROTEINS
In cheese manufacturing, a crucial role is played by added chymosin, which cleaves the glycomacropeptide off the κ-casein in milk acidified due to the activity of the starter lactic acid bacteria. The loss of the protective action of the hydrophilic glycomacropeptide brings about the formation of the casein coagulum. Endogenous and bacterial proteinases catalyze extensive modifications in milk proteins. Plasmin, the alkaline serine proteinase associated with casein micelles and fat milk membrane, attacks β-casein and αs1-casein. Several endogenous plasmin activators and inhibitors control the degradation of milk proteins. Enzyme activity is highest at pH 7.5, and is only slightly reduced during high-temperature, short-time pasteurization of milk. Extracellular proteinases produced by psychrotropic bacteria present in chilled milk hydrolyze different caseins and whey proteins. These enzymes release plasminogen and plasmine from casein micelles. Therefore, in cheese making, plasmine is not retained in the casein curd, but leaks into the whey. The bacterial proteinases are generally heat stable—some of them retain 90% of initial activity even after 9 minutes of heating at 121ºC. They can cause a bitter note in UHT milk, age gelation of sterilized milk, and flavor defects in fermented products. Also, proteinases of somatic cells present in milk, especially from cows in late lactation, may decrease the yield of cheese, lead to development of bitterness in pasteurized milk, and gelation in UHT milk after prolonged storage (Holt and Rogiński, 2001).
6.7.5 ROLE
OF
ENZYMES
IN
MUSCLE FOODS
The sensory quality of meat and seafood is significantly affected by the endogenous proteases. The lysosomes contain, among other enzymes, at least 12 cathepsins, which can exert a concerted action on proteins and peptides. The optimum pH for the activity of cathepsins is generally in a low acidic range. However, many enzymes also retain high activity at pH values 1 or 2 units away from the optimum. Cathepsins are released from the lysosomes in stored beef into the sarcoplasm. Most of the meat cathepsins hydrolyze at least some proteins of the myofibrils. Proteolysis by cathepsins has been regarded as one of the factors co-responsible for tenderization of meat. However, not all of these enzymes can have a significant effect on intact meat proteins in the usual temperature conditions of postslaughter handling and chilling of the animal carcasses. Furthermore, although they are able to hydrolyze myosin and actin, only insignificant degradation of these main proteins of the myofibrils takes place during tenderization of meat. They can, however, participate in the concerted action of various proteinases in aging meat and fish. The muscles also contain nonlysosomal proteinases capable of hydrolyzing several myofibrillar proteins responsible for the structural integrity of the muscle fibers, especially the cytoskeletal proteins. Calpains, the calcium-activated neutral metalloproteinases, require the cysteinyl thiol group for activation. The highest activity of calpains against isolated myofibrils is in the pH range 7.0 to 7.5, while at pH 6 and 4.5 the activity is about 80 and 40% of the maximum value, respectively. One form of the enzyme, called m-calpain, requires at least 0.1 to 0.5 mM Ca2+ for activation, the optimum concentration being 1 mM; the second form, µ-calpain, is
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activated at micromolar concentrations of Ca2+. Due to the loss of the Ca2+ retaining ability of the reticulum in the muscle postmortem, the concentration of Ca2+ in the sarcoplasm may increase up to about 0.1 mM. There is a cause-and-effect relationship between the activity of µ-calpain and the decrease of muscle strength (Purslow et al., 2001). The enzyme rapidly degrades the cytoskeletal protein desmin, causing depolymerization of the intermediate filaments that bind neighboring Z-discs in the myofibril. The major fragments resulting from the breakdown are further cleaved by cathepsins. The known variations in the rate of tenderization of various muscles are due to the differences in the activity of calpains and calpastatin—a family of endogenous inhibitor proteins that are strictly calpain specific and are located in the same cellular area as the enzyme. The meat tenderization process also involves degradation of other cytoskeletal proteins—titin, nebulin, and desmin (Jiang, 2000). The changes in proteins catalyzed by endogenous enzymes affect the sensory quality of various salted, cold-smoked, and marinated fish products. In lightly salted salmon, sturgeon, herring, anchovy, Baltic sprats, and fish of many other species, the hydrolytic processes lead to development of tender texture. The flavor becomes predominantly fishy and salty, with meaty, cheesy, and slightly rancid notes. The enzymes involved in the ripening of salted, uneviscerated fish like herring are mainly those of the pyloric appendages. The suitability of fish for salt ripening is affected by the activity of the endogenous proteases, which fluctuates seasonally and depends on the feeding intensity of the fish. Excessive proteolysis, in products stored too long at too high a temperature, leads to unacceptable softening of the flesh. On the surface of overripe salted fish a white “bloom” of crystallized peptides and a.a., mainly Tyr, may appear. In raw herring marinades the ripening occurs due to muscle cathepsins. High activity of muscle proteases during the spawning migration may bring about changes that increase the emulsifying ability of the meat of salmon during the spawning period (Kawai et al., 1990). Different endogenous proteinases are involved in the disintegration of the structure of fish gels known as modori, which often occurs due to slow cooking in the temperature range of 50 to 70°C. In such gels hydrolysis of myofibrillar proteins, particularly myosin, occurs. Early reports indicated the heat-stable alkaline proteinases, found in the muscles of fish of several species, were involved. The activity of these enzymes is usually not detectable below 50°C. Further investigations revealed that cathepins B, L, and L-like contribute to the disintegration of the gel structure by hydrolyzing the myosin heavy chain, light chains, actin, and troponins. Using cysteine proteinase inhibitors can limit these undesirable effects. Jiang (2000) presented a very thorough discussion of the role of different proteinases in modori softening. A high degree of proteolysis is involved in manufacturing edible fish sauces, silage for animal feeding, and hydrolysates for use as functional ingredients in foods and as bacterial peptones (Sikorski et al., 1995; Lopetcharat et al., 2001). Fish sauces belong to a group of traditional fermented products typical in Southeast Asia. They are manufactured by salting different small fish, mainly Stolephorus spp, Ristelliger spp., Sardinella spp., Engraulis spp., Clupea spp., Scomber colias, and Decapterus spp., according to a variety of recipes. In various recipes, the fish-to-salt proportions range from 1 to 6 and the fermentation time, usually at ambient temperature, from about 2 to 18 months. Endogenous and bacterial enzymes are involved in developing
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the typical flavor of the sauces. The undiluted filtrate of the autolysate is regarded as first-grade fish sauce; the brine extract of the nonhydrolyzed residue is of lower sensory quality and nutritional value. Various proteolytic enzymes from animal, plant, and microbial sources added in the process can increase the rate of proteolysis and even improve the quality of the products. Fish silage is typically made from by-catch fish and filleting offal by mincing, acidifying with formic acid or a mixture of formic and sulfuric acid, and proteolysis catalyzed by endogenous enzymes. After about 2 days at 30 to 40°C most of the tissues are solubilized. The silage containing 70 to 80% water can be used as such after removal of the solids and the fatty layer, or the liquid can be concentrated to the required degree. The product, manufactured commercially on a large scale, is used as a feed component for pigs, poultry, and fish. Fish hydrolysates are produced mainly from lean fish of underutilized species or filleting by-products in a process catalyzed by added proteinases of plant, animal, or microbial origin (Figure 6.8) The product, depending on its quality, can be used as a source of a.a. in growth media for microorganisms, as a milk replacement in animal feeding, or as a functional ingredient in foods. Fish Meat Water, 1:2 Endopeptidases NaOH or HCl
Mincing
Hydrolysis Optimum Temperature
Enzyme Inactivation 100oC
Filtration
Insolubles
Evaporation
Vapors
Drying
Vapors
Fish Peptone
FIGURE 6.8 A flow sheet of the process used for manufacturing fish hydrolysates.
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Partial autoproteolysis has been successfully utilized for producing in industrial conditions food-grade, thermally coagulated, frozen protein gel from Antarctic krill. The protein recovery is about 80% of the protein content in the whole krill (Kołakowski and Sikorski, 2000). Other possible applications of proteolytic enzymes in seafood processing include descaling of fish, pealing and deveining of shrimp, tenderizing of squid, isolating pigments from shellfish waste, and reducing the viscosity of fishmeal stickwaters. Squid muscles contain very active proteinases (Kołodziejska, 1999). High proteolytic activity causes extensive degradation of the myofibrillar proteins in the course of extraction of these proteins for analytical purposes. There are significant differences in the autoproteolytic activity in the muscles of squid of different species. Trimethylamine oxide demethylase, present in the muscles of many gadoid fishes, affects the functional properties of fish proteins indirectly by catalyzing the formation of formaldehyde, which may participate in cross-linking. These reactions are most important in frozen stored gadoid fish.
6.7.6 OTHER ENZYMATIC CHANGES
IN
FOOD PROTEINS
Phosphatases may dephosphorylate the caseins. Thiol oxidase may participate in the oxidation of SH groups to disulfides. Wheat lipoxygenase and soybean lipoxygenase, catalyzing oxidation of fatty acids, generate oxidized reaction products, which improve the dough-forming properties and baking performance of flour. A similar role is performed by polyphenol oxidase and peroxidase. Cyclic adenosine monophosphate dependent protein kinase is useful for phosphorylation of a.a. residues under mild conditions. The modification makes the soybean proteins soluble in media rich in Ca2+ and improves their emulsifying properties (Seguro and Motoki, 1990). In a papain-catalyzed reaction, at room temperature, proteins can be acylated and enriched in SH groups using N-acetyl-homocysteinethiolactone (Sung et al., 1983): O H3C
CO
NH
CH
C
CH2 C H2
S + H2N
Prot
papain pH 10
H3C
CO
NH
CH
CO
NH
Prot
(CH2)2 SH
REACTION 6.11
6.8 CHEMICAL MODIFICATIONS 6.8.1 INTRODUCTION Chemical additives and reactions are used to change the color and several properties of different foods and isolated proteins. Typical examples include the use of sodium nitrite in the curing of meat, chemical modifications of dough proteins for improving the texture of many baked goods, and applications of polyphosphates in the meat industry. The intended modification in the properties of proteins can be achieved by
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changing the charge, hydrophobicity, and steric parameters of the molecules due to cross-linking or alterations of the a.a. residues. Ascorbic acid and its oxidation product dehydroascorbic acid have been known as flour improvers in the baking of bread. Many experiments on chemical modification of a.a. residues study the structure–function relationship with respect to proteins in different food systems under the conditions prevailing during processing and storage.
6.8.2 ALKYLATION The carbonyl compounds formed due to autoxidation of lipids and in catabolic processes postmortem in muscles, or introduced with wood smoke can bring about undesirable effects by interacting with a.a. residues. On the other hand, some carbonyl compounds are used intentionally to modify proteins. Formaldehyde was previously used for hardening of the collagen dope in manufacturing sausage casings, protect fodder meals against deamination by the rumen microflora, and bind immobilized enzymes on supports. In reducing conditions, alkylation of amino, indole, thiol, and thioether groups, and binding of saccharides to a.a. residues is possible. 2Prot
NH2 + 4HCHO + NaCNBH3
pH 9
N(CH3)2 + NaHCNBO3 + H2O
2Prot
0 °C
REACTION 6.12
For alkylation of amino, phenol, imidazole, thiol, and thioether groups in a.a. residues of proteins reactions with haloacetates and haloamides can also be used. Prot
SH + ICH2COOH
Prot
S
CH2COOH + HI
REACTION 6.13 Prot — NH2 + ICH2CONH2
Prot
NH
CH2
CONH2 + HI
REACTION 6.14
The digestibility of such derivatives is generally somewhat lower than that of the unmodified proteins. Malondialdehyde, a typical lipid oxidation product, can form cross-links in proteins by reacting with two amino groups. O H
O C CH2 C
H
+ 2 Prot
NH2
Prot
N
CH
CH2
CH
N
Prot + H2O
REACTION 6.15
There is also a possibility that aliphatic monoaldehydes generated in lipid autoxidation can participate in cross-linking of proteins. Formaldehyde can react in food
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systems with amino, amide, hydroxyl, and thiol groups in a.a. residues of proteins, even at room temperature. Some of these reactions result in cross-linking of the proteins. In cheese, the biogenic formaldehyde reacts with the N-terminal histidine of γ2-casein to form spinacine. N N NH NH HCHO HN
CH2 CH H2N
COOH
COOH spinacine
REACTION 6.16
Other carbonyl compounds produced in cheese by the lactic acid bacteria form other derivatives in reactions with histidine (Pellegrino and Resmini, 1996).
6.8.3 ACYLATION Acylation of nucleophilic groups is intended to increase the hydrophobicity of the protein, introduce additional ionizable groups, or to contribute to cross-linking. O O O
+ H2N
Prot
-
OOC
(CH2)2 C
NH
Prot
O
REACTION 6.17
The acylating agents may react with amino, imidazole, hydroxyl, phenol, and thiol groups in a.a. residues. The rate of reaction depends upon the properties of the nucleophiles, pH, the steric factors resulting from the protein conformation, and the presence of inhibitors. The amino and tyrosyl groups can be acylated easily, while His and Cys derivatives readily hydrolyze. Acylation of hydroxyl groups in a.a. residues of proteins proceeds in the presence of an excess of acetic anhydride only after the amino groups have been acylated. N-carboxyanhydrides of a.a. are suitable for the formation of isopeptide linkages. The degree of acylation depends on the nature of the protein, the acylating agent, and the process conditions, and may reach up to 95% of Lys residues. Some food proteins are rich in phosphoric acid residues. The acid may either form ester bonds with Ser residues, as in caseins and in egg proteins, or may stabilize the native conformation of protein micelles by electrostatic interactions with negatively
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charged groups and Ca2+, as in the caseins. In soy proteins, Ser and Thr residues can be esterified and Lys amidated with cyclic sodium trimetaphosphate at pH 11.5 and 35°C (Sung et al. 1983): O Prot
Ser
O O-
OH
P +
Prot
Lys
O
Prot
Ser
O
P
O
O
P
O O
O + P2O 7 4 + H+
P O
O
O
NH2
pH 11 - 12 - H2O
-
pH 11 - 12
Prot
pH<5
Lys
H
O O-
N
P
O O
P
O
P
O
O
-
+ H+
O -
O
REACTION 6.18
The N ε-triphospholysine residues protect the NH2 group during heating in an alkaline environment and hydrolyze at pH lower than 5. For chemical phosphorylation other reagents are also effective, such as phosphorus oxychloride and phosphorus pentoxide. The reactivity of these compounds may, however, lead to undesirable cross-linking of the proteins: Prot
NH2 + POCl3
Prot HOOC
Prot
Prot
NH
NH
+
POCl2 + H + Cl
CO
-
Prot + Cl2PO 2- + H+
REACTION 6.19
Acylation of a.a. residues generally improves WHC, as well as the emulsifying and foaming capacity of the proteins. The pI shifts toward lower values, the solubility increases over that of the unmodified protein above the pI and decreases in the acidic range. This is important, for example, for wheat gluten, which has low solubility in the neutral pH range. The positive change in the functional properties is not in all cases achieved at the highest degree of modification because it depends on the change in surface hydrophobicity, which is affected by the nature of the protein and the acylating agent. The biological availability of acylated proteins depends on the kind of the acyl group and the extent of acylation. Isopeptides are generally well utilized, while the availability of other derivatives usually decreases with the increasing size of the acylating moiety and the degree of acylation.
6.8.4 N-NITROSATION The nitrous acid generated in foods at low pH from endogenous or added nitrites decomposes easily to yield the nitrosating agents nitrous anhydrate and the nitrosonium ion:
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2HO
171
O
N O
N O
N
O + H2O
+
H
O
N O
N
N
O
O + HO
N O
H
REACTION 6.20
Reactions of these compounds with the secondary and tertiary amines contained in many foods lead to carcinogenic N-nitrosoamines: -
O
N
O
NH + O
N
O
N
NO 2
O
or
R R
R
H N
R
O
or
O
H
N
N
O
+ HNO 2
+ -H
+
N
R R
N
N
O
REACTION 6.21
The rate of N-nitrosation increases with the pKa of the amine and depends on the pH; it is highest at a pH between 2 and 4. Compounds capable of binding the nitrosating agents can inhibit the reaction; in meat curing, sodium ascorbate is very effective. Foods low in amines and nitrites contain generally about 1 to 10 ppb, while cured and heavy smoked meat and fish have up to several hundred ppb of N-nitroso compounds. R R N+ N O R
R R N R
-
N O
O
N O
R R
N
N O + R O
N O
REACTION 6.22
6.8.5 REACTIONS
WITH
PHOSPHATES
Proteins may form protein–phosphate complexes of low solubility in an acid environment. The metaphosphates, which are less hydrated than the ortophosphates, form complexes that are less soluble than those with ortophosphates. This has been utilized for the modification of functional properties of protein concentrates and preparations, for separation of proteins in different food processing operations, and in the treatment of food plant effluents containing proteins. Polyphosphates improve the sensory quality of many food products. They prevent the separation of butter fat and aqueous phases in evaporated milk, or of the formation of gel in concentrated milk sterilized by HTST. They also stabilize the fat emulsion
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in processed cheese by disrupting the casein micelles, and thus enhance the hydrophobic interactions between lipids and casein. Polyphosphates are also used in meat processing for increasing the WHC and improving the texture of many cooked products. The mechanisms involved in different applications depend upon the properties of the phosphates and of the commodities, as well as on the parameters of processing. In meat products, the increase in WHC, texture improvement, and decrease in drip may be caused by increasing the pH, complexing of Ca2+ and Mg2+, binding of phosphates to proteins, rupturing the cross-links between myosin and actin, and dissolving some proteins. Phosphates facilitate the manufacture of meat products of low sodium content. To produce an emulsion-type sausage of acceptable quality, at least 2.5% salt is required. The salt content can be decreased to 1.5 to 2.0% without loss in product quality by adding phosphates in amounts of 0.35 to 0.5%. Generally, proprietary brands of several polyphosphates are used in order to provide the best quality and prevent precipitation of orto- and pyrophosphates in brines rich in Ca2+. Polyphosphates are added as cryoprotectants to minced frozen fish. They are also applied in the form of dips to fish fillets prior to freezing to prevent thaw drip losses, and to improve the texture of canned fish. Most common are 10% solutions of Me5P3O10 and Me4P2O7 used for 1 to 2 min. Different proprietary mixtures are also applied, such as Na4P2O7 + Na2H2P2O7 or Na3PO4 + Na4P2O7 + Na2H2P2O7.
REFERENCES Ashie, I.N.A. and Lanier, T.C., 2000, Transglutaminases in seafood processing, in Seafood Enzymes. Utilization and Influence on Postharvest Seafood Quality, Haard, N.F. and Simpson, B.K., Eds., Marcel Dekker, New York and Basel, chap. 6. Benjakul, S., Visessanguan, W., and Srivilai, C., 2001, Porcine plasma protein as proteinase inhibitor in bigeye snapper (Priacanthus tayenus) muscle and surimi, J. Sci. Food Agric., 81, 1039. Boatright, W.L. and Hettiarachchy, N.S., 1995, Soy protein isolate solubility and surface hydrophobicity as affected by antioxidants, J. Food Sci., 60, 798. Bramaud, C., Mimar, P., and Daufin, G., 1995, Thermal isoelectric precipitation of α-lactalbumin from a whey protein concentrate: influence of whey-calcium complexation, Biotechnology and Bioengineering, 47, 121. Chanyongvorakul, Y., Matsumura, Y., Monaka, M., Motoki, M., and Mori, T., 1995, Physical properties of soy bean and broad bean 11S globulin gels formed by transglutaminase reaction, J. Food Sci., 60, 883. Colas, B., Caer, D., and Fournier, E., 1993, Transglutaminase catalyzed glycosylation in vegetable protein. Effect on solubility of pea legumin and wheat gliadins, J. Agric. Food Chem., 41, 1811. Damodaran, S., 1989, Interrelationship of molecular and functional properties of food proteins, in Food Proteins, Kinsella, J. E. and Soucie, W. G., Eds., The American Oil Chemists’ Society, Champaign, IL, chap. 3. Doi, E., Shimizu, A., Oe, H., and Kitabatake N., 1991, Melting of heat-induced ovalbumin gel by pressure, Food Hydrocolloids, 5, 409. Doucet, D., Gauthier, S.E., and Foegeding, E.A., 2001, Rheological characterization of a gel formed during extensive enzymatic hydrolysis, J. Food Sci., 66, 711.
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Feng, Y. and Hultin, H.O., 2001, Effect of pH on the rheological and structural properties of gels of water-washed chicken-breast muscle at physiological ionic strength, J. Agric. Food Chem., 49, 3927. Fernández-Martin, F., Fernández José Carballo, P., and Jiménez-Colmenero, F., 2000, DSC study on the influence of meat source, salt and fat levels, and processing parameters on batters pressurization, Eur. Food Res. Technol., 211, 387. Friedman, M. and Cuq, J.L., 1988, Chemistry, analysis, nutritional value, and toxicology of tryptophan in food. A review, J. Agric. Food Chem., 36, 1079. Gómez-Guillen, M.C., Sarabia, Q.A.I., Solas, M.T., and Montero, P., 2001, Effect of microbial transglutaminase on the functional properties of megrim (Lepidorhombus boscii) skin gelatin, J. Sci. Food Agric., 81, 665. Haard, N.F., 2001, Enzymic modification of proteins in food systems, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, chap. 7. Haard, N.F., Simpson, B.K., and Pan, B.S., 1994, Sarcoplasmic proteins and other nitrogenous compounds, in Seafood Proteins, Sikorski, Z.E., Pan, B.S., and Shahidi, F., Eds., Chapman & Hall, New York, chap. 3. Hamm, R., 1960, Biochemistry of meat hydration, in Advances in Food Research, Vol. 10, Chichester, C.O., Mrak, E.M., and Stewart, G.F., Eds., Academic Press, New York, chap. 8. Hasler, C. M., 1998, Functional foods: their role in disease prevention and health promotion, Food Technology, 52(2), 57. Hayashi, R., Kawamura, Y., Nakasa, T., and Ohinaka, O., 1989, Application of high pressure to food processing: pressurization of egg white and yolk, and properties of gels formed, Agric. Biol. Chem., 53, 2935. Holt, C. and Rogiński, H., 2001, Milk proteins: biological and food aspects of structure and function, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, chap. 11. Hoseney, R.C. and Rogers, D.E., 1990, The formation and properties of wheat flour doughs, CRC Crit. Rev. Food Sci. Nutr., 29, 73. Hossain, M.I., Itoh, Y., Morioka, K., and Obatake, A., 2001, Contribution of the polymerization of protein by disulfide bonding to increased gel strength of walleye pollack surimi gel with preheating time, Fisheries Science, 67, 710. Howell, B.K., Matthews, A.D., and Donnelly, A.P., 1991, Thermal stability of fish myofibrils: a differential scanning calorimetric study, Inter. J. Food Sci. Technol., 26, 283. Jiang, S.T., 2000, Enzymes and their effects on seafood texture, in Seafood Enzymes. Utilization and Influence on Postharvest Seafood Quality, Haard, N.F. and Simpson, B.K., Eds., Marcel Dekker, New York, Basel, chap. 15. Jiang, S.T., Ho, M.L., and Chen, H.C., 1998, Purified NADPH-sulfite reductase from Saccharomyces cerevisiae effects on quality of ozonated mackerel surimi, J. Food Sci., 63, 777. Jost, R., 1993, Functional characteristics of dairy proteins, Trends in Food Science & Technology, 4, 283. Kączkowski, J., 2005, Transglutaminase—an enzyme group of extended metabolic and application possibilities, Pol. J. Food Nutr. Sci., 14/55, 3. Karamać, M., Flaczyk, E., Janitha, P.K., Wanasundara, P.D., and Amarowicz, R., 2005, Angiotensin I-converting enzyme (ACE) inhibitory activity of hydrolysates obtained from muscle food industry by-products—a short report, Pol. J. Food Nutr. Sci., 14/55, 133. Kato, A., Ibrahim, H.R., Takagi, T., and Kobayashi, K., 1990a, Excellent gelation of egg white preheated in the dry state is due to a decreasing degree of aggregation, J. Agric. Food Chem., 38, 1868.
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Kato, A., Ibrahim, H.R., Watanabe, H., Houma, K., and Koboyashi, K., 1990b, Enthalpy of denaturation and surface functional properties of heated egg white proteins in the dry state, J. Food Sci., 55, 1280. Kawai, Y., Hirayama, H., and Hatano, M., 1990, Emulsifying ability and physicochemical properties of muscle proteins of fall chum salmon Oncorhynchus keta during spawning migration, Nippon Suisan Gakkaishi, 56, 625. Kędzior, Z., 2004, Pea proteins and their functionality in cereal technology, Roczniki Akademii Rolniczej w Poznaniu, Rozprawy Naukowe, No. 348, 1–134 (in Polish). Kijowski, J. and Leśnierowski, G., 1999, Separation, polymer formation and antibacterial activity of lysozyme, Polish J. Food Nutr. Sci., 8, 49, 3. Killday, K.B., Tempesta, M.S., Bailey, M.E., and Metral, C.J., 1988, Structural characterization of nitrosylhemochromogen of cooked cured meat: implications in the meat-curing reaction, J. Agric. Food Chem., 36, 909. Ko, S., Janes, M.E., Hettiarachchy, N.S., and Johnson, M.G., 2001, Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes, J. Food Sci., 66, 1006. Kołakowski, E., 2001, Protein determination and analysis in food systems, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, chap. 4. Kołakowski E., 2005, Enzymes and their use for modification of food proteins, in Enzymatic Modification of Food Components, Kołakowski, E., Bednarski, W,. and Bielecki, S., Eds., Wydawnictwo Akademii Rolniczej w Szczecinie, Szczecin, chap.1 (in Polish). Kołakowski, E. and Sikorski, Z.E., 2000, Endogenous enzymes in Antarctic krill: control of activity during storage and utilization, in Seafood Enzymes. Utilization and Influence on Postharvest Seafood Quality, Haard, N.F. and Simpson, B.K., Eds., Marcel Dekker, New York, Basel, chap. 18. Kołodziejska, I., 1999, Enzymatic and functional properties of squid proteins. Characteristics and possibilities of utilization, Zeszyty Naukowe Politechniki Gdańskiej, Chemia, 44, 576, 1–72 (in Polish). Kristinsson, H.G., 2001, Evaluation of different methods to isolate cod (Gadus morhua) muscle myosin, Journal of Food Biochemistry, 25, 249. Kumar, S., Tsai, C.-J., and Nussinov, R., 2000, Factors enhancing protein thermostability, Protein Eng., 13, 3, 179. Kumazawa, Y., Numazawa, T., Seguro, K., and Motoki, M., 1995, Suppression of surimi gel setting by transglutaminase inhibitors, J. Food Sci., 60, 715. Kwok, K.C. and Niranjan, K., 1995, Review: effect of thermal processing on soymilk, Inter. J. Food Sci. Technol., 30, 263. Lampart-Szczapa, E., 2001, Legume and oilseeds proteins, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, chap. 14. Le Tien, C., Vachon, C., Mateescu, M.-A, and Lacroix, M., 2001, Milk protein coatings prevent oxidative browning of apples and potatoes, J. Food Sci., 66, 512. Lopetcharat, K, Choi, Y.J., Park, J.W., and Daeschel, M.A., 2001, Fish sauce products and manufacturing: a review, Food Reviews International, 17, 1, 65. Luo, Y.K., Kuwahara, R., Kaneniwa, M., Murata, Y., and Yokoyama, M., 2001, Comparison of gel properties of surimi from Alaska pollock and three freshwater fish species: effects of thermal processing and protein concentration, J. Food Sci., 66, 548. Matsumoto, J.J. and Noguchi, S.F., 1992, Cryostabilization of protein in surimi, in Surimi Technology, Lanier, T.C. and Lee, C.M., Eds., Marcel Dekker, New York, Basel, Hong Kong, chap. 15.
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Mohammed, Z.H., Hill, S.E., and Mitchell, J.R., 2000, Covalent crosslinking in heated protein systems, J. Food Sci., 65, 221. Nakai, S. and Li-Chan, S., 1988, Hydrophobic Interactions in Food Systems, CRC Press, Boca Raton, FL. Niwa, E., 1992, Chemistry of surimi gelation, in Surimi Technology, Lanier, T.C. and Lee, C.M., Eds., Marcel Dekker, New York, Basel, Hong Kong, chap. 16. O’Grady, M.N., Monahan, F.J., and Brunton, N.P., 2001, Oxymyoglobin oxidation and lipid oxidation in bovine muscle—mechanistic studies, J. Food Sci., 66, 386. Ohtsuka, T., Umezawa, Y., Nio, N., and Kubota, K., 2001, Comparison of deamidation activity of transglutaminases, J. Food Sci., 66, 25. Pellegrino, L. and Resmini, P., 1996, Evaluation of the stable reaction products of histidine with formaldehyde or with other carbonyl compounds in dairy products, Z. Lebensm. Unters. Forsch., 202, 66. Perez-Gago, M.B. and Krochta, J.M., 2001, Denaturation time and temperature effects on solubility, tensile properties, and oxygen permeability of whey protein films, J. Food Sci., 66, 705. Pietrasik, Z., Jarmoluk, A., and Shand, P.J., 2005, Textural and hydration properties of pork meat gels processed with non-meat proteins and carrageenan, Pol. J. Food Nutr. Sci., 14, 55, 145. Purslow, P.P., Ertbjerg, P., Baron, C.P., Christensen, M., and Lawson, M.A., 2001, Patterns of variation in enzyme activity and cytoskeletal proteolysis in muscle, in Congress Proceedings, Vol. 1, 47th International Congress of Meat Science and Technology, Kraków, Meat and Fat Research Institute, Warsaw, 38. Rodger, G., 2001, Production and properties of mycoprotein as a meat alternative, Food Technology, 55, 7, 36. Roy, S., Weller C.L., Gennadios, A., Zeece, M.G., and Testin, R.F., 1999, Physical and molecular properties of wheat gluten films cast from heated film-forming solutions, J. Food Sci., 64, 57. Seguro, K. and Motoki, M., 1990, Functional properties of enzymatically phosphorylated soybean proteins, Agric. Biol. Chem., 54, 1271. Shimizu, A., Kitabatake, N., Higasa, T., and Doi, E., 1991, Melting of the ovalbumin gels by heating: reversibility between gel and sol, Nippon Shokuhin Kogyo Gakkaishi, 38, 1050. Sikorski, Z.E., Gildberg, A., and Ruiter, A., 1995, Fish products, in Fish and Fishery Products. Composition, Nutritive Properties and Stability, Ruiter, A., Ed., CAB International, Wallingford, chap. 11. Sung, H.Y., Chen, H.J., Liu, T.Y, and Su, J.Ch., 1983, Improvement of the functionalities of soy protein isolate through chemical phosphorylation, J. Food Sci., 48, 716. Tani, F., Murata, M., Higasa, T., Goto, M., Kitabatake, N., and Doi, E., 1993, Heat-induced transparent gel from hen egg lysozyme by a two-step heating method, Biosci. Biotech. Biochem, 57, 209. Ternes, W., 2001, Egg proteins, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Lancaster, PA, chap. 12. Wrigley, C.W. and Bekes, F., 2001, Cereal-grain proteins, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Lancaster, PA, chap. 13. Zehetner, G., Bareuther, Ch., Henle, T., and Klostermeyer, H., 1995, Inactivation kinetics of gamma-glutamyltransferase during the heating of milk, Z. Lebensm. Unters. Forsch., 201, 336. Zhu, H. and Damodaran, S., 1994, Effects of calcium and magnesium ions on aggregation of whey protein isolate and its effect on foaming properties, J. Agric. Food Chem. 42, 856.
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7
Lipids and Food Quality Andrzej Stołyhwo
CONTENTS 7.1
General Description ..................................................................................... 178 7.1.1 Definition.......................................................................................... 178 7.1.2 Nonpolar or Simple Lipids .............................................................. 178 7.1.3 Polar or Complex Lipids ................................................................. 179 7.2 Isolation and Analysis of Lipids.................................................................. 179 7.2.1 Isolation from Natural Sources........................................................ 179 7.2.2 Separation into Classes .................................................................... 180 7.2.3 Determination of Fatty Acid Composition of Individual Lipid Classes by Gas Chromatography........................................... 181 7.3 Structures and Composition of Common Lipids ........................................ 182 7.3.1 Fatty Acids and Their Physical Properties ...................................... 182 7.3.2 Trans Fatty Acids in Natural and Partially Hydrogenated Trans Fats ......................................................................................... 184 7.3.3 Conjugated Linoleic Acid ................................................................ 188 7.4 Determination of the Trans Fatty Acid Content in Food Lipids ................ 189 7.4.1 Methods of Determination............................................................... 189 7.4.2 Biological Significance .................................................................... 192 7.5 Fatty Acid Composition of Common Fats .................................................. 193 7.6 Long-Chain Polyenoic Fatty Acids and Their Importance in Human Nutrition...................................................................................... 193 7.7 Triacylgylcerols............................................................................................ 197 7.7.1 Chemical and Functional Properties................................................ 197 7.7.2 Modifications.................................................................................... 199 7.7.3 Fractionation..................................................................................... 199 7.7.4 Lipolysis ........................................................................................... 200 7.7.5 Analysis of the Composition of Triacylglycerols ........................... 202 7.7.5.1 HPLC of Triacylglycerols ................................................ 202 7.7.5.2 GLC of Triacylglycerols................................................... 203 References.............................................................................................................. 206
177
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7.1 GENERAL DESCRIPTION 7.1.1 DEFINITION There are several definitions of the term lipids. Usually lipids are defined as a heterogeneous group of biological compounds almost insoluble in water but soluble in fats, hydrocarbon type and other fat solvents. Fats and oils are example of lipids. In older manuals lipids are defined as all substances which can be extracted from the investigated material with diethyl ether. In fact, the diethyl ether is a medium polar solvent, partially soluble in water, which is able to extract from slightly wet natural samples both so-called nonpolar and polar lipids.
7.1.2 NONPOLAR
OR
SIMPLE LIPIDS
Among naturally occurring nonpolar lipids one may find: •
• •
•
•
Alkanes or alkenes: hydrocarbons usually containing a straight saturated or unsaturated chain of up to 36 carbon atoms, which may be also branched, with one or more methyl groups in the side chain. In contrast with the diversity of methyl-branched alkanes found in insects, n-alkanes predominate in plants. Hydrocarbons with an odd number of carbon atoms (C15 up to C33) are predominant among the least polar components of plant surface lipids. The commonly known naturally occurring hydrocarbon is squalene C30H50, present, for example, in concentrations up to 12% in human serum, in lipids of human milk (0.12%), and in olive oil (0.6%). Considerable amounts of squalene are present in the livers of deep-sea sharks. Other hydrocarbons commonly occurring are carotenoids, such as beta-carotene, better known as provitamin A. Fatty alcohols: the aliphatic alcohols with saturated or unsaturated hydrocarbon chains, usually between C6 and C26, and one to three double bonds. Waxes: esters of fatty acids (FA) and long-chain alcohols. Esters of cholesterol and fatty acids in human serum are frequently investigated. Among naturally occurring waxes are: bees wax, carnauba wax, candelila wax, and many other well-known waxes. Sterols: a group of compounds known as phytosterols, which are present in plants. Cholesterol (CL), lanosterol, and other similar compounds are the main representatives of animal sterols. Both phytosterols or animal sterols may occur in a free state or esterified with fatty acids known as sterol esters; or esterified by sulfuric acid—sterol sulfates or linked to a glucide—known as steryl glycosides. Tocopherols: present in practically all naturally occurring plant oils. The largest concentration of tocopherols is found in germs. Tocotrienols are specific for some oils, like palm or amaranth oil. Both tocopherols and tocotrienols are generally known as vitamin E. Triacylglycerols (TAGs), in medicine called triglycerides, are triesters of glycerol and FAs. Natural
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plant oils or animal fats are 96 to 98% composed of TAGs, in which are dissolved other accompanying nonpolar and polar lipids. Mono- (MAGs) and diacylglycerols (DAGs): these are dissolved in natural oils or fats, and are the products of partial hydrolysis of TAGs. The hydrolysis may be catalyzed by enzymes—lipases present practically in all biological systems, for example, pancreatic lipase or lipases present in fungi such as Rhizopus javanicus. MAGs contain nonpolar fragments (hydrocarbon chain) soluble in fats, and polar (glycerol) fragments soluble in water. Thus they act as emulsifiers. MAGs are an important group of compounds manufactured commercially and used as emulsifiers for production of margarine, spreading fats, ice creams, cosmetics, and many other commercial products.
7.1.3 POLAR
OR
COMPLEX LIPIDS
These important lipids are widely distributed in plants, bacteria, and animals. They are known as the major constituents of cell membranes. Because of the large variety of complex lipids, their classification is a very difficult task. Usually the polar lipids are divided into three groups: •
•
•
Phospholipids (PLs): lipids with a phosphate residue, one glycerol, or an amino alcohol or a fatty alcohol, with or without one or two FA chains (exceptions are one inositol group, two phosphates, or four FA chains). Glycolipids: lipids containing a glycosidic moiety with a glycerol or an amino alcohol and with a FA chain or chains. Sometimes glycolipids also contain phosphate residue. Proteolipids: lipids containing one amino acid residue linked to long-chain alcohol or acids.
7.2 ISOLATION AND ANALYSIS OF LIPIDS 7.2.1 ISOLATION
FROM
NATURAL SOURCES
The methods for isolating lipids depend on the type of the sample. The nonpolar lipids are usually isolated by extraction with petroleum ether according to AOAC Method No. 920.39C. This applies mainly to the oil seeds. Lipids from milk and other dairy products are isolated using mainly the Rose Gottlieb method in which the lipids bound to proteins are first liberated with a solution of ammonia in ethanol, and then the extraction is carried out with diethyl ether. The exact analytical procedure is described in AOAC Method No. 932.06. The methods of isolating of fats from bakery, chocolate, or chocolate-like products or cosmetics are very drastic because some lipids are chemically bound to the matrix of the sample. The isolation of fats includes preliminary preparation by boiling of the sample in an 8M HCl solution, followed by filtration of the resulting mixture through a wetted filter paper. The fatty material retained on the filter paper is washed several times with warm water and loaded into a cellulose thimble, dried at 100 to
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101°C, and extracted with diethyl ether in Soxhlet glassware. The exact analytical procedure is described in AOAC Method No 922.06. The isolation of lipids from biological sources should not only be quantitative but also nondestructive with respect to the isolated sample. All isolated lipids must be protected against oxidation by solvents, oxygen, or enzymes in combination with temperature and light. The high sensitivity of the analytical methods needed for low amounts of extracted lipids requires the use of very pure solvents and clean glassware. For the isolation of lipids from organs or tissues, the Folch (1957) or Bligh and Dyer (1959) methods are used most frequently. In both methods a mixture of chloroform and methanol is used, which helps to tear apart weak physical bonds between polar lipids and the matrix of the sample. Because the above methods are not included in the AOAC analytical standards, for the convenience of the reader the Folch procedure is described in detail below. In the Folch procedure, one gram of a tissue is homogenized for 1 min in a highspeed blender with 20 ml of a 2:1 (v/v) chloroform and methanol mixture. After dispersion the mixture is allowed to stand at room temperature for 15 to 20 min and is occasionally agitated. The homogenate is filtered using soft filter paper into a 50 ml graduated cylinder equipped with a glass stopcock. The solid residue on the filter paper is carefully rinsed twice with 4 ml of a mixture of chloroform and methanol 2:1(v/v), which is added to the extract. To the final volume (V) of the extract in the graduated cylinder, 0.2 V of a 0.9% NaCl solution in water is added (the content of water in the sample before extraction should be taken into account. The cylinder is shaken for 2 to 3 minutes and allowed to stand in darkness to obtain exact separation of phases. If emulsions are formed, a small amount of powdered NaCl is carefully added to the cylinder to facilitate separation of the phases. Alternatively, the mixture is centrifuged at low speed (2000 rpm) to separate the two phases. The upper phase containing dissolved proteins is removed by siphoning. The lower chloroform phase containing lipids is dried over anhydrous sodium sulfate and evaporated under vacuum in a rotary evaporator or under a stream of nitrogen. The extracts should be stored under nitrogen in a freezer below –50°C or preferably in dry ice. In order to protect the extracted lipids against autooxidation, addition to the chloroform phase of small amounts (50 μg) of antioxidants (for example, nordihydroguaiaretic acid [NDGA] or butylated hydroxytoluene [BHT] is advisable).
7.2.2 SEPARATION
INTO
CLASSES
There are several methods of separation of extracted lipids in individual classes. A very efficient procedure of semipreparative separation by extraction to the solid phase (SPE) has been published by Kaluzny et al. (1985). The method for unskilled persons is slightly inconvenient, but its main feature is very good protection against the oxidation of sensitive components in the extracted lipids. Slightly less protective and selective, but very frequently used, is the method of semipreparative planar chromatography for the separation of extracted lipids into cholesteryl esters (CLE, TAG), CL, and PL. The procedure is as follows. Commercially available 20 × 20-cm plates covered with a 0.25-mm silica gel layer are activated before use at 120°C for 1.5 hrs. After
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cooling down in a dry desiccator a solution of about 30 to 50 mg of lipids in 0.5 ml of a chloroform and methanol solution (1:1) is applied on the plate in a form of continuous strips using an automatic mechanical applicator. After evaporation of the solvent using a fan, the chromatogram is developed in an unsaturated chamber with the mobile phase: hexane/diethyl ether/glacial acetic acid 80:20:1 (v/v/v). The development of the chromatogram is carried out up to the moment when the solvent front reaches about 2 cm below the upper edge of the plate. Then the plate is removed from the developing chamber and carefully covered with a glass plate leaving about 4 cm from one side of the plate uncovered. After drying the uncovered part of the plate with a cold air stream, the surface is sprayed with a solution of 2′,7′ dichlorofluoresceine in ethanol. The separated lipid classes are visualized under UV. The position of the bands of individual lipid classes is marked on a plate with a spatula. After the covering glass is removed, the corresponding (invisible) individual bands are scraped from the plate. The scrapped silica gel containing separated lipid classes are loaded into preferably glass syringes equipped in the bottom with a filtering disc or defatted cotton or glass wool. The lipids are eluted from the scrapped material with a mixture of chloroform and methanol (2:1, v/v).The unscraped part of the plate is sprayed with a 25% solution of sulfuric acid, heated at 110°C in order to char all organic material present on the plate. The charred plates may be scanned with an appropriate scanning system and transferred to the computer as a document. This type of separation is shown in Figure 7.1.
7.2.3 DETERMINATION OF FATTY ACID COMPOSITION OF INDIVIDUAL LIPID CLASSES BY GAS CHROMATOGRAPHY After separation into classes, usually the composition of the FAs in each class is determined. For that purpose the gas chromatographic (GC) method is used. Before Margin of the plate sprayed with 2′,7′ dichlorofluoresceine
CLE
TAG CL
PL
FIGURE 7.1 Example of semipreparative separation into classes of lipids extracted from rat liver by thin layer chromatography. (Abbr.: CLE = cholesterol esters; TAG = triacylglycerols; CL = cholesterol; PL = phospholipids.) Silica gel plates 0.25-mm layer thickness, mobile phase: hexane/diethyl ether/glacial acetic acid 80:20:1 (v/v/v). (Dzik and Stołyhwo 2004, unpublished data.)
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analysis by GC, the FAs present in each lipid class should be converted into their fatty acid methyl esters (FAME), which are more volatile than the corresponding free FAs. There are several methods of preparing FAMEs, and each method has same disadvantages. The FAMEs in milk fats and other dairy products, which contain butyric acids, are usually prepared by cold esterification in an alkaline medium. The most useful transesterifying agents are 1 to 2 M sodium or potassium methoxides in anhydrous methanol. These solutions are stable for several months at 4°C until a white precipitate of bicarbonate salt is formed. Glycerolipids are rapidly transesterified (2 to 5 min) at room temperature. This procedure protects against losses of very volatile methyl butyrate during preparation of FAMEs. However, great care must be taken to work with really dry samples, otherwise multiple artifacts are developed, which lead to unreliable results. The FAMEs of lipids extracted from human or animal tissues or from marine oils are usually prepared following the AOCS Method No. Ce 1b-89, according to which the lipids are saponified (under nitrogen) in 0.5N NaOH in methanol, and then the soaps are converted directly into FAMEs. In the case of samples containing marine oils or extracts from animal samples in which long-chain (C20 to C24) polyunsaturated FAs (LC-PUFA) are present, the addition of internal standard C23:0 is necessary because the response factors of FAMEs containing 3 and more double bonds differ substantially from those of saturated or monounsaturated FAMEs. For the convenience of the reader, the parameters of separation by GC and identification of chromatograms of FAMEs of rat liver lipids and of the mixture of Supelco 37 Component FAME Mix Cat No: 47885-U (Sigma-Aldrich) are presented in Figure 7.2.
7.3 STRUCTURES AND COMPOSITION OF COMMON LIPIDS 7.3.1 FATTY ACIDS
AND
THEIR PHYSICAL PROPERTIES
Carboxylic acids occur in many molecular forms; however, the majority of FAs found in lipids are those containing one carboxylic (–COOH) group. Several hundred forms of FAs have been identified, but the number occurring frequently in common lipids is much smaller—usually between 10 and 30 in plant oils and from 40 to over 100 in animal fats. FAs differ from each other by the length of the carbon chain, structure of the carbon chain (normal or branched), number of double bonds, their position in the hydrocarbon chain with respect to the carboxylic or terminal-methyl group, and the configuration cis or trans or conjugation of double bonds. In the systematic nomenclature, the position of the double bonds is related to the carboxylic group, for example, oleic acid C18:1 9c (octadecenoic acid-delta-9-cis). However in biochemistry, the more practical designation is that which relates to the end of the hydrocarbon chain—the methyl group. In such case the position of the double bond with respect to the terminal methyl group is designated as omega (in the case of oleic acid Ω-9) or as n-9.
Lipids and Food Quality
C22:5 n-3 C22:5 n-6
C20:5 n-3 C20:5 n-3
C23:0 C22:2
C20:3 n-6 C20:3 n-6
C22:0
C20:4 n-6 C20:2 C20:2
C20:3 n-9
C18:4 n-3 C21:0
C18:3 n-3 C18:3 n-3
C20:1 n-9 C20:1 n-9
C20:0 C20:0
C18:3 n-6 C18:3 n-6
C18:2 n-6 C18:2 n-6
C18:2 n-6 trans C18:2 n-6 trans
C18:1 n-9 trans C18:1 n-9 trans
C18:1 n-9
C18:0 C18:0
C17:0 C17:0
C16:1 n-7 C16:1 n-7
C13:0
C14:0 C14:0
C6:0 C6:0 C10:0 C11:0 C12:0
C16:0 C18:0
C18:1 n-9 C18:1 n-7
C17:1 n-7
C17:1 n-7
C15:1
FIGURE 7.2 Chromatograms of FAMEs of rat liver lipids compared to the mixture of Supelco 37 Component FAME Mix Cat No: 47885-U (SigmaAldrich). Parameters of separation: Column Rtx 2330 (Restek, Chromatographic Specialties, Canada), 100 m ; ID 0.25 mm, df = 0.2 μm; H2 flow 32 cm/s, split-splitless injection at 235°C; det. temp. 250°C; column temp: initial 155°C; hold 55 min; next rate 1.5°C/min; final temp. 210°C; next hold. Identification of peaks is given on chromatograms (author’s data).
B
C24:0 C24:0
C14:1 C15:0 C14:1 C15:0
FID1 A, (5890KUB/S3700000.D)
C22:1 C20:3 n-3 C20:4 n-6
C22:4 n-6 C24:1
A
C22:6 n-3 C22:6 n-3
FID1 A, (5890KUB/Z11W0000.D)
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TABLE 7.1 Saturated Fatty Acids Systematic name Butanoic Pentanoic Hexanoic Octanoic Nonanoic Decanoic Dodecanoic Tetradecanoic Hexadecanoic Heptadecanoic Octadecanoic Eicosanoic Docosanoic Tetracosanoic Hexacosanoic Octacosanoic Triacontanoic Dotriacontanoic
Shorthand designation
Melting point (°C)
Boiling pointa (°C)
4:0 5:0 6:0 8:0 9:0 10:0 12:0 14:0 16:0 17:0 18:0 20:0 22:0 24:0 26:0 28:0 30:0 32:0
7.9; 5.0a 3.2a 3.4; 4.0a 16.7; 16.0a 12.5a 31.6; 31.4a 44.2; 44.1a 53.9; 57.4a 63.1; 62.8a 61.3; 60.85a 69.6; 68.9a 75.3; 73.35a 79.9; 79.95a 84.2; 84.15a 87.7a 90.9a 93.6a 96a
163.4760 185.4760 204.9760 237.9760 255.6760 270.0760 298.1760 326.2760 351.5760 363.8760 361.1760 Destr. 318 30660; 26515 272.010 — — — —
Butyric Valeric Caproic Caprylic Pelargonic Capric Lauric Myristic Palmitic Margaric Stearic Arachidic Behenic Lignoceric Cerotic Montanic Melissic Lacceric
Note: Pressure indicated by superscript. Source: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm (2006). Source for items designated a; Doss, M.P., 1952, Properties of the Principal Fats: Fatty Oils, Waxes, Fatty Acids, and Their Salts, Technical Research Division of The Texas Company, New York.
Monocarboxylic FAs are classified according to the number of double bonds in the hydrocarbon chain as saturated; monounsaturated, di-unsaturated, and polyunsaturated. Systematic and common names of frequently occurring saturated and unsaturated FAs are given in Table 7.1, Table, 7.2, and Table 7.3.
7.3.2 TRANS FATTY ACIDS IN NATURAL HYDROGENATED TRANS FATS
AND
PARTIALLY
On January 1, 2006, all packaged foods that enter interstate commerce in the United States must list trans fat content on their nutrition facts labels. This means that from that date, trans FAs are considered as saturated fatty acids from a nutritional point of view, but their content in the composition of saturated FA must be listed separately. Much earlier in Europe the Danish Veterinary and Food Administration published Executive Order No, 160 on March 11, 2003, according to which, beginning in June 2003, the content of trans fatty acids in the oils and fats (including emulsions), “should not exceed 2 grams per 100 grams of oil or fat.” According to the same
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TABLE 7.2 Monounsaturated Fatty Acids Systematic name
Shorthand designation
Cis-2-octenoic Cis-4-decenoic Cis-8- decenoic Cis-9-decenoic Cis-5-lauroleic Cis-4-dodecenoic Cis-9-tetradecenoic Cis-5-tetradecenoic Cis-4-tetradecenoic Cis-9-hexadecenoic Cis-6-octadecenoic Cis-9-octadecenoic Trans-9-octadecenoic Trans-11-octadecenoic Cis-9-eicosenoic Cis-11-eicosenoic Cis-11-docosenoic Cis-13-docosenoic Trans-13- docosenoic Cis-15-tetracosenoic
10:1 (n-6) 10:1(n-6) 10:1(n-1) 10:1(n-1) 12:1(n-7) 12:1(n-8) 14:1(n-5) 14:1(n-9) 14:1(n-10) 16:1(n-7) 18:1(n-12) 18:1(n-9) 18:1(n-9)t 18:1(n-7)t 20:1(n-11) 20:1(n-9) 22:1(n-11) 22:1(n-9) 22:1(n-13)t 24:1(n-9)
Obtusilic Ioleocapric Lauroleic Linderic Oleomyristic Physeteric Tzuzuic Oleopalmitic Petroselinic Oleic Elaidic Vaccenic Gadoleic Gondoic Cetoleic Erucic Brassidic Nervonic
Melting point (°C)
6a to 0a 15a 1 –1.3a 4.5 a < 0a 18.0a–18.5a 0.5; 1 a 30; 28.6 a 16.2; 13.2 a 43.6 a 39; 43.5 a–44.5a 25; 25a 22a 32.5a–33a 33.4; 33.5 a 60 39; 39 a–39.5a a
Boiling pointa (°C) 12715 — 148–15023 155–716 164–512 170–213 1446 190–20015 185–813 218–22015 237–23815 234–515; 225–610 23415 1961–2 17801 — — 26415 282 30; 25610 —
Note: Pressure indicated by superscript. Sources: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm (2006). Source for items designated a; Doss, M.P., 1952, Properties of the Principal Fats: Fatty Oils, Waxes, Fatty Acids, and Their Salts, Technical Research Division of The Texas Company, New York.
executive order, products claiming to be free of trans fatty acids shall have less than 1 gram per 100 grams of the individual oil or the individual fat in the finished product. The industrial hydrogenation of plant or fish oils is carried out under slight overpressure of hydrogen at temperatures on the order of 180°C in the presence of a catalyst, usually nickel formate, which under conditions of hydrogenation parameters is decomposed into fine particles of metallic nickel. The hydrogenation of unsaturated FAs takes place on the active surface of the catalyst. The mechanism of the reaction is very complex, thus it will not be discussed in detail here. However, in spite of the saturation of the double bonds with hydrogen, isomerization of unsaturated FAs occurs during hydrogenation, as shown in Figure 7.3. Besides geometrical isomerization shown in Figure 7.3, during partial hydrogenation of unsaturated FAs, migration of the double bonds along the hydrocarbon chain also takes place resulting in formation of multiple positional FA isomers. Examples of structures of positional C16:114t and C16:1 3t isomers are shown in Figure 7.4.
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TABLE 7.3 Polyunsaturated Fatty Acids
Systematic name
Shorthand designation
MP (°C)
9,12-octadecadienoic
Linoleic(LA)
18:2(n-6)
5; 5 a5.2*
6,9,12-octadecatrienoic 8,11,14-eicosatrienoic
18:3(n-6) 20:3(n-6)
— —
5,8,11,14-eicosatetraenoic 7,10,13,16-docosatetraenoic 4,7,10,13,16-docosapentaenoic 9,12,15-octadecatrienoic
γ-linolenic (GLA) Dihomo-γ-linolenic (DGLA) Arachidonic (AA) — — α-Linolenic (ALA)
20:4(n-6) 22:4(n-6) 22:5(n-6) 18:3(n-3)
6,9,12,15-octadecatetraenoic 8,11,14,17-eicosatetrenoic 5,8,11,14,17-eicosapentaenoic 7,10,13,16,19-docosapentaenoic 4,7,10,13,16,19-docosahexaenoic 5,8,11-eicosatrienoic
Stearidonic (SDA) — EPA DPA DHA Mead acid
18:4(n-3) 20:4(n-3) 20:5(n-3) 22:5(n-3) 22:6(n-3) 20:3(n-9)
50 — — 11; 11.3* to 11* 57 — — — 44 —
MeEsters boiling point* (°C) 21520; 19810; 149.51 — — — — — 20714; 1551 — — — — — —
Note: Pressure indicated by superscript. Sources: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm (2006). Source for items designated a, Doss, M.P., 1952, Properties of the Principal Fats: Fatty Oils, Waxes, Fatty Acids, and Their Salts, Technical Research Division of The Texas Company, New York.
Oil (liquid)
180oC Catalyst (Ni)
Partially hydrogenated fat (solid)
H2
COOH H3C +2H Isomerization (geometrical cis-trans )
(Natural) Cis double bond
Hydrogenation (saturated fatty acid)
Trans double bond
FIGURE 7.3 Principal chemical reactions during partial hydrogenation of unsaturated FAs.
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COOH
H3C
COOH
H 3C
FIGURE 7.4 Examples of structures of positional C16:114t and C16:1 3t isomers. C === C 9 10
C 11
C === C 12 13 Linoleic acid C18:2 9cis- 12cis
C === C 9 10
C === C 11 12
C 13 Conjugated linoleic acid C18:2 9cis-11trans
+2H C 9
C 10
C === C 11 12
C 13 Vaccenic acid C18:1 11trans
C 11
C 13
+2H C 9
C 10
C 12
Stearic acid C18:0
FIGURE 7.5 Course of conversion of linoleic acid C18:2 9c12c to stearic acid C18:0 in the rumen caused by the Butyrivibrio fibrisolvens strain of bacteria.
As a result of industrial partial hydrogenation of plant or fish oils, most FAs, in particular C14:1, C16:1, C18:1, C20:1, C22:1, and C24:1, are converted into multiple positional isomers in the trans or cis configuration of the double bonds. The typical profiles of the composition of the C18:1 positional FA isomers in trans and cis configurations in partially hydrogenated soybean oil are covered in the next paragraph (Figure 7.8). The catalysts used for industrial hydrogenation of oils are usually very selective, which means that during hydrogenation, the rate of introduction of hydrogen into double bonds decreases with the number of double bonds in the FA molecule. Thus linolenic acid C18:3 is hydrogenated faster than LA C18:2, which in turn is hydrogenated faster than oleic acid C18:1. Consequently, in spite of different rates of hydrogenation of unsaturated FA in the end product, there are present small quantities of different geometric and positional isomers of PUFA, mainly trans-trans and cistrans positional isomers of LA. From the nutritional point of view, this fact creates serious problems because in the human body, the processes of desaturation and elongation of artificial c,t or t,t isomers of LA result in the creation of artificial LC-PUFA, which after incorporation into the structure of membranes, may potentially change their physical properties, including fluidity. The hydrogenation of unsaturated FAs also occurs in nature. The process is caused by the group of bacteria known as Butyrivibrio fibrisolvens that are present in the rumen of cows or other ruminants. The mechanism of biohydrogenation of unsaturated FA is totally different than that which is carried out in industry. Figure 7.5 illustrates the example of biohydrogenation of LA C18:2 9,12cc.
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As a result of the simplified schematic of the reaction presented in Figure 7.5, the intermediate products of biohydrogenation of linoleic acid are conjugated linoleic acid (CLA) C18:2 9c11t and vaccenic acid (VA) C18:1 11t. In nature, however, the process of biohydrogenation is more complicated. This is confirmed by the presence in milk fat of not only VA C18:111t, but also of other C18:1 trans-positional isomers, the presence of which indicates that side reactions also take place during biohydrogenation of unsaturated FA. The different mechanisms of industrial partial hydrogenation of oils and biohydrogenation of unsaturated FA in cow rumen results in an entirely different composition of FA positional trans and cis isomers. This is documented in Figure 7.8 in which the comparison of the profiles of the composition of C18:1 cis and trans isomers in industrially partially hydrogenated fats and in cow’s milk fat are shown.
7.3.3 CONJUGATED LINOLEIC ACID (CLA) CLA has attracted a lot of attention over the past few years. Many claims for the benefits of CLA have been made, from weight loss, to antioxidant, anticancer and, more recently, to activity in diabetes and cardiovascular disease as well. CLA is a mixture of positional and geometric isomers of LA, which is found predominantly in dairy products and meat of ruminants (Pariza et al., 2001). The two double bonds in CLA are primarily in position 9 and 11, or 10, and 12 along the carbon chain. There also can be other geometric or positional isomers (cis or trans configuration). Thus, at least 8 different CLA isomers of LA have been identified. Of these isomers, the C18:2 9c,11t form is believed to be the most common natural form with biological activity. Preliminary studies indicate that CLA is a powerful anticarcinogen in the rat mammary tumor model with an effective range of 0.1 to 1% in the diet. This protective effect of CLA is noted even when exposure to carcinogens is limited to the time of weaning (Ip et al., 1994). In turn, the isomer C18:2 10c,12t decreases the rate of biosynthesis of TAGs in preadipocytes; thus, according to some authors, the supplementation of diet with C18:2 10c,12t isomer strongly reduces body fat and body weight. There are also other opinions regarding the biological activity of CLA. According to Noone et al. (2002) the supplementation of the human diet with a blend of CLA had no effect on lipid metabolism in healthy human subjects. HOOC9c 11t
FIGURE 7.6 The structure of the C18:2 9c 11t CLA isomer.
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As the majority of studies have been carried out using mixtures of isomers in animal models, the observations regarding biological activity of CLA with respect to humans needs to be substantiated before they can be regarded as fact (Wahle et al., 2004).
7.4 DETERMINATION OF THE TRANS FATTY ACID CONTENT IN FOOD LIPIDS 7.4.1 METHODS
OF
DETERMINATION
As the content of FA trans isomers must be shown on labels in the United States since January 1, 2006, and the content of so-called trans fats present in food products in some European countries became strictly limited in July 2003, the need for an efficient method of determination of trans FA becomes obvious. Such determinations are difficult because the physical properties of FA positional isomers are very similar. In GC evaluations, some trans FA isomers with double bonds closer to the end methyl group overlap cis positional isomers with the double bond closer to the carboxylic group. Because of that fact, the existing method of determinations of trans isomer content according to AOCS Method 985.21 are strongly criticized. Using present state-of-the-art methods, it is rather impossible to separate in one chromatographic run all positional trans and cis isomers present in a sample of lipids isolated from any food product. However, the task, which is very laborious, becomes feasible with the application of some hyphenated techniques. The general procedure used in the author’s laboratory for the determination of FA trans isomer profiles is carried out in three stages. In the first stage the lipids are isolated from the investigated sample, usually with the Folch method, or extracted from a sample with diethyl ether. Next the extracts (if necessary) are fractionated into lipid classes or directly converted after saponification into FAME using 14% boron trifluoride (BF3) solution in methanol. In the second stage, the FAMEs are fractionated by silver ion HPLC using the ChromSphere Lipids 25-cm, 4-mm ID dp5-μm packing (Chrompack, Raritan, NJ) column; the mobile phase: the logarithmic gradient of methylene chloride (solvent B, more polar) in hexane (solvent A, as a less polar diluent). For the detection of separated components the laser light scattering detector (LLSD) is used (Stołyhwo et al., 1983, 1984). The silver ion HPLC is based on the ability of the silver ion to complex with unsaturated compounds. The stability of the complex increases with the increasing number of double bonds. The cis isomers are more stable than the trans isomers, and conjugated polyenes form less stable complexes than do those containing methylene-interrupted double bonds. FAs are resolved practically on the basis of the number and the configuration of their double bonds. Silver ion HPLC is very efficient in separating geometrical isomers. The trans isomers migrate ahead of the corresponding cis molecules. The migration order is tt > ct > cc for dienes, and ttt > ctt > cct > ccc for the trienes. The example of silver ion HPLC separation of FAMEs of partially hydrogenated soybean oil (Frede et al., 1997) is shown in Figure 7.7.
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FIGURE 7.7 Separation of FAME of refined (a) and partially hydrogenated soybean oil (b) into classes by HPLC/Ag+ (Frede, E., Buchheim,W., and Stołyhwo, A., 1997, New developments in milk fats, in Modern Developments in Food Lipids, Shukla, V.K.S. and Kochhar, S.P., Eds., CentreA/S, Lystrup, Denmark, pp. 171–191. With permission.)
Each fraction separated by silver ion HPLC (ChromSphere Lipids) is collected in microcollectors. In the next stage, all fractions are rechromatographed by GC using, connected in series, two 105-meter; 0.25-mm df = 0.2-μm Rt×2330 columns (Restek, Chromatographic Specialties, Canada). The chromatograms of separated cis and trans C18:1 positional isomers isolated from partially hydrogenated soybean oil are shown in Fig. 7.8. For comparison, the fraction of positional C18:1 trans isomers isolated from cow milk fat (Frede 1997) are included in the figure. As seen in Figure 7.8, the trans C18:1 positional isomers in conditions of analysis are eluted from the column before corresponding C18:1 cis positional isomers. The isomers with double bonds, which are closer to the carboxylic group, are eluted before positional isomers that are closer to the terminal methyl group. The physical properties of the positional 6t, 7t, 8t. 13t, and 14t are so similar that they cannot be
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t6, 7, 8 t9 t10 A
t11 t12 t13, 14 t5 t4
t15
t 16
c9 B
c6, 7, 8
c10 c11 c12 c13
c4
c14
c5
c15
t11
C
t10 t9 t6, 7, 8 t5
t12
t13, 14 t15
t16
FIGURE 7.8 Separation of individual positional FAME C18:1 isomers of hydrogenated fat, preliminarily separated (and collected) into classes (as in Figure 7.7). Parameters of separation: two 100-m ID 025-mm columns Rt× 2330 in series; column temp. 155°C, flow rate 28cm/s. A) C18:1 trans isomers of partially hydrogenated fat; B) C18:1 cis isomers of partially hydrogenated fat; C) C18:1 trans isomers of low milk fat.
separated to the base line even in conditions of analysis (as above) on the 200-meter GC column having almost one-half million theoretical plates. As seen in a comparison of Figures 7.8A and 7.8B, the retention times of some of the trans positional isomers overlap positional cis isomers. This fact indicates that existing methods of determining the content of trans isomers in food products (AOCS Method 985.21) are not sufficiently accurate to fulfill actual requirements regarding labeling of products containing trans isomers or limiting their content in food products
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to less than 2 g of trans isomers per 100 g of fat. Such methods seem to be inapplicable in the case when a given product is prepared using a mixture of partially hydrogenated fats and milk fat. Figures 7.8A, B, and C are also good examples of the differences in the mechanisms of industrial partial hydrogenation of oils carried out in autoclaves, and alternatively of biohydrogenation of unsaturated FA, which takes place in the cow’s rumen. Indeed, the profiles of trans isomers produced in both processes are totally different. In the case of biohydrogenation as seen in Figure 7.8C, the principal trans isomer produced in rumen is VA C18:1 11t, whose content in the composition of trans isomers accounts for 68% to 86% (author’s own data) in a season and a method of feeding of cows. In the fall, when the content of oil in the grass seed in the pasture is greatest, cows are consuming more unsaturated FAs, which results in a greater concentration of VA (and also of CLA) in the milk fat. The presence of positional trans C18:1 isomers, other than VA as referred to Figure 7.8C indicates that in the biohydrogenation in rumen, besides formation of VA, other side reactions take place. However, the rate of side reactions is much smaller because the content of other than C18:1 11t isomers is much smaller than of VA. Biohydrogenation of unsaturated FA in cow’s rumen forms small amounts of C18:113 cis isomers, and does not produce substantial amounts of any other C18:1 cis positional isomers. The industrial hydrogenation of plant oils results in production of multiple trans and cis positional isomers as shown in Figure 7.8A and B. In that case the principal C18:1 trans (as in Figure 7.8A) isomers are: 6t + 7t + 8t (24%); 9t: elaidic acid (17.1%); 10t (16.1%) and 11t: VA about 15% in the composition of all C18:1 trans isomers. In a parallel manner they produce multiple C18:1 cis isomers, the quantitative profile of which is as follows (author’s own data): 6c + 7c + 8c (16.8%); 9c (24.7%); 10c (16%); 11c (12.9%); 12c (8.6%); 13c (7.2%) 14c (4.5%); 15c (3.3%); and 116c (1.2%).
7.4.2 BIOLOGICAL SIGNIFICANCE The above data suggest a question: What is the nutritive value of all FA isomers produced by biohydrogenation or alternatively by industrial partial hydrogenation? At present there is official scientific information indicating that trans fats produced industrially are harmful with respect to human health. Trans fat is known to increase blood levels of low-density lipoprotein (LDL), or “bad” cholesterol, while lowering levels of high-density lipoprotein (HDL), known as “good” cholesterol. It can also cause major clogging of arteries, type 2 diabetes, and other serious health problems, and was found to increase the risk of heart disease. The trans FA may block the transport of LC PUFA across the human placenta resulting in a decrease of AA C20:4 (n-6) and DHA C22:6 (n-3) in the fetal brain (Stołyhwo 2000, unpublished) reducing the baby’s psychomotor abilities when delivered. Many food companies use trans fat instead of oil because it reduces cost, extends storage life of products, and can improve flavor and texture. VA produced by biohydrogenation plays a very important role in human organisms. It is believed that C18:1 11t undergoes delta 9–desaturation in women’s mammary
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glands, resulting in the formation of C18:2 9c, 11t, that is, CLA, which protects the breast against development of breast cancer. The industrially produced trans C18:1 trans isomers are mainly 6t + 7t + 8t; 9t; and 10t. Their delta 9–desaturation, as it results from their structure, is rather impossible. If delta 9–desaturation of VA in the human mammary gland can finally be proven, the difference between the nutritive value of trans isomers created by biohydrogenation or industrial partial hydrogenation will become evident.
7.5 FATTY ACID COMPOSITION OF COMMON FATS For the convenience of the reader, Table 7.4 and Table 7.5 cover the FA composition of common oils and fats used in the food industry. Because of seasonal variations, the above data should be considered as indicative only.
7.6 LONG-CHAIN POLYENOIC FATTY ACIDS AND THEIR IMPORTANCE IN HUMAN NUTRITION These FAs, also called PUFAs, have two or more cis double bonds, which are the most frequently separated from each other by a single methylene group. Typical members of this group are LA C18:2 9c 12c or ALA C18:2 9, 12, 15, all cis. The most common PUFAs are listed in Table 7.3. PUFAs, like saturated or monoenoic FAs, are the source of energy that is liberated during their oxidation in the human organism. The oxidation occurs on the side of the carboxylic group in such a manner that during each step of oxidation, two carbons (one carboxylic group and one methylene group) are cut out from the FA molecule. Thus, after each step of oxidation, the positions of the double bonds in the molecule of an FA, with respect to carboxylic acid, change. Moreover, independent of oxidation of FAs, in the human organism other important chemical reactions controlled by enzymes are taking place, that is, desaturation or elongation of hydrocarbon chains as shown in Figure 7.10 (see below). Again, after each step of elongation or desaturation, the position of the double bonds with respect to the carboxylic group in the FA molecule is changing. Thus in the biochemistry of lipids, the shorthand presentation of FA molecules becomes impractical. In the late 1950s, Ralph Holman from the Hormel Institute (University of Minneapolis, Austin, Minnesota) stated that in spite of changes in the FA molecule structure caused by oxidation or desaturation and elongations processes, the position of the double bonds in the skeleton of an FA, if counted from the terminal methyl group, does not alter. Consequently, Holman suggested counting the position of the first double bond in the FA molecule from the terminal omega (as this is the last letter in the Greek alphabet) methyl group, as shown in Figure 7.9. It was a revolutionary suggestion that resulted in the discovery of important families of omega-6 and omega-3 polyunsaturated FAs (as in Figure 7.10), which makes it easier to understand the metabolism of PUFAs in the human organism. Later the Committees on Chemical Nomenclature suggested the following shorthand notations for FAs from the omega-6 and omega-3 families: (n-6) and (n-3).
ND ND ND 0.1–0.5 0.5–1.5 38.0–43.5 ND–0.6 ND–0.2 ND–0.1 3.5–5.0 39.8–46.0 10.0–13.5 ND–0.6 ND–0.6 ND–0.4 ND ND–0.2 ND ND ND ND
C6:0 C8:0 C10:0 C12:0 C14:0 C16 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C22:0 C22:1 C22:2 C24:0 C24:1 ND ND ND 0.1–0.5 1.0–2.0 48.0–74.0 ND–0.2 ND–0.2 ND–0.1 3.9–6.0 15.5–36.0 3.0–10.0 ND–0.5 ND–1.0 ND–0.4 ND ND–0.2 ND ND ND ND
Palm stearin ND ND ND ND ND–0.2 1.5–6.0 ND–3.0 ND–0.1 ND–0.1 0.5–3.1 8.0–60.0 11.0–23.0 5.0–13.0 ND–3.0 3.0–15.0 ND–1.0 ND–2.0 >2.0–60.0 ND–2.0 ND–2.0 ND–3.0
Rapeseed oil ND ND ND ND ND–0.2 2.5–7.0 ND–0.6 ND–0.3 ND–0.3 0.8–3.0 51.0–70.0 15.0–30.0 5.0–14.0 0.2–1.2 0.1–4.3 ND–0.1 ND–0.6 ND–2.0 ND–0.1 ND–0.3 ND–0.4 ND ND ND ND ND–0.2 5.3–8.0 ND–0.2 ND–0.1 ND–0.1 1.9–2.9 8.4–21.3 67.8–83.2 ND–0.1 0.2–0.4 0.1–0.3 ND ND–1.0 ND–1.8 ND ND–0.2 ND–0.2
Safflower seed oil ND ND ND ND ND–0.1 7.9–10.2 0.1–0.2 ND–0.2 ND–0.1 4.8–6.1 35.9–42.3 41.5–47.9 0.3–0.4 0.3–0.6 ND–0.3 ND ND–0.3 ND ND ND–0.3 ND
Sesame seed oil ND ND ND ND–0.1 ND–0.2 8.0–13.5 ND–0.2 ND–0.1 ND–0.1 2.0–5.4 17.7–28.0 49.8–59.0 5.0–11.0 0.1–0.6 ND–0.5 ND–0.1 ND–0.7 ND–0.3 ND ND–0.5 ND
Soya bean oil
ND ND ND ND–0.1 ND–0.2 5.0–7.6 ND–0.3 ND–0.2 ND–0.1 2.7–6.5 14.0–39.4 48.3–74.0 ND–0.3 0.1–0.5 ND–0.3 ND 0.3–1.5 ND–0.3 ND–0.3 ND–0.5 ND
Sunflowerseed oil
194
Source: Codex Alimentarius Commission, Codex Standard for Named Vegetable Oils, Codex Standard 210.
Palm olein
Fatty acid
Rapeseed oil (low erucic acid)
TABLE 7.4 Fatty Acid Composition of Vegetable Oils as Determined by Gas Liquid Chromatography from Authentic Samples (expressed as percentage of total fatty acids)
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TABLE 7.5 Fatty Acid Composition of Animal Fats Lard Rendered pork fat
Premier jus tallow
< 0.5 in total 1.0–2.5 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 20–30 2.0–4.0 < 0.1 < 0.1 <1 <1 < 0.1 < 0.1 8–22 35–55 4–12 < 1.5 < 1.0 < 1.5 < 1.0 < 1.0 < 0.1 < 0.5
< 0.5 2–6 < 0.3 0.5–1.5 0.2–1.0 < 1.5
C10:0 C14:0 C14:ISO C14:1 C15:O C15:ISO C15:ANTI ISO C16:0 C16:1 C16:ISO C16:2 C17:0 C17:1 C17:ISO C17:ANTI ISO C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C20:4 C22:0 C22:1
20–30 1–5 < 0.5 < 1.0 0.5–2.0 < 1.0 < 1.5 15–30 30–45 1–6 < 1.5 < 0.5 < 0.5 < 0.1 < 0.5 < 0.1 not detected
Source: Codex Alimentarius Commission, Codex Standard for Named Animal Fats, Codex Standard 211-1999.
Terminal Omega – CH3 group t 19
17
15
13
11
9
7
5
3
H3C
1 COOH
FIGURE 7.9 The structure of EPA C20:5 5,8,11,14,17 all cis (C20:5 omega-3).
The discovery that unsaturated FAs are essential (Burr and Burr, 1929) was strongly enhanced by Hollman’s suggestion because it was possible to determine that for the human organism, there are only two essential unsaturated FAs—LA C18:2 (omega-6 or
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Chemical and Functional Properties of Food Components Diet
Present in human diet
C18:1(n-9) Oleic acid
C18:2(n-6) LA
C18:3(n-3) ALA
C18:2(n-9)
C18:3(n-6)
C18:4(n-3)
Metabolic processes Desaturase 6
-Linolenic GLA
SDA
C20:3(n-6) Dihomo- -linolenic DGLA
C20:4(n-3)
Elongase
C20:2(n-9)
Desaturase 5
C 20:3(n-9) Mead MA
C20:4(n-6) AA
C20:5(n-3) Timnodic EPA
Elongase
C22:3(n-9)
C22:4(n-6) Adrenic
C22:5(n-3) DPA
C22:5(n-6) Osmond
C22:6(n-3) DHA Cervonic
Desaturase
4
FIGURE 7.10 Metabolic pathways of n-3 and n-6 LC-PUFA in the human organism.
n-6) and ALA C18:3 (omega-3 or n-3). Neither LA nor ALA are synthesized in the human organism, thus they must be supplied with the diet. Other metabolites are not essential because they are created in the human organism as a result of the action of enzymes, that is, of adequate desaturases and elongases. The schematic of the metabolism of PUFAs in the human organism is shown in Figure 7.10. Both LA and ALA, as the “parents” of the n-6 or n-3 FA families, are converted in the human organism to their metabolites by the successive reactions of desaturation or elongation. Thus, both families compete for the appropriate enzymes. The most sensitive is the first step, delta 6–desaturation, because this step of FA metabolism is rate limiting. The increase in the concentration of FAs of one family, for example, the n-6 family caused by increased consumption of n-6 FAs (sunflower or maize, known as linoleic oils, do not contain n-3 FAs), causes a decrease in conversion of ALA into their metabolites and vice versa. Thus from the nutritional point of view, the proportions of n-6 and n-3 FA metabolites should be balanced as it is schematically shown in Figure 7.11.
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n-6 FA containing oils: sunflower (67% LA); maize (70% LA); safflower oil (up to 80% LA)
Flax seed oil (56% ALA); rapeseed oil (11% ALA); soybean oil (6–9% ALA) or fish oils
FIGURE 7.11 Lipid balance: the optimal proportions of n-6 and n-3 fatty acids in the human diet should be between 5:1 and 3:1.
The conversion of LA and ALA may be monitored in a human organism. The lipids isolated from human tissues by extraction according to the Folch or BlighDyer methods after separation into lipid classes may be converted to FAME and analyzed by GC. Individual LC PUFA may be easily identified on chromatograms as shown in Figure 7.12.
7.7 TRIACYLGYLCEROLS 7.7.1 CHEMICAL
AND
FUNCTIONAL PROPERTIES
The formal presentation of synthesis of TAGs is shown in Figure 7.13. The hydrocarbon chains R may be the same (though this is rare), two may be the same, or more usually they are different. Because there is a great variety in the chain length and structure of FAs, there is great scope for variation in the makeup of fats and oils. It is also possible that not all hydroxyl groups are esterified, thus other subgroups of acylglycerols may also be present in natural fats and oils. In MAGs, one FA is esterified to glycerol. Due to the orientation of that molecule, two isomeric forms exist as shown in Figure 7.14. R is a saturated or an unsaturated hydrocarbon chain. The external carbons of glycerol are frequently named as “α” and the central one as “β.” One part of a molecule of MAG contains hydrophilic –OH groups and another part lipophilic (hydrophobic) acyls of FA. Thus, the MAGs exhibit surface active properties and are commonly used in the food industry as emulsifiers. The main fields of application are margarines, mayonnaises, and ice creams. DAGs are FA diesters and occur in two isomeric forms (Figure 7.15). Their properties as surface active agents are much weaker than those of MAGs. In foods, DAGs are used as emulsifying agents in the production of fine baked goods, chewing gum, and in replacement of fats. Their main field of application is the cosmetic industry as one of the components of creams and shampoos.
C18:1 trans
n-3
C18:1 11
GLA
C18:3 (n-6)
LA
C18:2
C18:4 C20:4
E PA
DPA C22:5 (n-3)
C22:5
DPA
DHA
C22:5
C22:4
C22:4
C18:4 (n-3) EPA C20:5 (n-3)
A LA
DGLA C20:3 (n-6)
DGLA
AA
AA C20:4 (n-6)
FIGURE 7.12 Monitoring of LC PUFA transformations catalyzed by corresponding desaturases and elongases by GC (column: Rtx2330 100-m ID 0.25-mm; df 0.2-μm temp in 155 time 50; rate 1.5°C/min; fin 210°C, then hold. Inj. Split/splitless; hydrogen 32 cm/s) (author’s own data).
C16:2
C16:1 7
n-6
C18:0 C18:1 9
198
C12:0
C14:0
C16:0
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O
O H2C
OH
HC
OH
H2C
OH
+
HO
C O
R
HO
C O C
R
HO
Glycerol
R
H2C
O
C O
R
HC
O
C O
R
H2C
O
C
R
Fatty Acids
+ 3 H2O
Triacylglycerol
FIGURE 7.13 Formal presentation of synthesis of TAGs. α
1
β
2
α′
3
CH2 O CO R HO C H CH2 OH sn-1- or α-isomer
CH2OH CH O CO R CH2 OH 2- or β-isomer
FIGURE 7.14 Isomeric forms of MAGs.
CH2O CO R HO C H CH O CO R´ sn-1,3- or α, α′-isomer
3 CH2OH 2 CH O CO R 1 CH2 O CO R´ sn-1,2- or α, β-isomer
FIGURE 7.15 Isomeric forms of DAGs.
7.7.2 MODIFICATIONS TAGs can be modified by transesterification. This is a term covering a group of chemical reactions involving a fat and either an FA, an alcohol, or another ester. As a result of chemical interchange, a new ester is formed. In acidolysis, an FA in the TAG is replaced by another one according to the reaction in Figure 7.16. Alcoholysis is a reaction of TAGs with alcohol. A good example is the synthesis of MAGs in which one portion of TAGs containing only saturated FAs and 2 portions of glycerol are subjected to reaction at elevated temperature in the presence of a catalyst (NaOH, or ZnO) (Figure 7.17 ). In transesterification at an elevated temperature in the presence of catalysts, the acyl groups are exchanged between TAGs according to reaction shown in Figure 7.18. These reactions give rise to a great variety of possibilities in the industrial modification of the physical and physiological properties of fats and oils.
7.7.3 FRACTIONATION That some fats melt before others is obvious to anyone who has looked at butter in hot weather. Clear oil can be seen to be separating from the soft but not molten
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Chemical and Functional Properties of Food Components
CH2. OOCR1
CH2. OOCR
CH. OOCR2 + R. COOH
CH. OOCR2 + R1 COOH
CH2. OOCR3
CH2. OOCR3
FIGURE 7.16 Acidolysis: reaction of TAGs with free FA. CH2. OOCR1
CH2. OH
CH2. OOCR1 (or R2 or R3)
CH. OOCR2 + 2 CH. OH CH2. OOCR3
3CH. OH
CH2. OH
CH2. OH
or CH. OOCR1
CH2. OH
CH. OOCR2 + 3ROH
CH. OH + R1. COOR + R2. COOR + R3.COOR
CH2. OOCR3
CH2. OH
FIGURE 7.17 Alcoholysis: reaction of TAGs with alcohol. CH2. OOCR1
CH2. OOCR2
CH2. OOCR2
CH. OOCR2 + CH. OOCR3
CH.O OCR2
CH2. OOCR3
CH2. OOCR3
CH2. OOCR4
CH2. OOCR1 + CH. OOCR3 CH2. OOCR4
FIGURE 7.18 Transesterification: exchange of acyls between TAGs.
material. This is the basis of fractionation of fats. The melting points of different TAGs are shown in Table 7.6. The melting point (MP) of TAGs increases according to the chain length of FA and decreases according to the number of the double bonds in the TAG molecule. The MP of TAGs containing cis double bonds are much lower than corresponding TAGs with trans double bonds. In order to obtain good separation of TAGs with different MPs it is better to begin with molten fat and to cool it under strictly controlled conditions to solidify some parts (fractions). The solid fractions may be removed as they are formed by decantation, filtration, or centrifugation. The most popular fractionated fats on the market are palm oleine and palm stearine. Hydrogenated palm or rapeseed oils are also frequently subjected to fractionation in order to remove fractions of the TAGs with high melting points.
7.7.4 LIPOLYSIS In the human organism a TAG molecule cannot be directly absorbed across the intestinal mucosa. It must first be digested into a sn-2 (or β)–MAG isomer and two free FAs. The enzyme that performs this hydrolysis is pancreatic lipase, which is
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TABLE 7.6 Melting Points of Some Saturated and Unsaturated Triacylglycerols with Different Chain Lengths Triacylglycerol C6 C12 C14 C16 C18 C18:1 (cis) C18:1 (trans)
Melting point (°C) –15 15 33 45 55 –32 15
delivered into the lumen of the gut as a constituent of pancreatic juice. Pancreatic lipase catalyses the hydrolysis of FA from positions sn-1 and sn-3 (or α,α′) of TAGs to yield sn-2 (or β)- MAGs. Very little hydrolysis occurs at position sn-2 and there is limited isomerization to sn-1-MAGs, which may become substrates for further lipase action. The enzyme reacts with the TAG at the oil–water interface of large oil in water (O/W)) emulsion particles, but hydrolysis can occur only after both the enzyme and surface have been modified to allow interaction. Bile salt molecules accumulate on the surface of the lipid particles displacing other surface active constituents enabling pancreatic lipase to attack the TAG molecule. Schematically the enzymatic hydrolysis of TAG is shown in Figure 7.19. As far as human nutrition is concerned, very important conclusions can be drawn from Figure 7.19. Palmitic acid C16:0 is present in human milk or in human fat at a concentration of 30 to 32% in the FA composition. Almost 96% of palmitic acid in human fat is situated in position sn-2 of a TAG molecule. Positions sn-1 and sn-3 are usually occupied by short-chain (sn-1) FAs and unsaturated (sn-3) FAs. The hydrolysis of TAG molecules catalyzed by pancreatic lipase liberates short-chain or unsaturated FAs from sn-1 and sn-3 positions, which are absorbed directly into the bloodstream from the stomach. In the case of plant oils, the sn-2 position is usually occupied by LA, and the saturated FAs, including palmitic acid, occupy the external positions sn-1 or sn-3. Such plant oils are commonly added to infant formulas in order to increase the content of LA to the level of 9 to 11% in FA composition in order to mimic the FA composition of human milk. As a result of enzymatic hydrolysis saturated FAs are liberated in the infant’s gastric system. A part of the liberated saturated FA may react with the Ca2+ present in the gastric system. Especially in the case of newborn babies fed with infant formulas, difficult soluble calcium soaps are removed from the organism with the stool causing deficiency of Ca2+ in the organism. The stool of infants becomes very hard when the mother replaces natural feeding with formulas containing plant oils. Thus, because of differences of stereospecific structures of human and plant TAGs, addition of plant oils to infant formulas should be considered a poor solution for feeding of newborn babies.
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Chemical and Functional Properties of Food Components
R1 Saturated short chain
C O
sn-1
O
H 2C H
sn-2
C
R3 C
O
O
C
H 2C
R2 Palmitic saturated
Unsaturated
O
O
sn-3 Pancreatic lipase
OH
sn-1
H 2C H
sn-2
C
O
OH
C Palmitic
H 2C
R2
saturated
O
sn-3
FIGURE 7.19 Enzymatic hydrolysis of TAG molecule of human milk.
7.7.5 ANALYSIS
OF THE
COMPOSITION
OF
TRIACYLGLYCEROLS
7.7.5.1 HPLC of Triacylglycerols A great number of possible isomers and relatively large molecules are the reasons why analysis of the composition of the TAGs mixtures is a rather difficult task. In the case where di-, tri-, or more unsaturated FAs are present in a TAG molecule, the use of very efficient GC becomes problematic because high temperatures of columns much over 300°C must be used, which may be destructive with respect to the analyzed sample. In such cases, less efficient HPLC produces more analytical information and seems to be very useful. TAGs are classically named using three letters indicating the diversity of the acylated FA, for example, the homogeneous LLL, OOO, PPP, SSS or the heterogeneous OOL, SOO, or PSO: L for LA, O for oleic acid, P for palmitic acid, and S for stearic acid. Under isocratic conditions, the logarithm of the elution volume (or retention time) of a molecular species is directly proportional to the number of carbon atoms (CN), and inversely proportional to the total number of double bonds (DB) in the three fatty acyl chains (Stołyhwo et al. 1985). Furthermore, a linear relationship
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exists between the equivalent carbon number (ECN) and the carbon number of the TAG that showed the same unsaturation characteristics. The principal obstacle in the application of HPLC is the problem of detection. The refractive index (RI) detectors cannot be applied when gradient elution is to be used for the separation of natural mixtures of fats usually containing simultaneously free FA, MAGs, DAGs, and TAGs. The LLSD is the detector of choice for the analysis of lipid classes including TAGs (Stołyhwo et al. 1983, 1984). The advantage of LLSD lies in the fact that it is sensitive with respect to all relatively nonvolatile UV-absorbing or nonabsorbing substances. The mobile phase is evaporated before detection of analytes, thus any solvent (excluding buffer solutions), may be used in isocratic or gradient elution systems. Natural, unaltered TAGs do not absorb UV radiation at wavelengths over 220 nm, thus classical UV 254-nm detectors cannot be used for such purposes. The carbonyl group of the ester bond in TAGs absorbs UV at a wavelength below 218 nm, but this is the range at which there is a very limited number of solvents not absorbing UV. Thus, in practice for the analysis of TAGs, the mobile phases may be composed only of methanol, propanol-2, acetonitrile, and HPLC gradient-grade hexane. In spite of the drawbacks of UV, the use of a diode array detector (DAD) in several detection channels is very practical in identifying the substances dissolved in the analyzed sample, for example, carotenoids (detection at 450 to 465 nm); tocopherols (detection at 295 nm), FAs with conjugated double bonds (detection at 233 nm for CLA), and 268 nm for triunsaturated FAs with conjugated double bonds. Products of deterioration of lipids by autooxidation can also be detected, for example, peroxides, hydroperoxides, aldehydes, or epoxy acids (detection by UV at 235 to 250 nm). An example of the application of multichannel LLSD/DAD detection of TAGs of evening primrose oil (EPO) and of products of its autooxidation is shown in Figure 7.20 (a, b). With the use of LLSD it is possible to detect all relatively nonvolatile components of EPO. The signal S of the LLSD is dependent on the mass m of an analyte approximately according to the equation, S = km1.37, where k is the response factor (Stołyhwo et al., 1983, 1984). In channel DAD 215 nm, all analytes absorbing at 215 nm are detected but their quantities depend on the corresponding peak areas and on molecular coefficients of absorbance. Thus tocopherols are easily detected as well as products of autooxidation of TAGs. In channel DAD 268, the conjugated trienes as intermediate products of autooxidation of GLA C18:3(n-6) are detected with good sensitivity. The degree of deterioration of EPO caused by peroxidation may be estimated as the ratio of the areas 268 nm to 215 nm of the peaks corresponding to the LnLnLn and LnLnL. 7.7.5.2 GLC of Triacylglycerols The TAGs of fats containing more saturated FA, as, for example, of coconut oil, palm kernel oil, palm oil, cocoa butter, or milk fats may be analyzed by GC. In such
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Chemical and Functional Properties of Food Components
35.101
LnLnL
38
37.163
22.958
ADC1 A, ADC CHANNEL A of KURS\VITAMX50.D
nA
27.639
204
LLL
LLSD
36
LL
45.082 46.175
30.753
28.837 19.141
8.807
32
9.912 10.961 11.208
LnLnLn
47.743
LLP
34
30 20
35.314
6.438
20
15
50
min
DAD 215 nm
30.916
29.120
13.464
25
19.348
Products of autooxidation
30
40
37.378
11.161
10.145
Tocopherols 35
30 27.847
10
DAD1 A, Sig=218,5 Ref=550,100 of KURS\VITAMX50.D
23.174
0 mAU
10
5 0
10 20 DAD1 B, Sig=268,10 Ref=550,100 of KURS\VITAMX50.D
30
40
50
min
mAU
DAD 268 nm
35
22.835
30
Conjug. trienes
9.047
15
19.098
11.158
20
10
28.792
22.262
25
5 0
10
20
30
40
50
min
FIGURE 7.20 (a) HPLC of TAGs of natural evening primrose oil (EPO). Detection parallel in channels: LLSD, DAD1A at 218-nm bandwidth (BW) 5 nm; DAD1B at 268-nm BW 10 nm. Column: Hypersil ODS 5 μm; mobile phase: acetonitrile/propanol-2/hexane 66/21/13 v/v/v. Flow rate 0.8 mL/min, column temp. 18°C.
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*DAD1, 19.211 (180 mAU, mAU 9 8
*DAD1, 11.171 (434 mAU, mAU
268 nm
Conjug. trienes
Tocopherols
120
7
100
6
80
5
60
4
295 nm
40
3
20
2
0
1 230 240 250 260 *DAD1, 13.778 (22.5 mAU,
270
280
290
Products of peroxidation 235–250 nm
mAU 20 17.5
220 240 260 *DAD1, 23.291 (1131 mAU
nm mAU 14
300
nm
Conj. trienes 268 nm
12
15
280
10
12.5
8
10 6
7.5 5
4
2.5
2
0
0 220
240
260
280
300
320
nm
240
250
260
270
280
290
nm
FIGURE 7.20 (b) HPLC of TAGs of natural evening primrose oil (EPO). Detection parallel in channels: LLSD, DAD1A at 218-nm BW 5 nm; DAD1B at 268-nm BW 10 nm. Column: Hypersil ODS 5 μm; mobile phase: acetonitrile/propanol-2/hexane 66/21/13 v/v/v. Flow rate 0.8 mL/min, column temp. 18°C.
cases, instead of split/splitless injection, it is better to use a cool on column injection system. The TAGs dissolved in isooctane (conc. 0.04%) are injected (0.5 μl) directly into the capillary column at an inlet temperature of 77°C, and the temperature program is started. In the author’s laboratory for analysis of TAGs by GC, the Restek 65 TAG column (Restek, Chromatographic Specialties, Canada), 30-m 0.25-mm ID df 0.2-μm column is used. An example of the separation of TAGs of butter and of butter adulterated with 10% sunflower oil is shown in Figure 7.21. The TAGs of sunflower oil contain over 80% FAs with a chain length of 18 carbon atoms. Thus the carbon number (CN) of those TAGs is CN 54. Adulteration of butter with sunflower oil as shown in Figure 7.21 B is evident because natural butter contains only minute amounts (below 0.2%) of TAGs with CN 54.
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Chemical and Functional Properties of Food Components
CN46 38
CN32
CN 48
(a)
42
34 CN 30
CN 50
CN28 CL
CN 52
(b)
CN 54
FIGURE 7.21 Separation of TAGs of butter (a) and of butter adulterated with 10% sunflower oil (b). Column Restek 65 TG cool on column injection at 77°C. Temperature program in 80°C time 3 min, next rate 20°C/min, fin 240°C, next rate 3°C/min, fin temp 340°C, next hold 25 min. Flow rate: hydrogen 22 cm/s.
REFERENCES AOAC International, 1990, Method No. 920.39C, Crude fat or ether extract, Gaithersburg, MD. AOAC International, 1990, Method No. 932.06, Fat in dried milk (Roese Gottlieb Method IDF-ISO–AOAC), Gaithersburg, MD. AOCS (American Oil Chemist’s Society), 1993, Method No. 922.06, Acid hydrolysis method, Champaign, IL. AOCS (American Oil Chemist’s Society), 1993, Method 985.21, Total fatty acid isomers in margarines: Gas chromatographic method, Champaign, IL. AOCS (American Oil Chemist’s Society), 1993, Method No. Ce 1b-89, Fatty acid composition by GLC marine oils (modified), Champaign, IL.
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Bligh, E.G. and Dyer, W.J., 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Burr, G.O. and Burr, M.M., 1929, A new deficiency disease produced by the rigid exclusion of fat from the diet, J. Biol. Chem. 82, 345–367. Codex Alimentarius Commission, Codex Standard for Named Animal Fate, Codex Standard 211-1999, http://www.codexalimentarius.net/download/standards/337/cxs-211e.pdf Codex Alimentarius Commission, Codex Standard for Named Vegetable Oils, Codex Standard 210, http://www.codexalimentarius.net/downloads/standards/336/cxs-210e.pdf Folch, J., Lees, M., and Sloan-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226, 497–509. Frede, E., Buchheim,W., and Stołyhwo, A., 1997, New developments in milk fats, in Modern Developments in Food Lipids, pp. 171–191, Shukla, V.K.S. and Kochhar, S.P., Eds., Centre A/S, Lystrup, Denmark. Ip, C., Scimeca, J.A., and Thompson, H.J., 1994 (August 1). Conjugated linoleic acid. A powerful anticarcinogen from animal fat sources, Cancer, 74 (3 Suppl), 1050–1054. Kaluzny, M.A., Duncan, L.A., Merritt, M.V., and Epps, D.E., 1985. Rapid separation of lipid classes in high yield and purity using bonded phase columns, J. Lipid Res., 26, 135–140. Noone, E.J., Roche, H.M., Nugent, A.P., and Gibney, M.J., 2002. The effect of dietary supplementation using isomeric blends of conjugated linoleic acid on lipid metabolism in healthy human subjects, Br. J. Nutr. 88, 243–251. Pariza, M.W., Park, Y., and Cook, M.E., 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res., 40, 283–298. Stołyhwo, A., Colin, H., and Guiochon G., 1983. Use of light scattering as a detector principle in liquid chromatography, J. Chromatog., 265, 1–18. Stołyhwo, A., Colin H., and Guiochon, G., 1985. Analysis of triglycerides in fats and oils with the use of light scattering detector, Anal. Chem., 57, 1324– 354. Stołyhwo, A., Martin, M., Colin H., and Guiochon G., 1984. Study of the qualitative and quantitative properties of the light-scattering detector, J. Chromatog., 288, 253. Wahle, K.W., Heys, S.D., and Rotondo D., 2004. Conjugated linoleic acids: are they beneficial or detrimental to health? Prog. Lipid Res., 43(6), 553–587.
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8
Rheological Properties of Food Systems Anna Pruska-Ke˛dzior and Zenon Ke˛dzior
CONTENTS 8.1 8.2 8.3 8.4
Introduction .................................................................................................. 210 Scaling Time of Rheological Behavior of Materials .................................. 210 Types of Rheological Behavior in Simple Deformation............................. 211 Viscous Liquid ............................................................................................. 212 8.4.1 Introduction ...................................................................................... 212 8.4.2 The Shear-Dependent, Time-Independent Viscosity of Non-Newtonian Liquids .............................................................. 213 8.4.3 Materials with a Yield Value—The Plastic Behavior Model .......... 215 8.4.4 Non-Newtonian Time-Dependent Liquids....................................... 216 8.4.5 Temperature and Pressure Dependence of Viscosity ...................... 217 8.5 Viscoelasticity of Food Materials ................................................................ 217 8.5.1 Linear Viscoelasticity....................................................................... 217 8.5.2 Small Strain Experiments in Simple Shear ..................................... 219 8.5.2.1 Stress Relaxation after Sudden Strain.............................. 219 8.5.2.2 Creep and Creep Recovery Test....................................... 220 8.5.2.3 Dynamic Assay ................................................................. 221 8.6 Nonlinear Viscoelasticity—Normal Stress Differences .............................. 223 8.7 Rheological Properties of Food Macromolecular Systems......................... 224 8.7.1 Flow Behavior of Macromolecular Solution................................... 224 8.7.1.1 The Concentration Regimes Effect .................................. 224 8.7.1.2 Viscosity of Dilute Solution ............................................. 225 8.7.1.3 Viscosity of Semidilute and Concentrated Solutions ...... 227 8.7.1.4 Shear Dependence of the Viscosity.................................. 227 8.7.2 Linear Viscoelastic Properties of Food Polymer Systems .............. 229 8.7.3 Mechanical Spectra of Food Polymer Systems............................... 231 8.8 Structure–Rheology Relationship in Multiphase Food Materials............... 235 8.9 Elastic Solid ................................................................................................. 238 8.9.1 Introduction ...................................................................................... 238 8.9.2 Nonlinear Behavior of a Solid......................................................... 238 8.10 Summary ...................................................................................................... 240 References.............................................................................................................. 241 209
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8.1 INTRODUCTION Rheological investigations consist of mechanical excitation of a material under strictly defined conditions. Two basic types of deformation exist: shear strain and extension (elongation) strain. Depending on the stress or strain magnitude, the method of its application and the length of time the stress or strain acts on a material, shear flow or extensional flow of the material can occur. Deformation caused by an external force acting on the material can be large or small. If the deformation is small enough, the structure of the investigated material does not change. Large deformation of complex materials usually causes some changes in their macroscopicscale structure, which can be partially reversible or not. Rheological methods, like small and large-strain uni- and biaxial deformation in compression or elongation mode, as well as multifrequency small deformation methods in shear have been applied in food rheology.
8.2 SCALING TIME OF RHEOLOGICAL BEHAVIOR OF MATERIALS Common experience shows the complexity of the rheological properties of food materials when observed over a large time scale. When a stress or a strain is impressed upon a body, structural rearrangement on the supramolecular level, that is, 103 to 107 nm occurs inside the material as it responds to the imposed excitation. In any real material, these rearrangements (relaxation) necessarily require a finite time. Any natural material flows on some time-length scale. The Deborah number (De), a dimensionless quantity describing the “flowness” of a material, allows time scaling in rheology:
De =
Relaxation time Observation time
=
λ t
(8.1)
Accordingly, a material appears as a liquid if the Deborah number De is small, and as a solid if it is large, with respect to the observation time. When the changes (relaxation) take place so rapidly that time is negligible compared with the time scale of the observation (λ ≈ 0), De approaches 0 and the material behaves as a purely Newtonian viscous liquid. For example, characteristic relaxation time for water in a liquid state is 10–12 s. In the purely viscous material, all the energy required to produce the deformation is dissipated as heat. The time λ and De is infinite for the Hookean elastic solid. In a purely elastic material the energy of deformation is stored and may be recovered completely upon release of the forces acting on it. A material can appear solid-like either because it has a very long characteristic time λ or because the deformation process applied to its examination is very fast. Thus even mobile liquids with low characteristic times can behave like elastic solids in a very fast deformation process. Water behaves as a solid at very short times or equivalently at very high frequencies, that is, at time less than 10–8 s. By contrast, solid-like materials flow as liquids under a sustained stress at sufficiently
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Nonlinear viscoelasticity γyield
Elastic solid
2nd order fluid (η, ψ1, ψ2)
Newtonian fluid (η)
strain γ, strain amplitude γ0
Rheological Properties of Food Systems
Linear viscoelasticity (η, λ) . Deborah number = λ/t or λγ or γ0 λω
FIGURE 8.1 Rheological behavior of fluid systems over the time scale. (Adapted from Macosco, C.V., Rheology: Principles, Measurements, and Applications, VCH Publishers, New York, 1994. With permission.)
long times. For instance, crystallized honey flows at room temperature under gravity forces at times of an order 103 to 104 s. In principle, foods like mayonnaise, sauces, dough, meat, bread crumbs, and fruits, are viscoelastic bodies. Some energy may always be stored during the deformation of a material under appropriate conditions, and energy storage is always accompanied by dissipation of some energy. In a typically viscoelastic material, the time necessary for the material rearrangements to take place is comparable with the time scale of the experiment. For typically viscoelastic material, a Deborah number approaches unity. For many foods there are situations in which, relating to the time of experiments, liquids depart from purely viscous behavior and show viscoelastic and elastic properties (and vice versa) (Rao and Steffe, 1992). Figure 8.1 shows the types of rheological behavior occurring along the Deborah number scale: from Newtonian liquids to solid-state and linear viscoelasticity limits. Depending on the type of rheological behavior, the characteristic time t of the Deborah number is expressed as the inverse of deformation rate γ –1 in simple shear flow tests, or as the product of the amplitude of the oscillatory strain γ0 times its frequency ω, (γ0ω)–1 in dynamic tests. As demonstrated in Figure 8.1, strain γ or strain amplitude γ0 and the Deborah number allow us to characterize different main classes of rheological behavior of the continuous fluid phase.
8.3 TYPES OF RHEOLOGICAL BEHAVIOR IN SIMPLE DEFORMATION The response of linear polymers to simple shear and simple extensional or elongational forces are illustrated in Figure 8.2. The shear force causes both deformation and rotation, whereas the normal extension force results in only deformation without
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FIGURE 8.2 The response of linear polymer to simple shear (top) and simple extensional forces (bottom).
rotation. In shear flow, the overall shape of the viscosity curve is similar for linear and branched polymers, although the absolute magnitude of shear viscosity and its dependence on shear rate is somewhat different. The same polymer systems in extensional flow obey ideal extensional Trouton behavior, but only in the low-shear Newtonian viscosity range (cf. Figure 8.7). Extensional Trouton behavior is defined as ηE ≡ 3η, (η0), where η is Newtonian viscosity, η0 is zero-shear viscosity, and ηE is uniaxial extensional viscosity. When in a concentrated solution or polymer melt, the molecules are highly ordered by extensional flow, and the branching points of branched polymers can act as hooks, resulting in increasing the resistance to flow. Therefore, while linear polymer solutions or melts thin down under extensional forces, branched polymers tend to stiffen or experience strain hardening. For example, the strain-hardening phenomenon is commonly observed in wheat dough extensional behavior (Dobraszczyk and Morgenstern, 2003). The use of only steady-state shear rate viscosity appears to be sufficient in most cases of food processing (pipeline flow, extrusion, mixing, and molding), whereas the elongation properties become relevant for processing such as converging flow, fiber spinning, or film blowing. Although in most food processing the deformation and flow are very complex and involve uni- and multiaxial phenomena, generally the uniaxial measurements, in simple shear or simple extension, can satisfactorily estimate or predict many real process conditions (Bourne, 2002).
8.4 VISCOUS LIQUID 8.4.1 INTRODUCTION For Newtonian liquids, fundamental relations called constitutive relations between stress and strain rate are exhibited by Newton’s law of viscosity:
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σ = ηγ
(8.2)
where η, the Newtonian viscosity, is the coefficient independent of strain rate and time at a given temperature and density, σ is the shear stress, and γ = d γ dt is the shear rate or strain rate (≡ velocity gradient γ = dv dy in simple shear). The ideal Newtonian fluid is the basis for classical fluid mechanics. In accordance with Newton’s law, the stress is always directly proportional to shear strain but independent of the strain itself and time. Many real materials obey this ideal law. Most small molecule liquids like water, dilute true aqueous solutions (e.g., brine bath, vinegar), beverages (coffee, distilled liquors), and oils are generally Newtonian. Some Newtonian materials also have very high viscosity, such as liquid honey. The Newtonian fluid model may be adequate, although not exact, for dilute suspensions (e.g., clear fruit juice), emulsions (e.g., milk), and solutions of moderately long-chain molecules. Table 8.1 shows the viscosity range for some familiar fluid food materials.
8.4.2 THE SHEAR-DEPENDENT, TIME-INDEPENDENT VISCOSITY OF NON-NEWTONIAN LIQUIDS Many colloidal suspensions and polymer solutions do not obey a linear Newton’s relation. In this case, the coefficient of apparent viscosity (non-Newtonian viscosity) is dependent on strain rate η γ = σ γ ≠ const . Nearly all these materials give a viscosity that decreases with increasing velocity gradient in shear. Some concentrated suspensions show shear thickening behavior as illustrated in Figure 8.3. Liquids showing shear-thinning and shear-thickening non-Newtonian behavior are most often described using a power law liquid model or Cross model. The power law liquid model is used to represent the behavior of many liquid food polymer solutions and some colloidal suspensions and emulsions (Table 8.2). The equation for the power law liquid model may be written in the following form:
()
σ = k γ n
(0 < n < 1) .
(8.3)
TABLE 8.1 Newtonian Viscosity of Some Food Materials Material Water Cream, 10% fat Raw milk Homogenized milk Whey protein isolate, 10% water suspension Soybean oil Apple juice, 20° Brix
Temperature (°°C)
Viscosity η (Pa ⋅ s)
20 40 20 20 50
0.001 0.00148 0.00199 0.0020 0.0031
30 27
0.04 0.0021
Reference
Steffe, 1983
Walkenström et al., 1999 Steffe, 1983
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e
σ
d
b σ0
a c
•
γ FIGURE 8.3 Flow curves for model viscous fluid material. (a) Newtonian; (b) power law model n > 1; (c) power law model n < 1; (d) Bingham model; (e) Herschel-Bulkley model.
TABLE 8.2 Parameters of Rheological Models of Some Non-Newtonian Food Liquids Power law model
Temp. (°C)
Flow behavior index n
Consistancy coefficient k Pa ⋅ sn
Soybean milk
20
0.815
0.01898
Sugar beet molasses Brix (%) 81.46 Mustard
45 60 25 25 4 25 46 20 55 95
0.756 0.793 0.28 0.29 0.843 0.854 0.872 0.911 0.746 0.759
Material
Egg yolk (fresh, liquid) Whole liquid egg Cheese (Gouda)
19.51 9.23 17.30 35.91 1.683 0.421 0.148 0.0294 3.653 0.364
Casson model Casson plastic viscosity ηCk Pa ⋅ s
Casson yield stress σy Pa
0.0042 0.0106
15.05 31.07
0.0168
0.01
Reference Shin and Keum, 2003 Togrul and Arslan, 2004 Juszczak et al., 2004 Telis-Romero et al., 2006 Ahmed et al., 2003 Dimitreli and Thomareis, 2004
The viscosity of a shear-thinning liquid decreases when the shear rate increases. When the power law model, involving only two disposable constants, explains the behavior of a liquid adequately at a reasonably broad stress-shear rate range, it
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appears relatively easy to use in analytical or numerical calculation of liquid behavior in a complex flow situation (e.g., pipeline flow or mixing). The shear power law model (with n > 1) has often been used for shear-thickening materials, that is, materials that increase in viscosity as the shear rate increases (line c on Figure 8.3). Most shear-thickening materials, such as concentrated starch pastes and other swollen particulate systems (some caseinate suspensions), are shear thinning at low shear rates and suddenly become almost rigid as the shear rate is increased. Barnes et al. (1989) have reviewed work on shear thickening of suspensions. The Cross equation (8.4) has been proposed to describe the viscosity of complex liquids dependent on shear rate: η − η∞ 1 = η0 − η∞ 1 + γ γ 0
(
)
n
.
(8.4)
The equation relates the viscosity η to the shear rate γ , introducing two limits, η0 and η∞ with a characteristic shear rate γ 0 . A slope of log η versus log γ in the shear-thinning region is related to an exponent n. A physical meaning of the constant values η0 , η∞ and γ 0 will be explained further (see Section 8.7.1.4 and Figure 8.7). The Cross equation is a universal model suitable when considering colloidal dispersed and flocculated systems, polymer solutions and melts, and the systems containing particles interacting with each other in various ways.
8.4.3 MATERIALS MODEL
WITH A
YIELD VALUE—THE PLASTIC BEHAVIOR
Materials with a yield value do not flow before a certain stress is reached. Plastic materials are often dispersions where the dispersed particles form a network at rest. The stress level required to initiate flow is usually referred to as yield stress σy, and is related to the level of internal structure of the material, which must be destroyed before flow can occur. When plastic materials start to flow they can show a straight line or shear-thinning behavior. Three models of plastic material are most frequently used: the linear Bingham plastic model, and the Herschel-Bulkley and Casson nonlinear models. Line (d) on Figure 8.3 represents the ideal Bingham plastic model. The flow curve is linear with an intercept σy on the stress axis: σ – σy = ηpγ⋅
for σ ≥ σy
(8.5)
The constant parameter ηp is called the plastic viscosity. The Bingham model may represent, with reasonable accuracy, the behavior of some concentrated suspensions and emulsions, such as toothpaste, and food spreads such as margarine, mayonnaise, salad dressing, cheese spread, and ketchup. Nevertheless, behavior of almost all real materials departs significantly from the Bingham model because their flow curves are not linear, except within a very limited range of strain rates.
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The Hershel-Bulkley and the Casson models represent the behavior of a large number of nonlinear viscoplastic materials, and are widely used to develop pipeline design procedures. The Hershel-Bulkley model (Figure 8.3e) may be written as σ = σ y + k γ n
(8.6)
and the Casson model is expressed as σ1 2 = σ1y 2 + ( ηCA γ )
12
(8.7)
The Casson model appears to give an excellent fit to data of chocolate melt and confectionery products. Examples of non-Newtonian model parameters calculated for some food materials are shown in Table 8.2. Yield stress is a very important rheological parameter for predicting a product’s processing conditions, especially while it is being transported in a pipeline or being handled. It is also a significant factor of the product’s sensory perception characteristic, mainly its mouth feeling. Quantifying yield stress must be done carefully because the value obtained depends on the rheological technique used. Most food plastic products show relatively low-yield stress values and even sample collecting can destroy them. For example, yogurt is a highly structured material and extremely sensitive to external forces. Its characterization through the measurement of fundamental rheological properties demands using specially designed nondestructive devices.
8.4.4 NON-NEWTONIAN TIME-DEPENDENT LIQUIDS The viscosity of these kinds of liquids depends on shear rate magnitude and on the length of time the shear rate is applied. Time-dependent materials change the structure and therefore the viscosity with time, at a constant shear rate or constant shear stress. Depending on the way the structure is affected by shearing, the materials are divided into two groups: thixotropic and antithixotropic. Shear-thinning liquids are said to be thixotropic if, after a long rest, when γ (or σ) is applied suddenly and then held constant, the apparent viscosity is a diminishing function of the time of flow. The liquid completely recovers its initial state following a long enough interval after the cessation of flow. The reversible transition gel ⇔ sol is a classic example of thixotropy. Generally speaking, all multiphase and multicomponent foods show thixotropic behavior. Food suspensions (mustard, tomato concentrate), emulsions (soft butter, salad dressings, and mayonnaise) and foams (meat foams, fish foams, pancake batter, aerated desserts, such as crèmes and mousses, whipped cream) containing artificial dispersants or stabilizers, are time-dependent liquids and show thixotropic behavior. Water and fat content, fillings, thickeners, and emulsifiers are factors influencing apparent viscosity and thixotropic behavior of this media.
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Antithixotropy occurs when, under similar conditions, the apparent viscosity is an increasing function of the duration of flow, and the body recovers its initial state after a long enough interval at rest. When the body does not entirely recover its initial state, then partial thixotropy or partial antithixotropy takes place. Many food materials, like stirred yogurt, exhibit these intermediate properties, that is, partial thixotropy or partial antithixotropy when pumping, dosing, and storage. The existence of thixotropy or antithixotropy means that the flow history is an important factor in the proper design of mechanical processes, where the product’s viscosity continues to change for a long time at given shear conditions. The term rheopexy has been used with two different meanings: (1) solidification of a thixotropic system under the influence of a gentle and regular motion, or (2) progressive shear thickening. The first meaning is that for which the term was coined, and its use in the second sense is not recommended. Consequently, use of the term rheopectic fluid (or liquid) to describe a fluid with progressive thickening is not recommended.
8.4.5 TEMPERATURE
AND
PRESSURE DEPENDENCE
OF
VISCOSITY
The temperature dependence of viscosity can often be as important as its shear rate dependence for nonisothermal processing problems. For all liquids, viscosity decreases with increasing temperature and decreasing pressure. The viscosity of Newtonian liquids decreases with an increase in temperature following the Arrhenius relationship:
(
η = A exp − E η RT
)
(8.8)
where R = gas constant, T = absolute temperature, Eη = activation energy of viscous flow, and A = experimentally determined constant. It is often found that over quite a wide range, a graph of ln η against 1/T is linear. As far as temperature is concerned, for most industrial food applications involving aqueous systems, interest is confined from 0 to 100°C. Generally the viscosity of liquids increases exponentially with isotropic pressure. The changes are quite small for pressures differing from atmospheric pressure by about one bar (Macosco, 1994).
8.5 VISCOELASTICITY OF FOOD MATERIALS 8.5.1 LINEAR VISCOELASTICITY As a consequence of the structural rearrangements of a material taking place at a time scale comparable to that of the experiment in which they are observed (De ≈1), the relation between stress and strain or rate of strain cannot be expressed only by material constants. The rheological behavior of viscoelastic materials is characterized by time-dependent material functions. However, in the limit of infinitesimal deformation, viscoelastic behavior can also be described by linear differential equations with constant coefficients. Such behavior
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is termed linear time-dependent or linear viscoelastic behavior. In the most general case, the stress becomes a function of the strain, that is, it depends on the strain history. Supposing deformation of the material in simple shear, the linear constitutive equation is based on the principle that the effects of sequential changes in strain are additive (the Boltzmann superposition principle) (Ferry, 1980; Morrison, 2001). This additivity is an essential feature of linear behavior:
()
σ t =
t
∫ G (t − t ′ ) γ (t ′)dt ′
(8.9)
−∞
where γ = ∂γ ∂t is the shear rate, G(t) is called the relaxation modulus. The integration is carried out over all past times t′ up to the current time t. The strain can be expressed alternatively in terms of the history of the time derivatives of the stress using the following constitutive equation:
()
γ t =
t
∫ J (t − t ′ ) σ (t ′)dt ′
(8.10)
−∞
where σ = d σ dt , J(t) is called the creep compliance. Similar to Equation (8.9), the integration is carried out over all past times t′ up to the current time t. However, for a viscoelastic material J(t) ≠ 1/G(t) because of the difference between the two mechanical excitation time patterns. In practice it is found that most materials show linear time-dependent behavior even in finite deformation as long as the strain remains below a certain limit — the linear viscoelastic limit. This limit, that is, the magnitude of the strain above which linear viscoelastic behavior is no longer observed, varies and is a material property. For instance, the domain of linear viscoelastic behavior of wheat gluten extends usually up to about 5% strain amplitude in dynamic mode in the 10–3 to 100 rad/s frequency range, whereas wheat bread dough exhibits nonlinearity above strain values ∼0.1%. Linear viscoelastic response of nonstarch polysaccharide solutions extends up to 50% and higher in the same frequency range. The experiments designed to determine the linear viscoelastic response of a material to an instantaneous stress or strain base on the assumption that up to the time t0, i.e., the time of stress or strain application, the material remains in rest long enough to relax any prior excitations. A number of small strain experiments are used in rheology. Some of the more common experiments are transient experiments (stress relaxation, creep, and creep recovery), and the dynamic experiments in the harmonic regime (sinusoidal oscillation). Different small strain experimental methods are used because they may be more convenient or better suited for a particular material (viscoelastic solid or viscoelastic liquid) or because they provide data over a particular time range. A dynamic experiment at given frequency ω is qualitatively equivalent to the transient experiment at time t = 1/ω. Furthermore, it is often not easy to transform results from one type of linear viscoelastic measurement to another. Applications of each of these small strain methods and typical data for several rheologically different viscoelastic food materials will be considered below.
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8.5.2 SMALL STRAIN EXPERIMENTS
IN
219
SIMPLE SHEAR
8.5.2.1 Stress Relaxation after Sudden Strain In the stress relaxation test, an instantaneous deformation γ 0 is applied to a body. This can be done in shear, compression, or extension mode. Deformation or strain is maintained constant throughout the test, while the stress is monitored as a function of time: σ ( t ) = γ 0G ( t ).
(8.11)
Relaxation modulus G(t) is a time-dependent analog of equilibrium shear modulus of perfectly elastic solid G. For viscoelastic solid materials, the relaxation modulus decays to an asymptotic equilibrium modulus Ge. For viscoelastic fluid materials, the relaxation modulus decays to zero, usually after an extremely long time (Figure 8.4). The equation for the relaxation modulus as a function of time is usually expressed as the simplest modified Maxwell model describing a situation where the macromolecular system shows a very narrow profile of molecular weight distribution:
()
(
)
(
G t = Ge + G0 − Ge exp −t λ
)
(8.12)
where G0 is the initial modulus, Ge the equilibrium modulus, and λ is the relaxation time. In a real situation given the molecular polydispersity of a polymer tested, a group of n Maxwell elements in parallel represents a discrete spectrum of relaxation times, each time λi being associated with a spectral strength Gi (Ferry, 1980):
()
n
∑ G exp(−t λ ) . i
(8.13)
i
i =1
Relaxation modulus, G
G t =
Ge
Time, t
FIGURE 8.4 Stress relaxation after sudden strain. Solid line = viscoelastic solid; dotted line = viscoelastic fluid.
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Food gels, such as pectins, starch gel, thermally denatured globular proteins, and aggregated proteins from enzymatic or chemical action exemplify this kind of polydispersed viscoelastic solid system. The longest relaxation time of these systems tends to infinity, then the corresponding modulus contribution is the equilibrium modulus Ge. The stress relaxation experiment can be performed on both viscoelastic fluids and viscoelastic solids. 8.5.2.2 Creep and Creep Recovery Test Figure 8.5 gives a schematic representation of a typical creep and creep recovery experiment for a viscoelastic fluid. At time t = 0, a constant shear stress σ0 is applied to the sample and shear deformation γ is recorded as a function of creep time t long enough to reach a steady-state flow of material. At creep time t0, the stress is instantaneously set to zero and the recoverable part of deformation γr (t, t0) is measured as a function of creep recovery time. The combination of both experiments is called the retardation test. The time-dependent creep compliance in the linear viscoelastic regime can be calculated as
()
J t =
().
γ t
(8.14)
σ0
According to the linear viscoelasticity theory (Boltzmann superposition principle), the creep compliance J(t) can be written for polydispersed materials as a sum of instantaneous elastic compliance (glassy compliance Jg), discrete retardation spectrum 2N positive constants {τk, Jk} of a multiparameter Kelvin-Voigt model, and contribution of steady-state permanent viscous flow t η0:
()
J t = Jg +
N
∑ J (1 − exp ( −t τ )) + ηt k
k
(8.15)
0
k =1
where each retardation time τk is associated with a spectral compliance magnitude Jk; viscosity η0 is the steady-state viscosity. For food materials, Jg has a value of ~10–9 Pa–1 and can therefore be omitted. Assume that the real time of the experiment is long enough to reach a steadystate flow of material. According to Figure 8.5, for creep compliance at time t = t0 the following equation is valid:
( )
J t0 = Je0 +
t0 . η0
(8.16)
The Je0 is the steady-state compliance. As indicated in Figure 8.5, Je0 and η0 can be found graphically from creep test data analysis. The inverse of Je0 is a measure of total viscoelasticity of the material.
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FIGURE 8.5 Creep and creep recovery test of weak wheat gluten. (Adapted from PruskaKędzior, A., Application of Phenomenological Rheology Methods to Quantification of Wheat Gluten Viscoelastic Properties, The Agricultural University of Poznań Press, Poznań, 2006 [in Polish]. With permission.)
The recoverable compliance Jr(t, t0) (also called elastic compliance) follows from the creep or from the recoverable part of creep recovery experiments (see Figure 8.5) by subtracting the contribution of flow from total compliance
()
Jr t =
N
∑ J (1 − exp(−t τ )) . k
k
(8.17)
k =1
The retardation experiment is recommended for viscoelastic fluid materials such as bread dough, biscuit dough, vital wheat gluten, meat dough, meat stuffing, and pastry. 8.5.2.3 Dynamic Assay In dynamic assay a viscoelastic material is subjected to controlled sinusoidally variable excitation (stress or strain). Within the linear response of the material, variations of the noncontrolled variable (stress or strain) tend to become sinusoidal with a period identical with that of the controlled excitation, but with a different phase. This can be shown from the constitutive equation as follows: γ = γ 0 sin ω t
(8.18)
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(
σ = σ 0 sin ω t + δ
)
(8.19)
where γ indicates strain, σ the stress associated with it, ω is the angular frequency, and γ0 and σ0 are the amplitudes of the oscillations. The loss angle is the phase difference δ (ω) between stress and strain. The complex modulus is the complex number: G* =
σ0 exp iδ γ0
(8.20)
where i = −1 . The real part of the complex modulus is called the storage modulus G′, the imaginary part is called the loss modulus G″: G* = G ′ + iG ′′
(8.21)
G * = G ′ 2 + G ′′ 2 .
(8.22)
and
It is demonstrated that each dynamic measurement at a given frequency ω simultaneously provides two independent quantities G′ and G″ as well as tan δ, which is defined as tan δ = G ′′ G ′ .
(8.23)
The storage modulus G′(ω) is a measure of the mechanical energy stored and recovered per cycle of sinusoidal deformation, and the loss modulus G″(ω) is a measure of the energy dissipated per cycle as heat. G′(ω) is related to the elastic contribution to the reaction of a material on a deformation or stress, and G″(ω) is related to the viscous contribution. The data from sinusoidal experiments can also be expressed in terms of complex compliance: J * = 1 G* = J ′ − iJ ′′
(8.24)
where J′ is the storage, and J″ the loss compliance. The relationships among G′, G″, J′, and J″ are as follows: J′ =
G′ G ′ + G ′′ 2 2
(8.25)
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J ′′ =
G ′′ . G ′ 2 + G ′′ 2
223
(8.26)
Similarly, the complex viscosity η* of a viscoelastic material can be presented as η* = η′ – iη″. Functional relationships G*, G′, G″, η* = f(ω) represented graphically are referred to as mechanical spectra. Examples of viscoelastic food materials’ mechanical spectra as a functional relationship G′, G″ = f(ω) are shown in Figures 8.10 and 8.11 and are discussed in Section 8.7.3. From an industrial point of view, dynamic measurements provide knowledge about process-induced structure formation, which has to be considered for product formulation and process design. Dynamic measurements are required to follow the buildup and breakdown of structures as a function of the parameters of such processes as flow, deformation, heating, and cooling as well as combinations thereof.
8.6 NONLINEAR VISCOELASTICITY—NORMAL STRESS DIFFERENCES Much labor has been expended in the determination of the linear response of materials. There are many reasons for this. First, there is the possibility of elucidating the molecular structure of materials from their viscoelastic response. Second, the material parameters and functions measured in the relevant experiments often prove to be useful in industrial quality control. Third, a background in linear viscoelasticity is a helpful introduction to the much more difficult subject of nonlinear viscoelasticity. However, for some technological situations only a nonlinear viscoelasticity approach can bring satisfactory elucidation of processed material properties. Nonlinear viscoelasticity is a phenomenon in which shear flow normal stress appears significant. When a viscoelastic material is sheared between two parallel surfaces at an appreciable rate of shear, in addition to viscous stress σ12, there are normal stress differences N1 ≡ σ11 – σ22 and N2 ≡ σ22 – σ33. Here “1” is the flow direction, “2” is the direction perpendicular to the surfaces between which the fluid is sheared, and “3” is the neutral direction. The larger of the two normal stress differences is N1, and this difference is responsible for the processed material’s rod-climbing effect, termed the Weisenberg effect. For example, when cake batter is mixed with an egg batter, the material climbs up the rod rotating within it. Normal stress difference also appears important when food sensory properties are considered. It has been hypothesized that normal stresses generated by oral movements of semisolid food are sensed by the mouth and contribute to the perception of thickness. This implies that the actual force applied in the mouth to the semisolid food is equal to the force applied by the tongue muscle, say F0, minus the force representing the normal stress difference N1, exerted by the food (Terpstra et al., 2005). For isotropic materials, N1 has always been found to be positive in sign (unless it is zero). In a cone and plate rheometer, this means that the cone and plate
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surfaces tend to be pushed apart. N2 is usually found to be negative and smaller in magnitude than N1; typically the ratio –N2 / N1 lies between 0.05 and 0.3 (Macosco, 1994). At sufficiently low shear σ12 usually becomes linear in the shear rate, γ ; that is, the shear viscosity η ≡ σ12 γ becomes independent of γ . Similarly, N1 and N2 approach the limits, N1 ∝ γ 2, N 2 ∝ γ 2 at small γ , and thus the normal stress coefficients:
ψ1 =
σ11 − σ 22 γ 2
(8.27)
ψ2 =
σ 22 − σ 33 γ 2
(8.28)
approach constant values at a small γ . When the Deborah number is vanishingly small, the fluid can be described as Newtonian. When the Deborah number is small but not negligible and the flow is steady or near steady, nonlinear effects are weak and can be described by the secondorder fluid (Figure 8.1). The second-order fluid equation predicts the existence of normal stress differences in shearing (Ferguson and Kemblowski, 1991).
8.7 RHEOLOGICAL PROPERTIES OF FOOD MACROMOLECULAR SYSTEMS 8.7.1 FLOW BEHAVIOR
OF
MACROMOLECULAR SOLUTION
8.7.1.1 The Concentration Regimes Effect Important factors determining the rheological behavior of macromolecules are their size, shape, and flexibility. An appropriate macromolecular chain stiffness parameter is the ratio of the contour length L of the chain to the length b of the statistical segment unit comprising n monomers (Lefebvre and Doublier, 2004). A ratio L/b > 10 would be required for the polymer conformation to be regarded as a coil. Three concentration domains can be distinguished in solutions of polymers: dilute regime (c < c*), semidilute regime (c* < c < c**), and concentrated regime (c > c**) (Figure 8.6). In a very dilute solution, the volume available to each polymer molecule is much higher than that of the individual coil. The coils remain statistically far from each other, and encounters bringing them into contact are infrequent. The coils maintain the dimensions of an isolated chain. In this low-concentration region, the hydrodynamic forces can be neglected. Only the Brownian motion is acting against structural forces. This situation prevails up to the critical overlap concentration c*, semidilute regime, at which the coils fill the volume of the solution. When the polymer concentration is increased above c*, there is a progressive interpenetration of the coils,
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225
106 105 104
ηsp
103 102 101
c**
100
c*
10-1 10-2 10-2
10-1
100
101
102
c [η] FIGURE 8.6 Specific viscosity: the reduced concentration master curve for guar and hydroxyethyl cellulose solutions at 25°C. Guar = empty circles, five samples differing in molecular weight (450 ≤ [η] ≤ 1250 mL/g). Hydroxyethyl cellulose = filled triangles ([η] = 807 mL/g). The lines show the slopes 1.2 and ~5, relative to the dilute and to concentrated regimes, respectively. (Adapted from Lefebvre, J. and Doublier, J.-L., Rheological behavior of polysaccharides in aqueous systems, in Dumitriu. S. (Ed.), Polysaccharides: Structural Diversity and Functional Versatility, 2nd ed., Marcel Dekker, New York, 2004. With permission.)
concomitant with a contraction of their individual volume. The solution becomes a transient network of entangled chains. In a semidilute regime the coils still retain some degree of individuality. At certain concentrations (c** > c*) the polymer solution becomes an entanglement network where the chains have completely lost their individual character. Once the entanglement network forms, the only characteristic length in the system is now the mesh size of the network, which decreases as concentration increases, tending toward its limit value b in the polymer melt state. When low-energy interactions develop between chains in the regions of entanglements, the possibility of forming some junction zones appears. Junction zones exhibit lifetimes much longer than those of entanglements. The system has then shifted from the state of an entangled solution to that of a physical gel. Food polysaccharide solutions, for instance pectins, guar gum, locust bean gum, carob gum or carrageenans, and food proteins like whey proteins, bovine serum albumin, ovalbumin, collagen, actin, or myosin as well as polysaccharide-protein mixtures exhibit this dependence of the molecule’s state on solution concentration. 8.7.1.2 Viscosity of Dilute Solution The intrinsic viscosity of the macromolecule can be extracted from the viscosity measurement of the dilute macromolecular solution. The term intrinsic viscosity can be intuitively misleading. In fact, it is not a viscosity at all, but actually a measure
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TABLE 8.3 Intrinsic Viscosity of Some Polysaccharide Solutions
Material Starches of varying amylose content (solubilized in 0.2 M KOH) Amylopectin Waxy maize Potato Wheat Pea Amylose Galactomannans—tara gum (Caesalpinia spinosa) Galactomannans—locust bean gum (Ceretonia siliqua L.) crude purified Cassia javanica galactomannans Wheat water-soluble arabinoxylans (pentosans) Carboxymethylcellulose
Temperature (°C)
Intrinsic viscosity [η] (mL/g)
Reference Lourdin et al., 1995
25 25 25 25 25 25 25
121 121 270 210 250 161 1646
Sittikijyothin et al., 2005
25 25 25 25
1103 1496 1160 220–560
Goncalves et al., 2004 Dervilly-Pinel et al., 2004
25
675–5852
Kulicke et al., 1996
of the hydrodynamic volume of the coil in the case of noncharged polymer chains, or of the asymmetry of the particle in the case of rigid macromolecules. Most polymer solutions show non-Newtonian behavior as a result of the deformation and of the orientation of the polymer coil in flow. Therefore, the intrinsic viscosity is shear rate dependent. For practical reasons, in this section, viscosity and intrinsic viscosity will be referred to as measurements performed within the Newtonian domain, that is, at shear rates low enough for the dilute solution to display shear rate-independent viscosity. Therefore, the intrinsic viscosity is defined as η η = lim sp c →0 c
(8.29)
and is expressed in mL/g. The quantity ηsp = (η – η0)/η0 is called the specific viscosity of a solution. The specific viscosity is the ratio of the difference between the viscosity of the solution and that of the pure solvent under identical physical conditions. Table 8.3 shows examples of the intrinsic viscosity of some macromolecular food solutions. The relationship between intrinsic viscosity and the molecular weight of polymerlike macromolecules is usually expressed in the form of the empirical MarkHouwink equation: η = KM vis a
(8.30)
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where K and a are empirical parameters depending on the polymer and solvent properties and on the temperature; both are related to chain stiffness. M vis is the viscosity average molecular weight. The value of the exponent a gives information about the general conformation of the polymer. For flexible linear chains, a typically assumes values between 0.5 and 0.8. For instance, food galctomannans and arabinoxylans show a = 0.72 and 0.74, respectively, being characteristic of a random coil. Stiff chains display larger values of a, for example, values as high as 1.8 are reported for rodlike chain conformations. On the other end of the scale, a < 0.5 indicates some degree of coil collapse in the case of a linear chain (Lefebvre and Doublier, 2004). 8.7.1.3 Viscosity of Semidilute and Concentrated Solutions Flow behavior of semidilute and concentrated solutions is illustrated in Figure 8.6, where the data relative to two polysaccharides, guar gum and hydroxyethyl cellulose, differing in structure and in molecular weight, are plotted as specific viscosity ηsp versus reduced concentration c[η] (Lefebvre and Doublier, 2004). Specific viscosity reflects changes not only in conformation and size, but also in intermolecular interactions (association, aggregation), and in the physical state of the system. For c > c*, polymer solutions become non-Newtonian at moderate and even low shear rates; the viscosity values to be considered as those corresponding to low-shear Newtonian viscosity (η0), which are related to the equilibrium state of the solution at rest. The master curve (Figure 8.6) can be divided into three regions, limited by the critical concentrations c* and c**. Below c[η] ~ 1, that is, c < c*, the curve is linear, with the slope nd ~ 1.2. This is the dilute regime, where the increase in ηsp is attributable to the hydrodynamic interaction between polymer coils behaving as independent impermeable spheres. Above c = c*, the semidilute regime is reached. Progressive coil contraction and increasing entanglement density govern the rheological behavior of the solution, provided their molecular weight M > Mc. At the upper limit of the semidilute regime, the solution is about 100 times more viscous than the solvent because of the increase of entanglement density. This part of the curve is often approximated by a line segment with a slope nsd ~ 2.5. Finally, in the concentrated regime (c > c**), the experimental data can be fitted with a line segment with a slope nc ~ 4 to 5. Some differentiation in the values of c*[η] and c**[η] and shifts on the reduced viscosity scale as well as differences in the values of the slope and broadness of the semidilute regime c**/c*, because of differences in polymers’ structures (flexibility) and in interactions with the solvent were observed (Launay et al., 1997). 8.7.1.4 Shear Dependence of the Viscosity The non-Newtonian shear behavior is typical for polymer melts and polymer solutions at concentration c > c*. This kind of behavior is schematically illustrated by the flow curve in Figure 8.7, where the steady-state viscosity η is plotted versus the shear rate γ on bilogarithmic scales. Below a critical shear rate value, γ crit , the flow curve shows the low-shear Newtonian plateau, where the viscosity holds a constant value η0. Above critical shear rate value γ crit , a shear-thinning region (where the
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102
η (Pa·s)
101
Low-shear Newtonian viscosity
η0
100 Power law region Critical shear rate
High-shear Newtonian viscosity
10-1
η∞ 10-2 10-3
10-2
-1
10 γ·0
100
101
102
103
· γ (s–1)
FIGURE 8.7 Flow curve illustrating three regions of the typical macromolecular solution shear thinning behavior.
viscosity decreases as shear rate increases) follows. In this region a power law relation η ∝ γ − n accurately fits the data with usually n ~ 0.6 to 0.8. At high shear rates, the viscosity tends to a second plateau, η∞. Due to instrumental limitations or flow instability, this high-shear Newtonian plateau is rarely observed experimentally, and then in most cases, can be neglected. The flow curve of a concentrated polymer solution reflects the effect of shear rate on entanglement density. If γ is low enough, the system remains in its equilibrium fully entangled state ( η = η0 ). As γ increases, the entanglement density and the viscosity decrease. Each shear rate value corresponds to a given entanglement state resulting from the balance between the flow-induced disentanglement and reentanglement processes governed by Brownian motion. Concentrated solutions are viscoelastic by nature; therefore, they do not respond instantaneously to changes in shear rate or stress value. The constant equilibrium viscosity is reached at a given shear rate only after a long enough time, sometimes even after a few days. Many equations describing the shear rate dependence on viscosity of polymer solutions and melts have been proposed. The most applied expression is the Cross equation (Section 8.4.2). In the Cross model, the characteristic value of shear rate γ 0 obtained as the abscissa of the intersection of the line representing the low-shear Newtonian viscosity plateau η0 with the line fitted to the data of the power law region of the solution flow curve (see Figure 8.7.). The inverse of γ 0 gives the characteristic relaxation time of the solution λ sol = 1 γ 0 . The value γ 0 is taken instead as γ crit , because γ crit is difficult to determine in practice (Launay et al., 1997). Characteristic parameters η0 and λ sol depend on the nature of the polymer, its molecular weight, its concentration, and on temperature. They may change by several orders of magnitude from one system to another.
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100
η/η 0
η (Pas)
100
10–1
10–2
10-1 101
102
103
10-3
10-2
10-1
100
101
γ⋅ / γ⋅ 0
γ⋅ (1/s)
FIGURE 8.8 Flow curves of wheat water-soluble arabinoxylans (2% water solutions) (left) and the master curve (right). (Adapted from Pruska-Kędzior, A., Kędzior, Z., Michniewicz, J., Lefebvre, J., and Kołodziejczyk, P., in Fischer, P., Marti, I., and Windhab, E.J. (Eds): Proceedings of 3rd International Symposium on Food Rheology and Structure ISFRS—Eurorheo 200301, Laboratory of Food Processing Engineering, ETH, Zurich, 2003, p. 177. With permission.)
The flow curves of the polymer solution extend most often along several orders of magnitude of shear rate and of viscosity. Examples of flow curves described with the Cross model, representing a large range of variability of observed flow properties of a polysaccharide are presented in Figure 8.8. Despite large differences in viscosity, presented macromolecular solutions show the same type of behavior, which is proved by the flow master curve. The master curves are very useful in combining the data for different molecular weights, concentrations, temperatures or solvents. A master curve simplifies treatment of data for different systems and conditions. Discrepancies from the master curve allow determination of differences in polymer chain structures. Master curves are built either by graphically superimposing the individual flow curves η γ , by shifting them along the viscosity and the shear rate axes, or are calculated by using the reduced variables η/η0 versus γ γ 0 . For instance, a “universal” flow curve for polysaccharides has been thus published. This curve combines the data for a large number of different polysaccharides fitted with the Cross model (η∞ omitted) with n = 0.76 (Morris, 1990).
()
8.7.2 LINEAR VISCOELASTIC PROPERTIES SYSTEMS
OF
FOOD POLYMER
In the linear domain, viscoelastic behavior is a kind of “fingerprint” of the intact material microstructure, which responds in different way over a certain time scale without being changed. Mechanical spectra of viscoelastic materials differ qualitatively and quantitatively according to the nature of the sample tested. Almost all the data based on rotational shear rheometry have been obtained through small strain dynamic measurements carried out over a narrow, physically feasible frequency range, which practically extends from 0.001 to 200 rad/s. Thus, the experimental window frames only a section of the mechanical spectrum limited by the oscillation
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frequency. Therefore, within the same frequency window, only a part of viscoelastic response can be observed and rheological behaviors of the media often remain poorly characterized. Enlargement of the frequency window (time scale) can be easily achieved in the case of a polymer solution or melt displaying a thermorheologically simple behavior, that is, a case in which there is similar relaxation behavior with changes in temperature. The method involves application of the time-temperature superposition principle (TTS) to dynamic data obtained at different temperatures over a restricted frequency range. According to the principle, mechanical spectra are carried out at various constant temperatures, and then superimposed using a horizontal shift factor (aT) to form a single master curve. This principle was originally introduced by workers in synthetic polymers (William-Landau-Ferry [WLF] equation; Ferry, 1980). The TTS principle is only valid if its underlying assumptions are met. The first assumption is that a direct relationship between time (oscillation frequency) and temperature exists. Second, the polymer structure at the physical and molecular level is assumed to remain constant over the experimental temperature range. This assumption is fulfilled in entanglement polymer systems. An example of a typical mechanical spectrum based on TTS principle data of an entangled polymer melt is shown in Figure 8.9. Storage G′ and loss G″ moduli as well tan δ are plotted as a function of frequency ω (or time t = 1/ω) on bilogarithmic scales. In this case the spectrum extends over 16 frequency decades covering the entire range of rheological responses shown by viscoelastic liquids. The mechanical spectrum (Figure 8.9) comprises four distinct parts based on the intersection (crossover) of G′ and G″ moduli curves, which correspond to values of tan δ equal to one. First, in the low-frequency region viscous flow is predominant. This is a terminal region. In this region G′ and G″ are proportional to ω2 and ω, respectively, with G″ > G′. Slopes 2 and 1 in the terminal region are those expected for any linear viscoelastic liquid (Ferry, 1980). In the terminal region, a longestrange molecular motion (slow dissipative process) occurs. In the second zone, a viscoelastic (rubbery) plateau appears where G′ is nearly flat and G″ becomes smaller than G′, so that the plateau is delimited by moduli crossover in two points. In this region transient viscoelastic networked structure exists due to physical effects (topological entanglement or particle gel). In a plateau zone, G′ changes relatively slowly while G″ and the loss tangent (tan δ) pass through their minima. The energy losses are small here because the period of oscillation is long compared with the longest relaxation time of an entanglement network strand. However, it is short compared with any relaxation times for motion involving entanglement or junction zone slippages or disruptions. This characteristic plateau develops up to a high frequency region where G′ and G″ follow dependence on ω1/2. The third region is called the transition zone, at which the viscous response again becomes dominant (G″ > G′). In the transition zone, the viscoelastic behavior is a result of short scale time motions of segments of the chain, which are shorter than the distance between entanglements (so-called fast dissipative processes). Finally, at very high frequencies, the moduli cross over for a third time, entering the glassy zone. Glassy behavior corresponds to very high frequencies, so no
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1010
231 Transition region
Terminal region
Viscoelastic plateau
Rheological Properties of Food Systems
Glassy zone 106
104
1
102
102
10–2
tan δ
G' G" (Pa)
106
100 2
10–6 10–16
10-2 10–12
10–8
10–4
100
ω (rad/s)
FIGURE 8.9 Example of mechanical spectrum of entangled polymer system over a wide frequency range. (Adapted from Lefebvre, J. and Doublier, J.-L., Rheological behavior of polysaccharides in aqueous systems, in Dumitriu. S. (Ed.), Polysaccharides: Structural Diversity and Functional Versatility, 2nd ed., Marcel Dekker, New York, 2004. With permission.)
configuration rearrangements of the polymer backbone chain have time to take place. Energy dissipation occurs only through limited local motions. The polymer behaves as an amorphous or semicrystalline solid.
8.7.3 MECHANICAL SPECTRA
OF
FOOD POLYMER SYSTEMS
Rheological examination of the viscoelastic behavior of food polymers is limited experimentally to the finite range of the viscoelastic spectrum, but rheological properties of food polymers may span many orders of magnitude. No single experiment can cover the entire range. The application of the TTS principle to food biopolymers and real food systems is usually strongly restricted to a very narrow temperature range due to irreversible conformational changes of biological macromolecules at elevated temperatures, as protein denaturation, starch gelation or even starch (nonstarch) polysaccharides melt (Figure 8.13). Therefore, the extended mechanical spectra of these materials obtained using the TTS principle are also limited. Combining dynamic measurements with transient tests allows us to partially encompass this difficulty (Ferry, 1980; Tschoegl, 1989). For example, data of a retardation test can be converted from time domain to frequency domain and combined with dynamic test data. This permits us to significantly extend the mechanical spectrum of a material down to lower frequencies than those accessible
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104
G’,G" (Pa)
103
102
101
100 10–6
10–5
10–4
10–3
10–2
10–1
100
101
102
103
ω (rad/s) FIGURE 8.10 Example of the composite mechanical spectrum obtained by combining dynamic data and retardation test data converted according to the Kashta method. Material = wheat gluten obtained from a milling stream flour. Solid circles = G′, empty circles = G″, data converted from retardation test: solid line = G′, dashed line = G″. (From Pruska-Kędzior, A., Application of Phenomenological Rheology Methods to Quantification of Wheat Gluten Viscoelastic Properties, The Agricultural University of Poznań Press, Poznań, 2006 [in Polish]. With permission.) 104
G', G" (Pa)
101
Ge
G'
GN0
100
103
G" ↓
10–1
0 10–2
102 10–3
10–2
10–1
100
ω (rad/s)
101
102
10–3
10–2
10–1
100
101
102
ω (rad/s)
FIGURE 8.11 Mechanical spectra of chemically cross-linked gel (left, wheat water-soluble pentosans, 2% concentration) and physical gel (right; native wheat gluten).
for direct measurement in dynamic mode. Then, often lower crossover of moduli G′ and G″ and the beginning of the spectrum’s terminal region can be observed (Figure 8.10) (Lefebvre et al., 2003). Many polysaccharides, such as agar, κ-carrageenan, guar, gellan, locust bean gum, xanthan, and proteins such as bovine serum albumin or gelatin, polysaccharide–polysaccharide mixtures or polysaccharide–protein mixtures are commonly being used as structuring agents in the aqueous phase of emulsion food products and form gel matrices in many semisolid food products.
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Generally, mechanical spectra of protein and polysaccharide solutions obtained in the frequency range of 10–3 to 102 rad/s are restricted to the terminal region of the mechanical spectrum (see Figure 8.9). The study of the viscoelastic behavior of a dilute polysaccharide or protein solution is difficult and requires special instrumentation. Such studies have seldom been attempted. An exception is xanthan gum. Very high molecular weight and intrinsic viscoelasticity of this polysaccharide make this type of investigation available even at concentrations as low as 50 ppm (Lefebvre and Doublier, 2004). A true polysaccharide solution, in general, cannot be directly prepared at concentrations greater than about 2.5%. The master curve G′(ω) and G″(ω) can be built by a graphical shift procedure using different concentrations, as it can be carried out for the master flow curve. The experimental frequency window, extending over three logarithmic decades, encompasses the transition from the terminal region to the very beginning of the plateau region, stopping just beyond the first G′, G″ intersection (Figure 8.9). From certain threshold polysaccharide or protein solution concentrations, these macromolecules are able to form networked gel structures. Chemical cross-linking reactions or physical interactions can drive the gelation processes. In the case of chemical gelation of polymers, the process is directly controlled by the stoichiometry of the cross-linking. A classical example of chemical covalent gelation of macromolecules important in foods is transglutaminase-mediated crosslinking of proteins via isopeptide bonds and peroxidase-mediated cross-linking of water-soluble pentosans (arabinoxylans) via diferulic bonds. Chemical covalent gelation is irreversible in terms of sol-gel transition under physical action. All other food gels are considered as physical gels and can be subdivided into entanglement gels and particle gels. The entanglement network is formed by the simple topological interaction of polymers. They occur either in the melt or in solution when the concentration and molecular weight (Mr) becomes greater than some critical entanglement molecular weight Me (Ferry, 1980; Doi and Edwards, 1986). Their mechanical spectra in a usually accessible frequency range encompass a transient viscoelastic plateau (Figure 8.9). The entanglement-like structure of some polysaccharide gels, such as gellan in the presence of co-solutes (sucrose and corn syrup), follows the time-temperature superposition (TTS) principle. A single mechanical spectrum of 0.5% gellan gel constructed using TTS in a temperature range of 15 to 85°C covers a wide oscillatory frequency window in a plateau regime. Gelling polysaccharides modeled previously by TTS include κ-carrageenan and glucose, locust bean gum and sucrose, and gellan and glucose systems (Nickerson et al., 2004). Physical particle gels are structured due to the formation of junction zones with a finite energy and lifetime. Formation of junction zones is always associated with macromolecular ordered secondary structure. Relatively weak forces, like hydrogen bonds driving the formation of individual junction zones cause the structure of a physical gel to be strongly dependent on physical factors such as temperature, mechanical excitation, and time. Beta-lactoglobulin, bovine serum albumin, and collagen proteins represent models of simple protein systems gelling due to this mechanism, and wheat gluten represents the most complex networked system involving this mechanism.
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Mechanical spectra of chemical and physical gels differ significantly qualitatively. From a rheological point of view, chemical gel is a viscoelastic solid, and physical gel is a viscoelastic liquid. These differences are exemplified in Figure 8.11 showing the mechanical spectra of a chemical gel (2% wheat water-soluble pentosans gel) and wheat gluten as an example of a physical gel-like structure. The mechanical spectra of these two types of gels, recorded in dynamic measurements at the same range of oscillation frequency, encompass a part of a viscoelastic plateau. For a chemical gel, the G′ curve is almost flat (true rubbery plateau) tending toward an equilibrium modulus Ge at certain low oscillation frequency values, the largest relaxation time becomes infinite. Modulus G″ theoretically tends toward zero at some infinite time (infinitely low oscillation frequency). For a physical gel, G′ decreases with a decrease in oscillation frequency tending to some equilibrium state, while G″ passes through a minimum and tends, at lower oscillation frequencies, to cross over with G′; in the case shown in Figure 8.11, the crossover point is beyond the measurement frequency window. At frequencies below the crossover (in the terminal region) the material enters the steady-state permanent flow. Rheological properties of viscoelastic liquids and viscoelastic solids can be quantified with characteristic parameters. From mechanical spectra viscoelastic modulus, GN0 = 1/JN0, a quantitative parameter of the viscoelastic plateau for viscoelastic liquid and equilibrium elastic modulus, Ge, for the viscoelastic solid can be calculated. Some examples of moduli and characteristic parameters of food viscoelastic materials are shown in Table 8.4 and Table 8.5, respectively.
TABLE 8.4 Rheological Parameters of Some Viscoelastic Food Materials Measured at a Given Frequency
Material Cream cheesea Cream cheese lighta Caramelb
Egg whiteb (15% w/w protein conc.) Cooled from 90°C Xanthan cryogel (0.5%) c
Temp. °C 18 18 10 50 80 20 90 20 5
Yield stress σy Pa 7300 1300
Complex viscosity η* Pa ⋅ s 1588 292
16.1
Storage modulus G’ Pa
Loss modulus G” Pa
155739 28438 19324 434 108 < 0.01 12.2
30603 6787 12968 281 42
73.4 99.8
At frequency ω = 100 rad/s. At frequency ω = 1 rad/s. c Cryogelation at –20°C, values of moduli at frequency ω = 6.28 rad/s. a
b
Reference Kealy, 2006 Ahmed et al., 2006 Ngarize et al., 2004
15.5
Giannouli and Morris, 2003
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TABLE 8.5 Characteristic Rheological Parameters of Some Networked Food Systems Material WPI a,c WPI + 0.68% GM b WPI + 1.19% GM WPI cooled after heating at 80°C WPI a,c WPI + 0.68% GM b,d WPI + 1.19% GM d Wheat gluten Wheat gluten Wheat water-soluble pentosans gel (1) d Wheat water-soluble pentosans gel (2) d Wheat water-soluble pentosans gel (1) e Wheat water-soluble pentosans gel (2) e
Temp. °C
GN0 Pa
ω0 rad/s
80 80 80
820 320 230
— 150 14
— 0.5 0.5
20 20 20 20 20 20 20
1500 1300 770 3330 1721 244.3 11.9
100 4 0.20 2.37 0.68 62
0.37 0.37 0.32 0.45 0.58 0.67
20
13.5
25
20
12.8
100
20
14.9
50
n
Reference Goncalves et al., 2004
Lefebvre et al., 2000 Pruska-Kędzior et al., 2005 Pruska-Kędzior et al., 2003
0.61
β-lactoglobulin gels (11% w/w). GM—Cassia javanica galactomannans. c G′ at G″ minimum. d G 0 = viscoelastic plateau modulus; ω = characteristic frequency of loss peak; n = spread parameter N 0 related to the loss peak broadness—Cole-Cole fit. e Equilibrium modulus G , Winter and Charbon correlation. e a
b
The steady-state parameters of retardation test: compliance Je0, viscosity η0, and the longest retardation time τmax = Je0η0, are quantitative parameters of the lower end of the viscoelastic plateau for viscoelastic liquid (see Figure 8.5).
8.8 STRUCTURE–RHEOLOGY RELATIONSHIP IN MULTIPHASE FOOD MATERIALS The complex multiphase food systems are composed of dispersed and continuous phases. Liquid droplets compose the dispersed phase of emulsions, and solid particles form the dispersed phase of fine suspensions. Gas bubbles are usually present in dispersed phases and they represent an important volume fraction in aerated food products. The continuous phase also shows its inner structure due to the presence of macromolecules in colloidal dispersion. Physical and chemical interactions and bonding govern stabilization of the multiphase systems at rest. When these systems start to flow, their structure changes and reaches different equilibrium states depending on the shear rate (Figure 8.12).
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Chemical and Functional Properties of Food Components Dispersed Phase Cont. Ph. Ph. Dispersed Phase Cont.
η
Viscosity function for multiphase liquid systems
Yield stress σ ?0
Dispersed Phase Dispersed Phase
Cont. Ph. Cont. Ph.
Cont. Cont.
Dispersed Dispersed
η0
Dispersed Phase Cont. Ph. Dispersed Phase Cont. Ph. Dispersed Phase Dispersed Phase
η∞
Cont. Ph. Cont. Ph.
Structural forces
Hydrodynamic/viscous forces
γ⋅ 0
γ⋅ ∞
γ⋅
FIGURE 8.12 Shear rate dependency of multiphase food materials’ structure and rheological properties. (From Windhab, E.J., in Beckett, S.T. (Ed.), Physico-Chemical Aspects of Food Processing, Chapman and Hall, London, 1996. With permission.)
In the domain of the lowest shear rates (γ⋅ < γ⋅ 0) only the Brownian motion is acting against the structural forces. If the concentration of the dispersed phase, or concentration of the macromolecules in the continuous phase, is high, their strong interactions generate a yield value σy and the system shows plastic behavior. For low enough concentrations of structuring components, the viscosity η0 is independent of the shear rate (lower Newtonian regime). In the region of moderate and higher shear rate values (γ⋅ > γ⋅ 0) , hydrodynamic forces become of the same order of magnitude as the structural forces. This induces new structures, and if the time these shearing forces act is long enough, a new equilibrium structure is attained. Dispersed phase droplets or bubbles submitted to shear-induced deformations change their initial shape. Dispersed phase solid particles undergo ordering and aggregation–disaggregation processes. The continuous phase also changes its inner structure due to changes, rearrangements, and ordering of inter-macromolecular entanglement. The shear-induced state of arrangement and orientation of the system is unstable. Therefore, if this new structure or viscosity state is required for the product quality or for further processing, it must be fixed by physical or chemical means (for example, fixation of fine grain emulsions). The significance of determining the viscoelastic properties of the macromolecular components of food, such as starch and nonstarch polysaccharides and proteins, appears obvious when their state diagram is considered. Depending on the degree of macromolecular system hydration and its temperature, the system properties span from the glassy state to the flow state, passing through the viscoelastic (rubbery) state.
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FIGURE 8.13 State diagram of wheat starch and gluten proteins in the baking process. (From Cuq, B., Abecassis, J., and Guilbert, S., State diagrams to help describe wheat bread processing, Int. J. Food Sci. Technol., 38, 739–766, 2003. With permission.)
Figure 8.13 shows an example of a real technological process situation involving the degree of hydration and temperature evolution exemplified by the state diagram of wheat starch and protein in the baking process. Three states are distinguished on the state diagram: glassy, rubbery, and flow. Depending on the temperature and type of macromolecules, their properties in the rubbery state change from viscoelastic liquid to viscoelastic solid. At room temperature, structural changes of starches and proteins range from the glassy state (amorphous solid) for flour (approximately 14% water) to the viscoelastic rubbery state for dough (80% water). During baking, water content decreases from approximately 80% in dough to approximately 40% in the crumb. Meanwhile, temperature rises from approximately 30°C for dough to crumb final temperature in the oven of approximately 96°C. Next, the crumb is cooled down to room temperature. Proteins and starch behave differently following this water content and temperature evolution. Protein systems pass from viscoelastic liquid properties shown by the native gluten network, below protein thermal setting temperature, through viscoelastic behavior of the denatured gluten protein network at approximately 96°C (stabilized significantly by hydrophobic bonds that have replaced destroyed hydrogen bonds) to viscoelastic solid after cooling to room temperature. Starch granules behave as rubbery neo-Hookean bodies below gelation temperature, and act as a neutral filler of a viscoelastic gluten network. Above gelation temperature, swollen starch granules lose their integrity, and the liquid mixture of amylose and amylopectin enter the flow state above the starch melting point. Once the water content in the crumb diminishes, gelated starch reaches viscoelastic liquid properties at approximately 96°C, and then becomes a viscoelastic gel after the crumb cools to room temperature.
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8.9 ELASTIC SOLID 8.9.1 INTRODUCTION For solids, the fundamental relationship between force and deformation is Hooke’s law: σ = Gγ
(8.31)
where σ is a force per unit area (the stress), and γ is the relative length change or strain in shear. G is a constant of proportionality called the elastic modulus. This is an intrinsic property of solids. Hooke’s law is the basic constitutive equation of classical solid mechanics. Frequently, if a solid sample is subject to uniaxial tensile force (in extension or compression), deformation is described in terms of strain ε and the ratio of change in length to undeformed length l0: ε=
l − l0 l0
(8.32)
where l0 is the original length of the test material sample. Then Hooke’s law is written as σ11 = E ε
(8.33)
where σ11 is tensile (extensional) stress, perpendicular to the surface it acts upon, and E is called tensile or Young’s modulus. In the limit of the small strain region, the tensile modulus is three times that measured in shear for incompressible, isotropic materials, E = 3G. For compressible, isotropic materials, a parameter µ (Poisson’s ratio) is required to relate the tensile to shear modulus [E = 2G (µ + 1)], where µ ranges from 0.5 for the incompressible case to 0.
8.9.2 NONLINEAR BEHAVIOR
OF A
SOLID
Figure 8.14 explains the principle of rubbery material behavior in tension and in compression. It shows that for a small deformation near zero, the stress is linear with deformation, but at larger deformations, the stress becomes nonlinear and different from that predicted by Hooke’s law. These materials are described as neo-Hookean. In simple shear, the neo-Hookean model predicts the first normal stress difference that increases in square with strain (compare a second-order liquid, Equation 8.13): σ11 − σ 22 = G γ 2 .
(8.34)
Only one normal stress difference N1, for the neo-Hookean solid in shear exists (N2 = 0).
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25 (MPa)
ne oHo ok ea n
Tensile stress
20 15 10
Compressive stress
5
w
e’s la
Hook
0 –5 –10 0
2
4
6
8
Strain
FIGURE 8.14 Elastic response of a rubberlike body. (Adapted from Macosco C.V., Rheology: Principles, Measurements and Applications, VCH Publishers, New York, 1994. With permission.)
TABLE 8.6 Young’s Modulus of Some Food Solids Material High amylose maize starch foam a Waxy maize starch foam a Cassava starch film without plasticizers Cassava starch films with plasticizers Tomato powder c Bread crumb a Bread crumb b Soy protein isolate gel e Cheddar cheese Avocado pears—0 days postharvest Avocado pears—10 days postharvest a
Temperature (°C)
Young’s modulus E (MPa)
Ambient Ambient 25 25 21.5–79.2 d Ambient Ambient 20 20 25 25
24.7 16.5 1200–2800 <1200 100–1000 0.132–0.851 0.280–0.440 0.0039–0.0042 0.4–1.79 480 48
Foam or crumb cell wall material, Young’s modulus. Foam or crumb Young’s modulus related to material density. c Young’s modulus at the glass transition temperature. d Depending on the powder dry matter content (89–96% dm). e Depending on the pH and ionic strength. b
Reference Lourdin et al., 1995 Mali et al., 2005 Palzer, 2005 Zghal et al., 2002 Renkema, 2004 Hort et al., 1997 Baryeh, 2000
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The neo-Hookean model has been applied to large strain deformation problems. In a number of polymer-processing operations, such as blow molding, film blowing, and thermoforming, deformations are rapid and the polymer melt behaves more like a cross-linked rubber than a viscous liquid. In many real situations, as the time scale of experiment is shortened, the viscoelastic liquid looks more and more like a neoHookean solid; thus in many cases of rapid deformations the simplest and often most realistic model for the stress response of these polymeric materials is the elastic solid. Meat protein elastin is considered as a neo-Hookean elastic protein. Many real food materials, from fruits and vegetables, to cheese, meat, and sausages, can be considered as neo-Hookean. Some examples of Young’s moduli E values of food materials are shown in Table 8.6.
8.10 SUMMARY The main aspects of practical applications of rheological methods based on shear are summarized in Figure 8.15. Large deformation tests are dedicated to designing industrial processes involved in food pipeline transport, mixing, stirring, or centrifugation. These tests are also applied to food production online quality control Rheology of food macromolecules and colloid dispersed particles in shear
Large deformation tests
Steady-state flow behavior test
.
Viscosity η(γ) Yield stress σ y . Thixotropy η(γ,t) Normal stress N1, N2
Small deformation tests
Storage modulus G'(ω) Loss modulus G"(ω) Dynamic viscosity η'(ω) Phase angle δ (ω)
Application
Flow Pumping Mixing Stirring
Transient test
Dynamic steady shear test
Food quality control Food stability Food structure
Relaxation test
Creep and creep recovery test (retardation test)
Relaxation modulus G(t) Relaxation time λ(t)
Compliance J(t) Retardation time τ(t)
Application
Microstructure Particle size and shape Morphology
Food quality control Food stability Food structure
FIGURE 8.15 Rheological methods applied to investigation of food materials.
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considering its structure formation or preservation. Small deformation tests are useful particularly for raw material characterization and designing rheological properties of formulated final products in the research and development stage when their microstructure, including macromolecular, has to be described. These tests can also be applied in industrial quality control from the point of view of food product structure and stability control, including texture and sensory appreciation. However, existing rheological methods still do not answer all demands, while properties of food macromolecular systems are considered and new techniques are developed. Optorheological methods involving direct circular dichroism and birefringence measurements during material mechanical excitation are introduced for transparent systems. Rheoacoustic methods extracting rheological parameters from analysis of ultrasound propagation within opaque material are examined. Integrated rheo-NMR methods are applied to studying the intramolecular response of material to mechanical excitation. Image analysis of complex materials and correlation of structural fractal dimensions of the components with the rheological parameters of the material is being tested. Surface rheology is applied to explain rheological properties of food components at interfaces.
REFERENCES Ahmed, J., Ramaswamy, H.S., and Pandey, P.K. (2006) Dynamic rheological and thermal characteristics of caramels, Lebensm. Wiss. u.-Technol., 39, 216–224. Ahmed, J. et al. (2003) Effect of high pressure on rheological characteristics of liquid egg, Lebensm.-Wiss. u.-Technol., 36, 517–524. Barnes, H.A, Hutton, J.F., and Walters K. (1989) An Introduction to Rheology, Elsevier Science Publishers, Amsterdam. Baryeh, E.A. (2000) Strength properties of avocado pear, J. Agric. Eng. Res., 76, 389–397. Bourne, M. (2002) Food Texture and Viscosity: Concept and Measurement, 2nd ed., Academic Press, New York Dervilly-Pinel, G., Tran, V., and Saulnier, L. (2004) Investigation of the distribution of arabinose residues on the xylan backbone of water-soluble arabinoxylans from wheat flour, Carbohydr. Polym., 55, 171–177. Dimitreli, G. and Thomareis, A.S. (2004) Effect of temperature and chemical composition on processed cheese apparent viscosity, J. Food Eng., 64, 265–271. Dobraszczyk, B.J. and Morgenstern, M.P. (2003) Rheology and the breadmaking process, J. Cereal Sci., 38, 229–245. Doi, M. and Edwards, S.F. (1986) The Theory of Polymer Dynamics. Oxford University Press, Oxford. Ferguson, J. and Kemblowski, Z. (1991) Applied Fluid Rheology, Elsevier Applied Science, London. Ferry, J. (1980) Viscoelastic Properties of Polymers, 3rd ed., John Wiley and Sons, Inc., New York. Giannouli, P. and Morris, E.R. (2003) Cryogelation of xanthan, Food Hydrocolloids, 17, 495–501. Goncalves, M.P. et al. (2004) Rheological study of the effect of Cassia javanica galactomannans on the heat-set gelation of a whey protein isolate at pH 7, Food Hydrocolloids 18, 181–189.
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Hort, J., Grys, G., and Woodman, J. (1997) The relationship between the chemical, rheological and textural properties of cheddar cheese, Lait, 77, 587–600. Juszczak, L. et al. (2004) Rheological properties of commercial mustards, J. Food Eng., 63, 209–217. Kealy, T. (2006) Application of liquid and solid rheological technologies to the textural characterisation of semi-solid foods, Food Res. Int., 39, 265–276. Kulicke, W.-M. et al. (1996) Characterization of aqueous carboxymethylcellulose solutions in terms of their molecular structure and its influence on rheological behaviour, Polymer, 37, 2723–2731. Launay, B., Cuvelier, G., and Martinez-Reyes, S. (1997) Viscosity of locust bean, guar and xanthan gum solutions in the Newtonian domain: critical examination of the log (ηsp)0 –log C[η]0 master curves, Carbohydr. Polym., 34, 385–395. Lefebvre, J. and Doublier, J.-L. (2004) Rheological behaviour of polysaccharides aqueous systems, in Polysaccharides, Structural Diversity and Functional Versatility, 2nd ed., Dumitriu, S., Ed., Marcel Dekker, New York, chap. 13. Lefebvre, J. et al. (2000) Temperature-induced changes in the dynamic rheological behavior and size distribution of polymeric proteins for glutens from wheat near-isogenic lines differing in HMW glutenin subunit composition, Cereal Chem., 77, 193–201. Lefebvre, J. et al. (2003) A phenomenological analysis of wheat gluten viscoelastic response in retardation and in dynamic experiments over a large time scale, J. Cereal Sci., 38, 257–267. Lourdin, D., Della Valle, G., and Colonna, P. (1995) Influence of amylose content on starch films and foams, Carbohydr. Polym., 27, 261–270. Macosco, C.V. (1994) Rheology: Principles, Measurements and Applications, VCH Publishers Inc., New York. Mali, S. et al. (2005) Water sorption and mechanical properties of cassava starch films and their relation to plasticizing effect, Carbohydr. Polym., 60, 283–289. Morris, E.R. (1990) Shear thinning of “random coil” polysaccharides: characterization by two parameters from a simple shear plot, Carbohydr. Res., 13, 85. Morrison, A. (2001) Understanding Rheology, Oxford University Press., New York. Ngarize, S., Adams, A., and Howell, N.K. (2004) Studies on egg albumen and whey protein interactions by FT-Raman spectroscopy and rheology, Food Hydrocolloids, 18, 49–59. Nickerson, M.T., Paulson, A.T., and Speers, R.A. (2004) Time-temperature studies of gellan polysaccharide gelation in the presence of low, intermediate and high levels of cosolutes, Food Hydrocolloids, 18, 783–794. Palzer, S. (2005) The effect of glass transition on the desired and undesired agglomeration of amorphous food powders, Chem. Eng. Sci., 60, 3959–3968. Pruska-Kędzior, A. et al. (2003) Rheological properties of water-soluble wheat pentosans solutions and gels obtained from various cultivars of common wheat, in Proceedings of 3rd International Symposium on Food Rheology and Structure ISFRS—Eurorheo 2003-01, February 9–13. 2003, Zurich, Switzerland, Fischer, P., Marti, I., and Windhab, E.J., Eds., Laboratory of Food Processing Engineering, ETH, Zurich, 175–179. Pruska-Kędzior, A. et al. (2005) Application of dynamic rheology methods to describing viscoelastic properties of wheat gluten, Electronic J. Polish Agric. Univ. Food Sci. Technol. Series, 8(2), http://www.ejpau.media.pl/volume8/issue2/art-33.html. Rao, M.A. and Steffe, J.F. (1992) Viscoelastic Properties of Foods, Elsevier Applied Science, New York.
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Renkema, J.M.S (2004) Relations between rheological properties and network structure of soy protein gels, Food Hydrocolloids, 18, 39–47. Shin, S. and Keum, D.-Y. (2003) Viscosity measurement of non-Newtonian fluid foods with a mass-detecting capillary viscometer, J. Food Eng., 58, 5–10. Sittikijyothin, W., Torres, D., and Goncalves, M.P. (2005) Modelling the rheological behaviour of galactomannan aqueous solutions, Carbohydr. Polym., 59, 339–350. Steffe, J.F. (1983) Rheological properties of liquid foods, ASAE Paper No. 83-6512, ASAE, St. Joseph, MI. Telis-Romero, J. et al. (2006) Rheological properties and fluid dynamics of egg yolk, J. Food Eng., 74, 191–197. Terpstra, M.E.J. et al. (2005) Modeling of thickness for semisolid foods, J. Text. Studies, 36, 213–233. Togrul, H. and Arslan, N. (2004) Mathematical model for prediction of apparent viscosity of molasses, J. Food Eng., 62, 281–289. Tschoegl, N.W. (1989) The Phenomenological Theory of Linear Viscoelastic Behavior. An Introduction, Springer Verlag, Berlin, pp. 433–435. Walkenström, P. et al. (1999) Effects of flow behaviour on the aggregation of whey protein suspensions, pure or mixed with xanthan, J. Food Engineering, 42, 15–26. Zghal, M.C., Scanlon M.G., and Sapirstein H.D. (2002) Cellular structure of bread crumb and its influence on mechanical properties, J. Cereal Sci. 36, 167–176.
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9
Food Colorants Jadwiga Wilska-Jeszka
CONTENTS 9.1 9.2
Introduction .................................................................................................. 245 Carotenoids................................................................................................... 246 9.2.1 Structure ........................................................................................... 246 9.2.2 Occurrence ....................................................................................... 249 9.2.3 Carotenoids Used as Food Colorants .............................................. 251 9.2.4 Physical and Chemical Properties ................................................... 252 9.2.5 Biological Activity ........................................................................... 253 9.3 Chlorophyll................................................................................................... 255 9.4 Heme Pigments ............................................................................................ 259 9.5 Anthocyanins................................................................................................ 260 9.5.1 Occurrence and Structure ................................................................ 260 9.5.2 Chemical Properties ......................................................................... 262 9.5.3 Biological Activity ........................................................................... 264 9.5.4 Stability of Anthocyanins in Food................................................... 264 9.6 Betalains ....................................................................................................... 265 9.7 Quinone Pigments ........................................................................................ 268 9.8 Turmeric and Curcumin (E 100) ................................................................. 268 9.9 Riboflavin (E 101) ....................................................................................... 269 9.10 Caramel (E 150)........................................................................................... 270 9.11 Melanoidins.................................................................................................. 270 9.12 Melanins ....................................................................................................... 271 9.13 Synthetic Organic Colors............................................................................. 272 References.............................................................................................................. 274
9.1 INTRODUCTION Color is an important indicator of food quality. The consumer associates food color with good processing and safety. However, color cannot be studied without considering the human sensory system. Perception of color is related to three factors: spectral composition of the light source, physical object characteristics, and eye sensitivity. Fortunately the human eye views color in a fairly uniform manner and it is not difficult to replace the eye, in food analysis, with some sensor instrument or photocell. 245
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The color of food is the result of the presence of natural pigments or of added synthetic organic dyes. The definition of a natural colorant is variable from one country to another, but generally, natural colorants include the pigments occurring in unprocessed food, and those that can be formed upon heating, processing, or storage. The natural pigments may be divided into four, not necessarily homogenous, groups: • • • •
Isoprenoid derivatives—carotenoids Porphyrins—chlorophylls and hemes Phenolics—anthocyanins, betalains, quinones, curcuminoids Miscellaneous naturally occurring colorants—riboflavins, caramels, melanoidins, and melanins
All natural pigments are unstable and participate in different reactions, so the food color is strongly dependent on conditions of storage and processing. The use of synthetic organic colors has been recognized for many years as the most reliable and economical method of restoring some of the food’s original shade to the processed product. Synthetic colors are superior to natural pigments in tinctorial power, stability, ease of application, and cost effectiveness. However, from the health safety viewpoint they are not accepted by consumers, so over the past years increasing interest in natural food colorants has been observed.
9.2 CAROTENOIDS 9.2.1 STRUCTURE Carotenoids are the most widespread and important group of pigments in nature. They play a crucial role in plants as accessory pigments for light harvesting and in prevention of photooxidative damage, as well as acting as attractants for pollinators. Carotenoids comprise a group of structurally related colorants that are mainly found in plants, algae, and several lower organisms. Nature produces around 108 tons per year of carotenoids. Most of them are found in marine algae and green leaves. At present, about 600 carotenoid compounds have been identified. Their basic structure is a symmetrical tetraterpene skeleton, formed by head-to-tail condensation of two C20 units, which is modified by cyclization, addition, elimination, rearrangement, and substitution, as well as oxidation. Based on their composition, carotenoids are divided into two classes: •
•
Carotenes, which are pure polyene hydrocarbons; they contain only carbon and hydrogen atoms, including acyclic lycopene and bicyclic β-, α- and λ-carotene (Formulas 9.1 to 9.4) Xanthophylls containing oxygen in the form of hydroxy (lutein), epoxy (violaxanthin), and oxo (canthaxanthin) groups (Formulas 9.5, 9.6, and 9.9)
Carotenoids with fewer than 40 carbon atoms are also known. The compounds derived by the loss of one or both end fragments are called apocarotenoids, for example, β-apo-8′-carotenal or diapocarotenoids such as bixin (Formulas 9.10 and 9.11).
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FORMULA 9.1
FORMULA 9.2
FORMULA 9.3 4' 7
14'
15
12'
10'
8'
6'
1 8 3
5
10
12
14
15'
2'
7'
Lycopene
FORMULA 9.4 OH
HO
Lutein
FORMULA 9.5
All carotenoids contain a system of conjugated double bonds that influence their physical, biochemical, and chemical properties. In principle, each of the polyene chain double bonds could exist in a cis or trans conformation, thus creating a number of isomers. In practice, however, only a few isomers exist. The reason for this is
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OH O O HO Violaxanthin
FORMULA 9.6
HO
FORMULA 9.7 OH O
OH
Capsanthin
FORMULA 9.8 O
O
Canthaxanthin
FORMULA 9.9
steric hindrance, making cis isomers less stable than the trans form. Thus, the vast majority of natural carotenoids are in the all-trans configuration. The carotenoids owe their characteristic yellow, orange, or red colors to the absorption of light in the 400- to 500-nm range as a result of the presence of the chromophore of conjugated double bonds. A minimum of seven conjugated double bonds are required for the yellow color to appear. The increase in the number of double bonds results in a shift of the major adsorption band to the longer wavelengths. In lycopene and β-carotene there are 11 conjugated double bonds, but in some molecules, such as canthaxanthin, two carbon–oxygen double bonds extend the chromophore chain. Food processing and storage can cause isomerization of carotenoids and affect the color because increasing the number of cis bonds results in gradual lightening of the color The cis isomers not only absorb less strongly
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CHO
FORMULA 9.10
CH3O O Bixin
C HO O
FORMULA 9.11
than the all-trans isomers, but they also show a so-called cis peak at 330 to 340 nm (Barua et al., 2000).
9.2.2 OCCURRENCE It is generally considered that de novo biosynthesis of carotenoids is observed only in photosynthetic higher plants, mosses, and algae, and nonphotosynthetic bacteria and fungi. All photosynthetic tissues contain carotenoids: higher plants in the chloroplasts, although the color is masked by the chlorophylls, and some bacteria in photosynthetic membranes. The chloroplasts, present in green unripe fruits, in most cases gradually change into chromoplasts on ripening, and carotenoid synthesis, often of novel pigments, is greatly stimulated. Typical examples are the tomato and red pepper. Oxygen but not light is required for carotenoid synthesis. In all photosynthetic organisms, carotenoids have two major functions: as accessory pigments for light harvesting and in the prevention of photooxidative damage. In plants they are essential components of the light-harvesting antennae, where they absorb photons and transfer the energy to chlorophyll. The other function of carotenoids is to protect against photooxidation processes that are caused by the excited triplet state of chlorophyll. Carotenoid molecules, with nine or more conjugated carbon–carbon double bonds, can absorb triplet-state energy from chlorophyll, and thus prevent the formation of harmful singlet oxygen. The main carotenoids of green leaves are lutein, violaxanthin, cryptoxanthin, and β-carotene; the others are produced in smaller quantities. Common carotenoids in fruits are β-carotene, lycopene, and different xanthophylls. The last are usually present in esterified form. Some of the carotenoids, such as β-carotene, lycopene, and zeaxanthin, are widely distributed and so become important as food components. However, the content of carotenoids usually does not exceed 0.1% of dry weight (Table 9.1). In plant products, carotenoids may occur as simple or as very complex mixtures; some of the most complex are found in citrus fruits. The simplest carotenoids usually exist in animal products because the animal organism is limited in
a
Violaxanthin 5.81 3.04 2.36 0.07 0.13 0.12 — 0.18 — 0.06 0.003 0.51 0.02 0.005 0.22
Lutein 18.6 9.54 2.92 0.08 0.25 0.41 0.21 0.8 0.36 0.65 0.04 0.98 0.04 0.02 0.02
Lycopene — — — — 3.45 — 11.44 — — — — — — 2.77 —
α-Carotene 0.15 0.09 0.04 — — 0.01 0.15 — 4.89 0.02 0.0002 0.14 0.02 — 0.006
Total 34.76 17.31 8.48 0.25 30.37a 0.70 12.7 1.56 15.99 0.90 0.05 2.40 1.13 3.50 0.40
X-Cryptoxanthin 0.12 — 0.03 0.002 1.01 0.002 — 0.011 — 0.008 0.0005 0.08 0.06 0.012 0.05
Including 13.94 mg/100 g capsanthin/capsorubin and 5.0 mg/100g of capsolutein. (Adapted from H. Muller, Z. Lebensm Unters Forsch A 204, 88–94, 1997.)
8.68 3.68 1.68 0.034 3.78 0.11 0.89 0.32 9.54 0.13 0.006 0.40 0.90 0.59 0.013
β-Carotene
250
Kale Spinach Lettuce White cabbage Red paprika Green paprika Tomato Broccoli Carrot Blackberry Strawberry Nectarine Apricot Grapefruit Orange
Vegetable or fruits
TABLE 9.1 The Quantites of Different Carotenoids in Some Vegetables and Fruits (mg/100 g edible portion)
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its ability to absorb and deposit them. The compounds, which have been isolated only from animal tissues, mainly xanthophylls, are the result of metabolic changes, generally oxidative, in the ingested carotenoids. Crude vegetable oils contain carotenoids, but bleaching and hydrogenation leads to almost complete degradation of these pigments. Particularly rich in carotenoids (0.05 to 0.2%) is crude palm oil containing mainly α-carotene and β-carotenes. Egg yolk contains only xanthophylls, mainly lutein, zeaxanthin, and cryptoxanthin (0.3 to 8.0 mg/kg).
9.2.3 CAROTENOIDS USED
AS
FOOD COLORANTS
The most commonly used natural carotenoid extracts for foodstuffs are annatto, paprika, and saffron. Many other sources, including alfalfa, carrot, tomato, citrus peel, and palm oil, are also utilized. Annatto [E 160(b)] is the orange-yellow, oil-soluble natural pigment extracted from the pericarp of the seed of the Bixa orellana L. tree. The major coloring component of this extract is the diapocarotenoid bixin (Formula 9.11), Several other pigments, mainly degradation products of bixin, are also present, including transbixin, norbixin, and trans-norbixin. Bixin is a methyl ester of a dibasic fatty acid, which on treatment with alkalis is hydrolyzed to water-soluble norbixin. Two types of annatto are therefore available: the extract in oil containing bixin, and a powdered, water-soluble form containing norbixin. Paprika oleoresin [E 160(c)] is the orange-red, oil-soluble extract from sweet red peppers (Capsicum annum). The major coloring compounds are xanthophylls: capsanthin (Formula 9.8), capsorubin as their dilaurate esters, and β-carotene. The presence of characteristic flavoring and spicy pungency components limits application of this extract in foodstuffs. Raw, unrefined palm oil contains 0.05 to 0.2% carotenoids with α- and β-carotenes, in a ratio of 2:3, as the main constituents. It is particularly used as a colorant for margarine. Saffron, an extract of flowers of Crocus sativus, contains the water-soluble pigment crocin, the digentiobioside of apocarotenic acid (crocetin), as well as zeaxanthin, β-carotene, and characteristic flavoring compounds. The yellow color of this pigment is attractive in beverages, cakes, and other bakery products. However, use of this colorant is restricted by its high price. Carrot extracts (E 160a), carrot oil, and related plant extracts are also available on the market. Their main components are β- and α-carotenes (Formulas 9.1 and 9.2, respectively). Processes for the commercial extraction of carotene from carrots have been developed. Purified crystalline products contain 20% α-carotene and 80% β-carotene. They may be used for coloring fat-based products, such as dispersion of microcrystals in oil. Individual carotenoid compounds—β-carotene, β-apo-8′-carotenal (Formula 9.10), β-apo-8′-carotenoic acid ethyl ester, and canthaxanthin (Formula 9.9)—are synthesized for use as food colorants for edible fats and oils. Their properties are given in Table 9.2. The carotenoid pigments, in combination with surface active agents, are also available as microemulsions for coloring foods with a high water content.
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TABLE 9.2 Properties of Carotenoids Used as Food Colorants Solubility [g/100 cm3] w 20°C Carotenoid
Color
β-Carotene β-Apo-8′-carotenic acid ethyl ester β-Apo-8′-carotenal Canthaxanthin
yellow yellow to orange orange to red red
Oils
Ethanol
λmax
Vitamin A activity (IU/mg)
0.05–0.08 0.7
0.01 0.1
455–456 448–450
1.67 1.2
0.7–1.5 0.005
0.1 0.01
460–462 468–472
1.2 0
Source: Adapted from Klaui, H. and Bauernfeind, J.C., Carotenoids as Food Colors in Carotenoids as Colorants and Vitamin A Precursors, CRC Press, Inc., Boca Raton, FL, 1981.
9.2.4 PHYSICAL
AND
CHEMICAL PROPERTIES
Carotenoids are extremely lipophilic compounds that are almost insoluble in water. In an aqueous medium they tend to form aggregates or adhere to surfaces. They are soluble in nonpolar organic solvents such as hexane, halogenated hydrocarbons, or tetrahydrofurane. In oils their solubility is rather low, particularly in a pure crystalline state. In the organism they are located in cellular membranes or in lipophilic compartments. In some plants hydroxylated carotenoids are esterified with various fatty acids, which makes them even more lipophilic. With their large number of conjugated double bonds, the carotenoids contain a reactive electron-rich system, which is susceptible to reaction with electrophilic compounds. This structure is responsible for the high sensitivity of carotenoids to oxygen and light. The central chain of conjugated double bonds can be oxidatively cleaved at various points, giving rise to a family of apocarotenoids. Most carotenoids (but not vitamin A) are singlet oxygen quenchers. Singlet oxygen 1O2, interacts with the carotenoid to give triplet states of both molecules. The energy of an excited carotenoid is dissipated through vibrational interaction with the solvent to recover the ground state. Carotenoids are the most efficient naturally occurring quenchers of singlet oxygen. They may also participate in the propagation step of the oxidation process as chain-breaking antioxidants that scavenge reactive peroxyl radicals. However, carotenoids serve this function best at low oxygen tensions. At higher oxygen levels, a carotenoid intermediate radical might add oxygen to form carotenoid peroxyl radicals such as Car-OO, which could act as prooxidants, initiating the process of lipid peroxidation [Berg et al., 2000]. On the other side, radicals and peroxides, occurring in food as a result of lipid oxidation, accelerate oxidative degradation of carotenoid pigments, which leads to formation of epoxides located at the β-ionone ring, β-apo-carotenones, and β-apocarotenals of different chain lengths. Lipoxygenase involved in the decay of vegetable matter may also cause the destruction of carotenoids. Antioxidants, including ascorbic acid, and their derivatives, tocopherols, and polyphenolics are used to suppress this oxidative degradation.
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Due to oxidative degradation of carotenoids, aroma compounds are also formed, including β-ionone with an odor threshold value of 14 ng/g in water. The formation of β-ionone in dehydrated carrots causes an undesirable off-flavor with an odor like violets. Unsaturated ketones derived from carotenoid degradation are readily further oxidized. The stability of carotenoids in frozen and heat-sterilized foods is quite high, but it is poor in dehydrated products, unless the products are packaged in inert gas. Dehydrated carrots fade rapidly.
9.2.5 BIOLOGICAL ACTIVITY A wide range of carotenoid chemical reactivity makes it obvious that they can exert bioprotective effects through a variety of mechanisms. According to Berg et al. (2000), carotenoids have diverse biological functions and actions, the most important of which are summarized below: Provitamin A activity Antioxidant Cell communication Immune function enhancers UV skin protection Macula protection
β-carotene, α-carotene, β-cryptoxanthin All carotenoids β-carotene, canthaxanthin, cryptoxanthin β-carotene β-carotene, lycopene Lutein, zeaxanthin
Some carotenoids are precursors of a separate class of bioactive compounds, the retinoids, which can regulate cell growth and differentiation in various cell types through interaction with ligand-dependent transcription factors. But of the 600 carotenoids that have been identified, only about 30 are believed to have some provitamin A activity, especially β-carotene. One mole of β-carotene can theoretically be converted, by cleavage of the C 15 = C 15′ double bond, to yield two moles of retinol (Reaction 9.1). However, the physiological efficiency of this process appears to be only 50%. The observed average efficiency of intestinal β-carotene absorption is only two thirds of the total content. Thus, a factor of 1/6 is used to calculate the retinol equivalent (RE) from β-carotene, but only 1/12 from the other provitamin A carotenoids in food (Combs, 1992). Pathological processes, including cancer and cardiovascular disease, are commonly associated with an oxidative stress condition. Carotenoids have a considerably high antioxidant activity, which has most often been the focus of its role in preventing disease initiation and propagation. Excessive production of oxygen radical species and particularly hydroxyl radicals, can affect lipid cell membranes to produce lipid peroxides and reactive oxygen species (ROS), which are linked to a variety of degenerative diseases and acceleration of the aging process. Carotenoids are membranal pigments: apolar carotenes are immersed in membranes and show limited mobility, whereas polar xanthophylls have a variable position and mobility in membranes. As can be deduced, carotenes have high antioxidant activity against radicals generated inside the membranes. The structure of carotenoids, in particular the length of the polyene chain, significantly influences its antioxidant properties.
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15 15'
CHO
Retinal alcohol dehydrogenase
retinal aldehyde reductase
CH2OH
Retinol
REACTION 9.1 Formation of vitamin A from β-carotene.
Different methods have been used for determination of antioxidant activity. One of the frequently used methods is based on the ability to quench the colored 2,2azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS). The comparison of antioxidant activity of some carotenoids, determined with this method and calculated as the Trolox equivalent of antioxidant capacity (TEAC), is given in Table 9.3. Carotenoids have also been reported to have immunomodulatory effects, such as a reduction in UV-induced immunosuppression, and an increase in natural killer (NK) cell activity after dietary supplementation with β-carotene. Cell-to-cell communication by gap junction is another important physiological function, playing a role in morphogenesis and cell differentiation. Epidemiological studies have also shown an inverse association between carotenoid intake and the risk of cataract and age-related macular degeneration. Lutein and zeaxanthin are the principal components of macular pigments. Their content can be modified by diet modification or lutein supplementation. Generally it was observed that people with low carotenoid intake or low blood levels, have an increased risk of degenerative diseases. In a number of these diseases, free radical damage plays a role in the pathophysiology of the disease. Earlier studies focused mainly on the protective effects of β-carotene and lycopene against prostate
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TABLE 9.3 Antioxidant Activities of Some Carotenoids TEAC (mM) Lycopene β-Cryptoxanthin β-Carotene Lutein Zeaxanthin α-Carotene
2.9 2.0 1.9 1.5 1.4 1.3
TEAC = Trolox equivalent antioxidant capacity. Source: Adapted from Rice-Evans, C., Sampson, J., Bramley, P.M., and Holoway, D.E., Free Rad. Res., 26, 381, 1997.
and lung cancer, but there is as yet no definitive proof for a causal relationship, nor for a beneficial antioxidant effect of carotenoids. The cancer-preventing effect of β-carotene has been investigated in several intervention trials. However, in only one of these studies, the so-called Linxian study, a protective effect was found for a combined β-carotene, vitamin E, and selenium supplementation. In other studies no protective effect was found, and in some cases a higher risk of lung cancer has been observed after high doses of β-carotene. Therefore the conclusion from these studies is that high-dose supplements of β-carotene are contradicted mainly for heavy smokers, but could have a beneficial effect on individuals with a poor baseline carotenoid status. Probably a diet rich in high-carotenoid-containing fruits and vegetables is more efficacious than individual carotenoid compounds, because it represents a lower, regular intake of various constituents with several mechanisms of action, as opposed to a high intake of one constituent with limited functions (Berg et al., 2000).
9.3 CHLOROPHYLL Chlorophyll is the most widely distributed natural plant pigment. It is known that chlorophyll has existed on Earth for at least 2.6 billion years. Over that period, innumerable plant species have come and gone, while the constitution and action of chlorophyll has remained, despite the powerful forces of natural selection. Its annual production is estimated to be 1.2 billion tons worldwide, of which one quarter is terrestrial and the rest of marine origin (Humphrey, 2004). In higher plants and algae, except the blue-greens, chlorophyll is found in chloroplasts, while in blue-green algae and photosynthetic bacteria, it is located on intracellular lamellae. In living plant tissues, in chloroplasts, chlorophyll is complexed with polypeptides, phospholipids, and tocopherols and held within hydrophobic membranes. Chlorophylls are derivatives of dihydroporphirin chelated with a centrally located magnesium ion (Formula 9.12). They are diesters: one carbonyl group is esterified
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CH3
CH
1
CH2
2
H3C
N
8 H
N Mg N
7
N
O O
3
R
4
CH2CH3
CCH2CH2 H 6
C
5
O
OCH3
CH3 O
Chlorophyll a R= —CH 3 Chlorophyll b R= —CHO
FORMULA 9.12
with methanol and the other with a C20 monounsaturated isoprenoid alcohol–phytol, which make the chlorophyll molecule hydrophobic. There are two chlorophylls: a (blue-green) and b (yellow-green), occurring in plants in a ratio about 3:1. Chlorophyll b differs from chlorophyll a in that the methyl group on C3 is replaced with an aldehyde. This is a stable ring-shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll captures the energy of sunlight. In plant photosynthesis, incoming light is absorbed by chlorophyll and other accessory pigments in the antenna complexes of photosystems I and II. The antenna pigments are predominantly chlorophyll a, chlorophyll b, and carotenoids. Each antenna complex
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has between 250 and 400 pigment molecules, and the energy they absorb is shuttled by resonance energy transfer to specialized chlorophyll a at the reaction center of each photosystem. When either of the two chlorophyll a molecules at the reaction center absorb energy, an electron is excited and transferred to an electron-acceptor molecule, leaving an electron hole in the donor chlorophyll. The resulting chemical energy is then captured in the form of ATP (adenosine triphosphate) and reduced nicotinamide adenine diphosphate (NADPH) and is ultimately used to convert CO2 to carbohydrates. Chlorophyll a is common to all eucariotic photosynthetic organisms, and due to its role in the reaction center, it is essential for photosynthesis. The accessory pigments, such as chlorophyll b and carotenoids, are not essential. Typical leaf material contains about 2.5 mg/g total chlorophylls, 0.3 mg/g xanthophylls, and 0.15 mg/g carotenes (Humphrey, 1980). In many fruits, chlorophyll is present in the unripe state, and gradually disappears during ripening as the yellow and red carotenoids take over. Fruit and vegetable quality and freshness are strongly associated with color. Chlorophyll pigments are unstable and may be used as an indicator of health and ripeness of different plant material as well as of processing condition. Chlorophyll degradation may occur within a few or over several weeks. The degradation process is strongly dependent on the pH and temperature. The most important transformations of chlorophylls are: • • •
Easy loss of Mg in dilute acids or replacement of Mg by other divalent metals Hydrolysis of the phytyl ester in dilute alkalis or transesterification by lower alcohols Hydrolysis of the methyl ester and cleavage of the isocyclic ring in stronger alkalis
Removal of Mg gives olive-brown pheophytin a and b. Replacing Mg by Fe or Sn ions yields grayish-brown compounds, while Cu or Zn ions retain the green color. Upon removal of the phytyl group by hydrolysis in dilute alkali, or by the action of chlorophyllase, green chlorophyllin are formed. Removal of Mg and the phytyl group results in olive-brown pheophorbide formation (Figure 9.1). Chlorophylls and pheophytins are lipophilic due to the presence of the phytol group, while chlorophyllins and pheophorbids without phytol are hydrophilic. The copper complexes of both pheophytin and pheophorbid have the metal firmly bound; it is not liberated even by the action of concentrated hydrochloric acid, and not removed to any appreciable extent, on metabolism; thus it is acceptable for the coloration of foodstuffs. The chlorophyll preparations for the food colorant market are mainly obtained from alfalfa (Medicago sativa) and nettles (Urtica dioica). Brown seaweeds, which are the commercial source of alginates, are also an interesting source of chlorophyll because as in single-cell phytoplankton, they contain chlorophyll c, which is more stable than chlorophyll a and chlorophyll b (Delgado-Vargas and Paredez-López, 2003). Acetone, methanol, ethanol, and chlorinated solvents are used as extracting
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Chemical and Functional Properties of Food Components
CHLOROPHYLID (green)
chlorophilase
phytol
CHLOROPHYLL (green)
phytol
acid Mg2+
methanol
alkali Mg2+
PHEOPHYTIN (olive-green)
acid CHLOROPHYLLIN (green)
phytol phytol
Mg2+
alkali acid PHEOPHORBIDE (olive-brown)
FIGURE 9.1 Transformation of chlorophyll pigments.
vehicles. The yield of extraction is around 20% in which chlorophylls, pheophytins, and other degradation products are included. Chlorophyll and its metabolites—pheophytin, pyropheophytin, pheophorbide, as well as chlorophyllin—have demonstrated in vitro antimutagenic and anticarcinogenic effects against some substances known or suspected to cause cancer, such as polycyclic aromatic hydrocarbons found in smoke, heterocyclic amines found in cooked meats, as well as aflatoxin B1. The mechanism of these activities of chlorophyll and chlorophyllin are unknown, but it is speculated that one possible mechanism is the formation of complexes between the mutagen or carcinogen and the chlorophyll or chlorophyllin through strong interaction between their planar unsaturated cyclic rings. Such complexes would effectively inactivate the mutagen or carcinogen. Chlorophylls are approved in Codex legislation as food additives for different applications. However, the Na and K salts of Cu-chlorophylls are approved by the U.S. Food and Drug Administration (FDA) only in preparation of dentifrices and drugs, but not as food additives (Delgado-Vargas and Paredez-López, 2003). The oil-soluble chlorophylls, as well as the water-soluble chlorophyllins, a semisynthetic mixture of Na and Cu salts derived from chlorophyll, have good stability in light and heat, and moderate stability with respect to both acid and alkalis. Their application as food colors is in canned products, confectionery, soups, and dairy products.
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9.4 HEME PIGMENTS Heme is a metal-containing cofactor that consists of an Fe atom contained in the center of a large heterocyclic ring called porphyrin. There are three biologically important kinds of heme called a, b, c. The proteins that contain heme a or c are cytochromes, and the most abundant heme b is present in both hemoglobin and myoglobin. The main function of heme is the retention of O2 and delivery of it for enzymatic reaction. Heme is made up of four heterocyclic pyrrole rings connected at their corners by methylene bridges. The four nitrogen atoms bind the Fe(II) or Fe(III). However, only Fe(II) can bind O2. Each myoglobin molecule contains one heme prosthetic group inserted into a hydrophobic pocket. Myoglobin is a monomeric heme protein found mainly in muscle tissue, where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bound oxygen, which is then used for metabolic purposes. Hemoglobin consists of four protein chains, each about the size of a myoglobin molecule, which fold to give a structure that looks very similar to myoglobin. Thus hemoglobin has four separate heme groups that can bind a molecule of O2 each. The cooperative interaction between binding sites makes hemoglobin an unusually efficient oxygen-transport protein. It binds oxygen in the pulmonary vasculature and releases it in tissues, where the situations are reversed. Myoglobin is the main pigment of meat. In fresh meat, in the presence of oxygen, the reversible reaction of myoglobin (Mb) with oxygen occurs and bright red-colored oxymyoglobin (MbO) is formed. Due to oxidation of Fe(II) the brownish metmyoglobin (MMb) is formed. MbO2 red
Mb purplish red
MMb brownish
REACTION 9.2 Basic transformations of myoglobin.
Oxymyoglobin and myoglobin exist in a state of equilibrium with oxygen. The ratio of these pigments depends on oxygen pressure. In meat there is a slow oxidation of heme pigments, mainly myoglobin to metmyoglobin, which cannot bind oxygen. Heating of meat results in the denaturation of the globin linked to iron, as well as in the oxidation of iron to the ferric state, and formation of different brown heme pigments named hemichrome. In the curing of meats, the heme pigments react with the nitrite of the curing mixture and the red colored nitrite–heme complex nitrosomyoglobin is formed, but it is not particulary stable. More stable is the pigment with a denatured globin portion, called nitrosylhemochrome, found in meat heated at a temperature of more than 65°C. The reactions of heme pigments in meat and meat products are summarized in Figure 9.2. In the presence of thiol compounds as a reducing agent, in the reversible reaction myoglobin may form a green sulfmyoglobin. Other reducing agents, such as ascorbate, lead to the formation of cholemyoglobin. This reaction is irreversible.
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dMMb brownish
heating
heating
MbO2 red
MMb brownish
oxidation
-O2
NO
reduction
reduction
NO
Mb purplish red
+O2
MMbNO bright red
MbNO bright red
heating
dMbNO bright red
FIGURE 9.2 Transformation of myoglobin in meat. Mb = myoglobin, MMb = metmyoglobin, MbO2 = oxymioglobin, MbNO = nitroxymioglobin, dMMb = denatured metmyoblobin.
A potential source of red and brown heme pigments is animal blood and its dehydrated protein extracts, which mainly consist of hemoglobin; these may be used as red and brown colorings in meat products. However, in most countries, their use as food coloring agents is not permitted.
9.5 ANTHOCYANINS 9.5.1 OCCURRENCE
AND
STRUCTURE
Anthocyanins are among the most important groups of plant pigments. They are present in almost all higher plants and are the dominant pigments in many fruits and flowers, giving them red, violet, or blue color. They play a definite role in attracting animals in pollination and seed dispersal. They may also have a role in the mechanism of plant resistance to insect attack. Anthocyanins are part of the very large and widespread group of plant constituents known as flavonoids, which possess the same C6–C3–C6 basic skeleton. They are glycosides of polyhydroxy and polymetoxy derivatives of 2-phenylbenzopyrylium or flavylium cation (Formula 9.13). Differences between individual anthocyanins include (1) the number of hydroxyl groups in the molecule; (2) the degree of methylation of these hydroxyl groups; (3) the nature, number, and the position of
R3' 2'
HO 8 7 6 5
O + 4
4' 5' 6'
2 3
OH OH FORMULA 9.13
OH
3'
R5'
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glycosylation; (4) and the nature and number of aromatic or aliphatic acids attached to the glucosyl residue. From about 20 known naturally occurring anhocyanidins, only six occur most frequently in plants (Table 9.4). Substitution of the hydroxyl and methoxyl groups affects the color of the anthocyanins. An increase in the number of hydroxyl groups tends to deepen the color to a more bluish shade. An increase in the number of metoxyl groups increases redness. Because of the possibility of various ose and acid substitutions at different positions, the number of anthocyanins is 15 to 20 times greater than the number of anthocyanidins (Mazza and Miniati, 1993) The ose molecules most commonly bonded to anthocyanidins are glucose, galactose, rhamnose, arabinose, and di- and trisaccharides, formed by combinations of these four monosaccharides. The most common classes of anthocyanins are 3-monosides, 3-biosides, 3,5-diglycosides, 3,7-diglycosides; however, glycosylation of the 3′-, 4′-, and 5′-hydroxyl group is also possible. The composition and content of anthocyanins in fruits are very diversified (Table 9.5).
TABLE 9.4 Naturally Occurring Anthocyanidins Antocyanidin Pelargonidin (Pg) Cyanidin (Cy) Peonidin (Pn) Delphinidin (Dp) Petunidin (Pt) Malvidin (Mv)
R3’
R5’
λmax[nm]
Color
H OH OCH3 OH OCH3 OCH3
H H H OH OH OCH3
520 535 532 546 543 542
orange orange-red orange-red bluish-red bluish-red bluish-red
TABLE 9.5 Anthocyanins in Some Fruits—Composition and Content Fruits
Main anthocyanins
Blackberry Bilberry Black current Chokeberry Cranberry Strawberry Red grapes
Cy 3-Glu, Cy 3 Rut Dp 3-Gal and Arab, Mv 3-Gal and Arab, Pt 3-Gal and Arab Cy 3-Rut and Glu, Dp 3-Rut and Glu Cy 3-Gal, Cy 3-Arab, Cy 3-Glu, Cy 3-Xyl Cy 3-Gal and Arab, Pn 3-Gal and Arab Pg 3-Glu and Cy 3-Glu Mv, Dp, Pt, Pn 3-Glucosides, 3-acetylglucosides, and 3 p-coumarylglucosides
Total anthocyanins (mg/100 g) 83–326 250–490 250 520–800 78 7–30 30–750
Source: Adapted from Macheix, J.J., Fleuriet A., and Billot J., Fruit Phenolics, CRC Press Inc., Boca Raton, FL, 1990; and Wang H., Coa G., and Prior R.L., J. Agric. Food Chem. 45, 304, 1997.
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9.5.2 CHEMICAL PROPERTIES In aqueous media, most of the natural anthocyanins behave like pH indicators: red at low pH, bluish at intermediate pH, and colorless at high pH. According to Brouillard (1982), in acidic and neutral media, four anthocyanin structures exist in equilibrium: the red flavylium cation (AH+), blue or red quinonoidal base (A), colorless carbinol pseudobase (B), and colorless chalcone (C) (Reaction 9.3). The pH of the medium plays a particularly important role in the equilibrium between these different anthocyanin forms, and consequently in color modification. In a strongly acid solution, at a pH below 2, the red cation AH+ is the dominant form. As the pH is increased, a rapid proton loss occurs to yield the red or blue quinoidal base A, usually existing in two forms (Figure 9.3). On standing, a further reaction occurs, that is, hydration of flavylium cation AH+ to yield a colorless carbinol pseudobase B. Relative amounts of form AH+, A, B, and C at equilibrium vary with both pH and the structure of anthocyanins. For the common anthocyanin 3-glycosides or 3,5-diglycosides, the principal product formed on raising the pH above 3 is the colorless carbinol pseudobase B (Figure 9.3). At this pH, however, small amounts of the blue quinoidal base and the colorless chalcones are also present and increase with increasing pH. Between pH 4 and 6, very little color remains because the amounts of the colored forms AH+ and A are very small (Reaction 9.3). By varying the substitution pattern of the flavylium ring, anthocyanidin, which exists primarily in the colored form, can be prepared (Jacobucci and Sweeny, 1983), but such anthocyanin compounds are not found in plants. The color of anthocyanin-containing media depends on different factors. The most important are structure and concentration of anthocyanin pigments; pH; presence of copigments and metallic ions, which influence the color shade; as well as temperature, presence of oxygen, phenoloxidase, ascorbic acid, and sulfur dioxide, which influence the anthocyanins’ degradation rate and color stability. OH
OH O
HO
A
B
+
O
HO
OH
OH
OH O
HO
+
- H /H2 O
C
O Gl
O Gl
O Gl
OH
OH
AH+: Flavylium cation
OH
CE: E-Chalcone
B: Carbinol pseudobase -H+
+H+
+
+H
- H+
O
OH
OH
HO
OH O
HO
O
O
O OH
Gl
O
O Gl
Gl
O
OH
OH
A: Quinonoidal bases
CZ: Z-Chalcone
REACTION 9.3 Structural anthocyanin transformations in aqueous media.
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100 AH+
% of total
B
50 A
C
0
1
2
4
3
5
6
pH FIGURE 9.3 Distribution of anthocyanin structures as a function of pH.
The structure of the anthocyanin molecule has a marked effect on color intensity and stability. The increase of the number of hydroxyl groups in the B-ring shifts the absorption maximum to longer wavelengths, and the color changes from orange to bluish-red. Methoxyl groups replacing hydroxyl groups reverse this trend. The hydroxyl group at C-3 is particularly significant because it shifts the color from yellow-orange to red. The same hydroxyl, however, destabilizes the molecule; the 3-deoxyanthocyanidins are much more stable than the other anthocyanidins. Similarly, the presence of a hydroxyl group at C-5 and substitution at C-4, both stabilize the colored forms. Glycosylation also affects the stability of these pigments; the half-life of anthocyanidins is significantly lower than their corresponding 3-glucosides. Anthocyanins containing two or more aromatic acyl groups, such as cinerarin or zebrinin, are stable in neutral or weakly acidic media. This is possibly a result of hydrogen bonding between phenolic hydroxyl groups in anthocyanidins and aromatic acids. Brouillard (1981) observed that diacylated anthocyanins are stabilized by sandwich-type stacking caused by hydrophobic interaction between the anthocyanidin ring and the two aromatic acyl groups. The increase in anthocyanin concentration results in an increase in absorbance at λmax, which is greater than expected according to the Beer-Lambert law. It is probably connected with anthocyanins’ self-association. Intermolecular copigmentation of anthocyanins with other flavonoids, some phenolic acids, alkaloids, and other compounds, including anthocyanins themselves, increases the color intensity (hyperchromic effect) and causes a shift in the wavelength (batochromic shifts), giving purple to blue colors. The intensity of the copigmentation effect depends upon several factors, including type and concentration of anthocyanins and copigments, pH, and temperature of the solvent (Brouillard et al., 1991). The pH value for maximum copigmentation effect is about 3.5, and may vary slightly depending on the pigment–copigment system. Color intensification by copigmentation increases with an increasing ratio of copigment to anthocyanins. Increasing temperature strongly reduces the color-intensifying effect.
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The copigmentation phenomenon is widespread in nature, and also occurs in fruit and vegetable products, such as juices and wines. In the presence of metals, anthocyanins can form purplish-blue or slate-gray pigments called lakes. This reaction may induce color changes if fruit products, during processing or storage, are in contact with metals such as Sn, Al, or Fe.
9.5.3 BIOLOGICAL ACTIVITY Anthocyanins are natural colorants that belong to the large family of phenolic compounds—flavonoids. They are known to display different pharmacological and biological effects, such as those that are vasoprotective, antiinflammatory, and radioprotective, as well as prevention of cholesterol-induced arteriosclerosis and heart disease. However, the most important properties of anthocyanins seem to be their activity as potent free-radical scavengers and powerful chain-breaking antioxidants. Similar to other flavonoids, they can react with ROS and lipid peroxyl radicals, and inhibit lipid peroxidation at the early stage. It is very important because in vivo lipid peroxidation has been implicated as the primary cause of coronary heart disease, arteriosclerosis, cancer, and aging. The antioxidant effectiveness of anthocyanin pigments is structure dependent, but for the main aglicons, it is about two times higher than that of vitamins C and E. Recently increasing attention has been focused on anthocyanins as natural antioxidants because of their ubiquitous presence in plant products, particularly in fruits, juices, and red wine. The daily intake of anthocyanins in humans is as high as 200 mg/day. Despite a relatively high potential intake in humans, the physiological impact of the anthocyanins is not well recognized because of the lack of pure compounds and their low stability. Anthocyanin-enriched materials, of different purity, from the skin of grapes and other by-products of wine and juice manufacturing are produced in different countries. One of the better known, commercially available anthocyanin concentrates is extract of Vacciniun myrtillus—bilberry (VMA), which contains largely glycosides of delphinidin and cyanidin, and is used to treat various microcirculation diseases resulting from capillary fragility (Wang et al., 1997). Red wine, much more than white wine and grape juice, is also recognized as a rich source of natural antioxidants. It has been demonstrated that red wine has superoxide radical-scavenging potential, and is able to effectively inhibit oxidation of low-density lipoprotein in vitro and in vivo. However, in all these products, not only anthocyanins but also other phenolic antioxidants, such as flavonols, and flavanols are present and may influence the biological and antioxidant activity. Therefore the consumption of fruits rich in anthocyanins and red wine appears to be a good means of impeding the lipid oxidation process responsible for different diseases and aging.
9.5.4 STABILITY
OF
ANTHOCYANINS
IN
FOOD
Anthocyanins appear to have low stability in all products manufactured from fruits. This limits the use of these pigments as food colorants. Two main groups of factors are responsible for the stability of anthocyanin color in fruits during processing:
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• •
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Initial composition of the fruit, with regard to anthocyanins and other constituents including enzymatic systems Processing factors such as temperature, light, and the presence of oxygen
Several enzymes and, in particular, phenolases, peroxidases, and β-glucosidases, can decrease the quality of the initial product during the extraction of fruit juices or the preparation of processed products, leading to browning and to loss of color by enzymatic degradation of anthocyanins. Anthocyanins themselves are not good substrates for o-diphenol oxidase, but they are instead oxidized by the chlorogenic acid ↔ chlorogenoquinone redox shuttle phenomena. Thus enzymatic oxidation of chlorogenic acid may by combined with nonenzymatic oxidation and polymerization of anthocyanins. The same phenomenon has been observed in the presence of catechins. Ascorbic acid may have a protective effect with regard to anthocyanins because it reduces the o-quinones formed before their polymerization. However, ascorbic acid as well as products of its degradation increase the anthocyanin degradation rate. Sulfur dioxide, widely used in fruit processing at concentrations as low as 30 mg/kg, inhibits the enzymatic degradation of anthocyanins. At moderate concentrations on the order of 500 to 2000 mg/kg, it forms a colorless SO2–anthocyanin complex. This is a reversible reaction: after removal of SO2 the color turns red again. Regardless of the favorable action of high temperature on the blockage of enzymatic activities, anthocyanins are readily destroyed by heat during processing and storage. A short-time, high-temperature process was recommended for best pigment retention. For instance, in red fruit juices heated 12 min at 100°C, anthocyanin losses appear to be negligible. The reactions described above can proceed at different rates and can bring about different changes in color depending on the composition of food products. Most frequently the red color slowly turns brown. However, in correctly processed and stored fruits, the color changes are so slow that they only slightly affect consumer appreciation of fruit products. Anthocyanin concentrate may be used as a food colorant at a pH less than 4.
9.6 BETALAINS Betalains, a class of water-soluble red-violet or yellow pigments, occur only in 10 families of the order Caryophyllales (old name for Centrospermae) and some fungi species, such as Amanita muscaria. The presence of betalain in plants is mutually exclusive of occurrence of anthocyanins, which are more widely distributed in the plant kingdom. About 50 betalains have been identified, which are found in different plant organs and are accumulated in cell vacuoles, mainly in epidermal and subepidermal tissues. However, only red beetroot (Beta vulgaris L. Chenopodiaceae), cactus fruits (genera Opuntia and Hylocereus) are the only edible source containing these pigments. Betalains consist of red-violet betacyanins (λmax ~ 540 nm) and yellow betaxanthin (λmax ~ 480 nm). The betanin (Formula 9.14), glucoside of betanidin, is the main betalain pigment in red beetroot and accounts for 75 to 95% of total pigments in red
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GlO
GlO
H +
N
HO
COO
H +
-
N
HO
-
H
HOOC H
COO
N
HOOC
COOH
N
COOH
H
H
Betanin
Isobetanin
FORMULA 9.14
R O COO
-
+NH
HOOC H
N
COOH
H R = —NH2 vulgaxanthin I R = —OH vulgaxanthin II
FORMULA 9.15
beets. The other red pigments are isobetanin (C-15 epimer of betanin), prebetanin, and isoprebetanin. The latter two are sulfate monoesters of betanin and isobetanin, respectively. Unlike anthocyanins, betanins cannot be hydrolyzed to aglycone by acid hydrolysis without degradation. The major yellow pigments are vulgaxanthin I and II (Formula 9.15). High betalain content in beetroot, on average 1% of the total solids, makes these vegetables a valuable source of food colorants. Cactus fruits from the genera Opuntia and Hylocereus are the other edible sources of betalain pigments. There are important differences in the chemical composition and visual appearance of these fruits. The color shade of the red juice of Opuntia ficus-indica cv. Rossa is similar to that of beet preparations, the juice from Opuntia ficus-indica cv. Gialla displays a yellow tonality, whereas the juice from Hylocereus polyrhizus is
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characterized by purplish hues. Betacyanin contents in these juices are 74, 1.3, and 525 mg/dm3, respectively, whereas betaxantins amounted to 36, 48, and 5.3 mg/dm3 (Stintzing, 2003). The other source of betalains is the family Amarantahaceae recognized as a rich source of diverse and unique betacyanins. They consist of six simple (nonacetylated) and ten acetylated betacyanins, including eight amaranthine-type pigments, six gomphrenin-type pigments, and only two betanin-type pigments. Total betacyanin pigment content ranges from 0.08 to 1.36 mg/g of fresh weight. The main pigment in this family is amaranthine (betanidin 5-O-β-glucuronosylglucosides), whose content averages about 91% of total betacyanins. The effect of acyl groups on the color intensity and stability of betacyanin pigments is not clear (Cai et al., 2001). The color stability of betanin solution is strongly influenced by pH and heating. Betanin is stable at pH 4 to 6, but thermostability is greatest between pH 4 and 5. As a result of betanin degradation, cyclo-DOPA and betalamic acid are formed (Reaction 9.4). This reaction is reversible (Czapski, 1985). Light and air have a degrading effect on betanin. These effects are cumulative, but some protection may be offered by antioxidants such as ascorbic acid. Small amounts of metal ions increase the rate of betanin degradation. Therefore, a chelating agent can stabilize the color. Many protein systems present in food products also have some protective effect. In the last 10 years, several studies have presented data showing that red beet is a good source of antioxidants. In a test of linoleate peroxidation by cytochrome c, betanin exhibits ten times higher antioxidant activity than tocopherol, and three times higher than catechin. These compounds are cationized, which increases their affinity to membranes, a great beneficial attribute for antioxidants (Kanner, 2001). Beetroot red (E162), available as liquid beetroot concentrate and as beetroot concentrate powders, is suitable for products of relatively short shelf life, which do not undergo severe heat treatment, for example, meat and soybean protein products, ice cream, and gelatin desserts. Betalains possess high molar extinction coefficients and their coloring power is comparable to that of synthetic colorants. Compared with the latter, betalains are not toxic, nor do they cause allergic reactions. GlO
H +
N
HO
COO
O H
-
+ H2O - H2O
GlO
H
+ HO
N H
COOH HOOC H
HOOC H
N
COOH
H N
COOH
H
Betanin
Cyclodopa glucoside
REACTION 9.4 Degradation of betanin.
Betalamic acid
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9.7 QUINONE PIGMENTS These pigments are widely distributed. They are the major yellow, red, and brown coloring materials of roots, wood, and bark. They also occur at high levels in certain insects. The largest group is that of anthraquinone pigments. The most important qininoid pigments commercially available for use in foodstuffs are cochineal and cochineal carmine. Cochineal (E 120) is the red coloring matter extracted from the dried bodies of female insects of the species Dactylopius coccus Costa or Coccus cacti L. These insects are cultivated on cactus plants in Peru, Equador, Guatemala, and Mexico. The major pigment of cochineal is polyhydroxyanthraquinone C-glycoside, carminic acid (Formula 9.16), which may be present at up to 20% of the dry weight of the mature insects. Cochineal extract or carminic acid is rarely used as a coloring material for food, but are usually offered in the form of their lakes. Aluminum complexes (lakes) can be prepared with various ratios of cochineal and Al varying from 8:1 to 2:1, having corresponding shades from pale yellow to violet. CH3
O
OH Gl
HOOC
OH
HO O
OH
FORMULA 9.16
Cochineal carmine is insoluble in cold water, dilute acids, and alcohol, and slightly soluble in alkali giving a purplish-red solution. The shade becomes bluer at higher pH. Cochineal carmine is stable in light and heat, but the stability in the presence of SO2 is poor. In powdered form this pigment can be used for coloring various instant foodstuffs in alkaline solutions, and in ammonia for coloring different foods, including baked products, yogurts, soups, desserts, confectionery, and syrups. It is currently the most robust red colorant among the authorized natural colorants, and its affinity for proteins means that it is an excellent colorant for dairy products, though it has the disadvantage of high production costs.
9.8 TURMERIC AND CURCUMIN (E 100) Turmeric is native to South and Southeast Asia. It is cultivated in China, India, South America, and the East Indies. The annual production is more than 240,000 tons, about 90% of which is produced and consumed in India. Turmeric belongs to the Curcuma genus of the Zingiberaceae family. Several species of Curcuma are known, but only the Curcuma longa L. represents the turmeric of commercial importance (Delgado-Vargas, 2003). Turmeric is important as a spice and coloring agent. References to it can be found in ancient Indian Vedic texts. The yellow color is the principal functional
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269 O
O OCH3
H3CO
HO
OH
Curcumin
FORMULA 9.17
property of turmeric. The main compounds involved in color are curcumin (Formula 9.17), demethoxycurcumin, and bisdemethoxycurcumin. The commercial forms are turmeric powder and oleoresin, as well as purified curcumin—a yellow, crystalline, odorless powder. Turmeric and curcumin are insoluble in water but soluble in alkalis, alcohols, and glacial acetic acid, and are used mainly as food color and secondarily as a spice. The use of turmeric depends on the food item and the part of world. Generally turmeric powder is used in mustard paste and curry powder as a colorant and for aroma. Oleoresin is added to mayonnaise, to breading of fish and potato croquettes, and to nonalcoholic beverages. However, curcumin is added to products where turmeric is incompatible, such as cheese, butter, ice cream, and some beverages. All turmeric pigments have good heat stability, but they are light sensitive. Curcumin is used not only as a food colorant and spice, but also as a healthpromoting food supplement. It has been shown that curcumin has a wide range of therapeutic actions: it protects against free radical damage because it is a very effective antioxidant; it reduces inflammation by lowering histamine levels; it protects the liver from different toxic compounds; and it keeps platelets from clumping together, which may protect against atherosclerosis.
9.9 RIBOFLAVIN (E 101) Riboflavin (vitamin B2, Formula 9.18) is a yellow pigment present in many products of plant and animal origin. Milk and yeast are the best sources of riboflavin. It is an orange-yellow crystalline powder, intensely bitter tasting, which is very slightly soluble in water and ethanol, affording a bright green-yellow fluorescent CH2OH (HCOH)3
Riboflavin
CH2 H 3C
N
H3C
N
N
NH O
FORMULA 9.18
O
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solution. Riboflavin is stable under acid conditions, but unstable in alkaline solution and when exposed to light. Reduction produces a colorless leuco form, but color is regenerated again in contact with air. Riboflavin-5′-phosphate sodium salt is much more soluble in water than unesterified riboflavin, and is not so intensely bitter. It is one of the physiologically active forms of vitamin B2. It is rather more unstable to light than riboflavin. Both forms can be used as coloring and as an enriching food additive to cereal, dressing, and cheese.
9.10 CARAMEL (E 150) Caramels are the brown to brown-black viscous liquids or hygroscopic powders resulting from carefully controlled heat treatment of different carbohydrate sources, such as dextrose, invert sugar, malt syrup, or lactose in the presence of a selected accelerator. A good source must have high levels of glucose because caramelization occurs only via the monosaccharide. Heating induces several chemical reactions resulting in the generation of a complex mixture of polymeric substances (>3000 Da) and low-molecular-weight compounds (<1000 Da) (Delgado-Vargas and Paredez-López, 2003). The composition and coloring power of caramel depend on the type of raw material and processing conditions. Both Maillard and caramelization reactions are involved, and the commercial products are extremely complex in composition. Four classes of caramels are recognized depending on the accelerator used: burnt sugar, caustic, ammonia, and ammonium sulfite. The first is used mainly as a flavoring additive, and the other as food colorants. Caramel is the colorant soluble in water and insoluble in organic solvents. It not only imparts color but also has important functional properties: it stabilizes colloidal systems and prevents the haze formation in beers, facilitating the dispersion of water-insoluble components; inhibits enzymatic browning; and retards the flavor changes and preserves the color of beverages exposed to light. The U.S. Food and Drug Administration (FDA) permits the use of caramel color in food in general. Over 80% of the caramel produced in the United States is used to color soft drinks, particularly colas and root beers.
9.11 MELANOIDINS Melanoidins represent an important group of brown pigments formed as a consequence of food processing. Formation of this pigment is favored by heat treatment and includes a number of complex reactions, mainly Maillard reactions. It is a type of nonenzymatic browning that involves the reaction of simple sugars and amino acids or proteins. They begin to occur at lower temperatures and at higher dilutions than caramelization, and occur in most food upon heating and also take place in the human body. The Maillard reaction has three basic phases: 1. The initial reaction is the condensation of the carbonyl group of reducing sugars with a free amino acid group, which loses a molecule of water, to form N-substituted glycosylamine.
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2. The unstable glycosylamine undergoes the Amadori rearrangement to form ketosamines. 3. The ketosamine can then react in different ways: One is simply dehydration (loss of 2 H2O molecules) into reductones and dehydroreductones, which are powerful antioxidants. A second is the production of shortchain hydrolytic fission products, such as diacetyl, acetol, and pyruvaldehyde. A third path is the Schiff’s base or furfural path. This involves the loss of 3 H2O molecules and then a reaction with amino acids and water. All these products react further with amino acids to form the brown nitrogenous polymers and copolymers called melanoidins (Nicoli and Manzocco, 2002). Because the Maillard reaction produces water, a high water activity environment inhibits the reaction. In practice, this reaction occurs most rapidly at intermediate aw (0.5 to 0.8). The temperature increase or time of heating results in increases in color development. Pentoses react more readily than hexoses, which in turn are more reactive than disaccharides (e.g., lactose). Different amino acids produce different amounts of browning. Of all amino acids, lysine results in the most color in the Maillard reaction due to its free ε-amino group. Therefore, foods containing protein rich in lysine, such as milk, are likely to brown readily. Much of the color that develops when foods are heated is due to the occurrence of melanoidins which, depending on their molecular weight, may be divided into two classes: low-molecular weight below 1000 Da, consisting of up to four linked rings, and high-molecular weight up to 150,000 Da (Nicoli and Manzocco, 2002). Melanoidins formed in the advanced steps of the Maillard reaction are responsible for a great variety of changes in food quality attributes and stability. The most important are browning, typical flavor formation, viscosity and water activity increase, enzyme activity inhibition, antimicrobial activity, and lipid and pigment oxidation slowdown. Typical effects of Maillard reactions may be observed as changes in the color of dried and condensed milk, browning of bread into toast, as well as the color of beer, chocolate, or maple syrup. Most of the functional and technological properties attributed to melanoidins trace their origin to the ability of these compounds to act as antioxidants able to act as chain breakers, reducing compounds, and metal chelators. However, food subjected to severe heat treatment may generate, via Maillard reaction, low but effective levels of mutagenic and carcinogenic amines.
9.12 MELANINS Melanins are water-insoluble polymers of various types of phenolic and indolic compounds, mainly derived from the tyrosine enzymatic oxidation. In general they are not homopolymers, and form large and complex macromolecules. Melanins are responsible for the black, gray, and brown colors of animals, plants, and microorganisms, where they are usually complexed with protein and often with carbohydrates. Because melanins are the aggregates of smaller component molecules, there are a number of differing types of melanins with differing proportions and bonding
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patterns of these component molecules. Eumelanin is the most abundant melanin in animals and humans, and is responsible for the black and brown colors of skin and hair. This is the melanin form most likely deficient in albinism. In mammals, eumelanins are accumulated with proteins and metals, such as Fe, Cu, or Zn. In seeds, spores, and fungi, another group of melanins—allomelanins (quinons)—is present. Melanins form but also scavenge free radicals; they dissipate energy, and thus provide protection from the damaging effects of radiation, electronic energy, and sunlight. They are able to bind metals and consequently, if any metal is toxic to the organism, melanins can prevent its entry into cells. In conclusion, melanins are not important for normal growth and development, but very important for the survival of many organisms.
9.13 SYNTHETIC ORGANIC COLORS Until the mid-19th century all dyes were obtained from plant or animal extracts. The textile industry used natural pigments, such as cochineal, turmeric, wood madder, or henna. In 1856, H. Perkin established the first factory of organic synthetic dyes to produce mauve. A few years later the discovery of diazotization and a coupling reaction by Peter Griess was the next major breakthrough for development of the color industry. In the 19th century, synthetic organic dyes were developed, creating a more economical and wider range of colorants. Since then their quality has been improved due to extensive research and development. The economic importance of the color industry is clearly reflected in the large number of synthesized compounds; as many as 700 colorants are currently available. Toward the end of the 19th century, when synthetic colors were first adopted for use on a large scale, they were hailed as a significant technological breakthrough. The term synthetic was associated with the idea of progress and synthetic colorants were actually considered safer in food than the naturals, as they were tinctorially much stronger and hence a smaller quantity was needed to achieve a specific colored effect. Synthetic colorants were used in foods, medicines, and cosmetics, but through the years their importance diminished. This retrenchment of synthetic colorants started about five decades ago. All synthetic food components suffered severe criticism, including synthetic additives and particularly food pigments. Color additives were one of the first man-made products regulated by law. Today, all food color additives are carefully regulated by federal authorities to ensure that foods are safe to eat and accurately labeled. Government regulation of food colorants is complex, and consequently legislation differs around the world. As an example, some colorants are permitted in the United States and by the World Health Organization (WHO): alura red, brilliant blue, fast green, but not by the European Union (EU); others are permitted by the EU and WHO: carmoisine, ponceau 4R, patent blue V, but not in the United States; still other pigments have wide acceptability around the world, such as sunset yellow, tartrazine, indigotine and erythrosin (Delgado-Vargas and Paredez-López, 2003). The main certifiable synthetic colorants, permitted for use in foods, drugs, and cosmetics (FD&C), according to their chemical structure, are:
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1. Azo dyes: a. Tartrazine (E 102); C.I. food yellow 4; bright orange-yellow powder; freely soluble in water b. Sunset yellow (E 110); C.I. food yellow 3; FD&C yellow No 6; orangered crystals soluble in water, slightly soluble in ethanol c. Alura red (E 129); C.I. food red 17; FD&C red No. 40; monoazo compound; soluble in water and in 50% alcohol 2. Triarylmethane dyes: a. Brilliant blue (E 133); C.I. food blue 2; FD&C blue No. 1; reddish-violet powder or granules with a metallic luster; soluble in water and ethanol b. Fast green; C.I. food green 3; FD&C green No. 3; red to brown-violet powder or crystals; soluble in water, slightly soluble in ethanol 3. Indigoid dyes: a. Indigotin (E 132); C.I. food blue 1; FD&C blue No. 2; dark blue powder with coppery luster; sensitive to light and oxidizing agents; soluble in water, slightly soluble in alcohol, insoluble in organic solvent 4. Xanthene dyes: a. Erythrosine (E 127); C.I. food red 14; FD&C red No. 3; brown powder soluble in water to cherry red solution (Delgado-Vargas and ParedezLópez, 2003) They can also be divided into water-soluble, oil-soluble, insoluble (pigment), and surface-marking colors. Water solubility is conferred on many dyes by introducing to the molecule at least one salt-forming group. The most common is the sulfonic acid group, but carboxylic acid residues can also be used. These dyes are usually isolated as sodium salts. They have colored anions and are known as anionic dyes. The other dyes containing basic groups, such as –NH2, –NH–CH3, or –N(CH3)2, form water-soluble salts with acids. These are the cationic dyes and their colored ion is positively charged. If both acidic and basic groups are present, an internal salt is formed. Oil-soluble or solvent-soluble colorants lack salt-forming groups. Precipitation of water-soluble colors, with Al, Ca, or Mg salts (generally with Al), forms water-insoluble lakes. Lakes may be prepared from all classes of watersoluble food colors, and they are one of the most important groups of food color pigments. The stability of synthetic colors in food processing conditions depends upon product composition, temperature, and time of exposure. Generally they are resistant to boiling and baking, but light has a destructive effect on all such colors. Certifiable synthetic color additives are preferred over natural additives for their coloring ability. They need to be added in smaller quantities, are more stable, provide a wide range of hues, and do not impart undesirable flavors to foods. The use of synthetic colors is the most reliable and economical method of coloring those products that have little or no natural color present, such as dessert powders, dairy-based desserts, beverages, dry powder drinks, candy and confectionery products, table jellies, and sugar confectionery.
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REFERENCES Barua, A.B. et al., Vitamin A and Carotenoids, in Modern Chromatographic Analysis of Vitamins, A.P. De Leenheer, W.E. Lambert, and J.F., Van Bocxlaer, Eds., Marcel Dekker, Inc., New York, 2000, p. 8. Berg, H. et al., The potential for improvement of carotenoid levels in food and the likely systematic effect. J. Sci. Food Agric., 80, 880, 2000. Brouillard, R., Origin of exceptional color stability of the Zebrina anthocyanin, Phytochemistry, 20, 143, 1981. Brouillard, R., Chemical structure of anthocyanins, in Anthocyanins as Food Colours, P. Markakis, Ed., Academic Press, New York, 1982, p. 1. Brouillard, R. et al., pH and solvent effects on the copigmentation reaction of Malvin by polyphenols, purine and pyrimidine derivatives, J. Chem. Soc. Perkin Trans. 2, 1235, 1991. Cai, Y. et al., Identification and distribution of simple and acetylated betacyanins in Amaranthaceae, J. Agric. Food Chem., 49, 1971, 2001. Combs, G.F., Jr., The Vitamins—Fundamental Aspects in Nutrition and Health, Academic Press, Inc., San Diego, CA, 1992, 121. Czapski, J. The effect of heating conditions on losses and regeneration of betacyanins, Z. Lebensm. Unters. Forsch., 180, 21, 1985. Delgado-Vargas, F. and Paredes-Lopez, O., Natural Colorants for Food and Nutraceutical Uses, CRC Press, Boca Raton, FL, 2003. Humphrey, A.M., Chlorophyll, Food Chem., 5, 57, 1980. Humphrey, A.M., Chlorophyll as a color and functional ingredient, J. Food Sci. 69, 425, 2004. Jacobucci, G.A. and Sweeny J.G., The chemistry of anthocyanins, anthocyanidins and related flavylium salts, Tetrahedron, 39, 3005, 1983. Kanner, J. et al., Betalains—A new class of dietary cationized antioxidants, J. Agric. Food Chem., 49, 5178, 2001. Klaui, H. and Bauernfeind, J.C., Carotenoids as food colours, in Carotenoids as Colourants and Vitamin A Precursors, Academic Press, New York, 1981, p. 47. Macheix, J.J., Fleuriet, A., and Billot, J., Fruits Phenolics, CRC Press, Boca Raton, FL, 1990, p. 239. Mazza, G. and Miniati, E. Anthocyanins in Fruits Vegetables and Grains, CRC Press, Boca Raton, FL, 1993, p. 1. Muller, H., Determination of the carotenoid content in selected vegetables and fruits by HPLC and photodiode array detection, Z. Lebensm. Unters. Forsch. A 204, 88–94, 1997. Nicoli, M. and Manzacco, L., Functional and technological properties of food melanoidins, International Congress on Pigments in Food, Lisbon 11–14, VI, 2002. Rice-Evans, C. et al., Why do we expect carotenoids to be antioxidants in vivo, Free Rad. Res., 26, 381, 1997. Stintzing, F.C. et al., Evaluation of color properties and chemical quality parameters of cactus juices, Eur. Food Res. Technol., 216, 303, 2003. Wang, H. et al., Oxygen radical absorbing capacity of anthocyanins,. J. Agric. Food Chem., 45, 304, 1997.
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10
Food Allergens Barbara Wróblewska
CONTENTS 10.1 10.2
Introduction................................................................................................ 275 Causes of Allergies .................................................................................... 276 10.2.1 Atopy ........................................................................................... 276 10.2.2 Exposure to Allergens ................................................................. 276 10.2.3 Environmental Factors ................................................................ 276 10.3 Mechanisms of Allergic Reactions to Food.............................................. 277 10.4 Allergen Terminology................................................................................ 278 10.5 Basic Food Allergens................................................................................. 278 10.5.1 Cow’s Milk Allergens ................................................................. 278 10.5.2 Egg Allergens .............................................................................. 280 10.5.3 Fish Allergens.............................................................................. 281 10.5.4 Crustacea Allergens .................................................................... 281 10.5.5 Nut Allergens............................................................................... 282 10.5.6 Peanut (Arachis hypogaea) Allergens......................................... 283 10.5.7 Soy (Glycine max) Allergens ...................................................... 284 10.5.8 Wheat Allergens (Triticum aestivum) ......................................... 285 10.6 Other Plant Materials as Sources of Food Allergens................................ 285 10.7 Food Additives........................................................................................... 286 10.8 Effect of Processing on Allergenicity of Foods........................................ 287 10.9 Allergen Cross-Reactions .......................................................................... 289 10.10 Antibiotic Contamination in Food ............................................................ 291 Glossary of Basic Terms Used in Immunology and Allergology ........................ 291 References.............................................................................................................. 292
10.1 INTRODUCTION Allergy is a very expansive disease. According to data obtained by the European Academy of Clinical Allergology and Immunology, 35% of the total population displays symptoms of allergy. A significant increase in allergy incidence concerns: • • •
Seasonal allergic inflammation of nasal mucosa Atopic bronchial asthma Atopic skin inflammation (especially in the female population in the form of allergy to nickel—over 25% of patients) 275
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A considerable increase in the incidence of allergic diseases has been recently attributed to the so-called hygienic hypothesis, which says that a small number of bacterial infections and exposure to bacterial endotoxins in early childhood are held responsible for directing immunological reactions toward allergy phenotypes. Children from large families with a low standard of living are at low risk for development of allergic diseases. The inhabitants of highly developed and industrialized countries are more exposed to allergens of dust, mold, and domestic animals because they spend more time in air-conditioned rooms (Jahnz-Różyk, 2004). The most common allergenic agents are pollens (49%), house dust (19%), chemicals (17%), food (17%), animal hair (16%), drugs (16%), nickel (13%), molds (6%), and latex (2%). Recently, particular attention has been paid to an increasing occurrence of food allergies, that is, an abnormal reaction of the organism with immunological background caused by consumption of food(s) or food additives. A number of allergen sources have been found making the so-called big eight of food allergens, which account for 90% of all IgE-dependent food allergies, namely, peanuts, nuts, milk, egg, soy, fish, crustaceans, and wheat.
10.2 CAUSES OF ALLERGIES 10.2.1 ATOPY The mechanisms of atopy, heredity, localization, and sequence of genes responsible for allergies have not been fully recognized yet. The probability of inheriting allergies when both parents are allergic is 60 to 70%; when only one parent is allergic, it is 30 to 40%; there is also a 10 to 15% chance that a child of nonallergic parents will be allergic. Genetic factors are estimated to be responsible for approximately 50% of all allergy cases. Because no changes were found within the human genome in the last century, a lot of attention is presently being paid to recognizing environmental factors.
10.2.2 EXPOSURE
TO
ALLERGENS
A necessary condition for hypersensitive reactions is contact with an allergen. The most common allergens are pollens, molds, house dust, mites and their excreta, animal epidermis, and insect venom. The degree of allergic symptoms depends mainly on individual sensitivity. Allergic response is caused by proteins and glycoproteins contained in pollen particles. Combining allergens with specific antibodies activates mastocytes which, as a result of lysis, excrete mediators such as histamine, leucotrienes, and prostaglandins, which are responsible for the clinical picture of disease.
10.2.3 ENVIRONMENTAL FACTORS An increase in allergy incidence is related to nonspecific factors with adjuvant character. These factors are:
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•
•
•
•
•
277
Ozone: This has an irritating effect on the respiratory tract and conjuctival mucosa. An increase in the amount of ozone is caused by exhaust fumes, industrial fumes and, indoors, by copiers and laser printers. Ozone causes so-called oxygen stress, that is, the condition of an elevated concentration of free radicals, which are responsible for destroying cells and structures significant for functioning of the organism. Formic aldehyde: This is released by paints, glues, synthetic carpets, and chipboards, causing damage to the ciliary apparatus of the mucosa responsible for clearing the respiratory tract of pollen, bacteria, viruses, and other substances. SO2, NO, NO2, CO2: These compounds are released during combustion of coal, gas, and engine fuels. In a humid environment they turn into sulfuric and nitric acid. They have a damaging effect on the respiratory tract mucosa, and by irritating nerve endings in the bronchi, cause inflammations. An increase in the number of people living in the vicinity of highways and suffering from dust diseases has been observed. Tobacco smoke: Both in the case of active and passive smokers, this brings about changes in the structure of the mucosa cilia of the respiratory tract, thus depriving it of the ciliary apparatus, which is directly related to causing allergies. Tobacco smoke is also claimed to be responsible for oxygen stress. Respiratory tract infections: Frequent bronchial infections in childhood are a cause of allergic responses at later ages. Asthma is likely to develop at a later age in 80% of people who had bronchitis as children. Household chemicals: Detergents, washing powders, liquid cleaners, bath soaps, and cosmetics, as well as alcohol and UV radiation can also be a cause of oxygen stress.
Also, changes in lifestyle, mainly hygienic conditions, ways and styles of feeding (e.g., bottle nursing from birth, abrupt introduction of a vegetarian diet), as well as frequent infections combined with inappropriately taken drugs are significant.
10.3 MECHANISMS OF ALLERGIC REACTIONS TO FOOD There are four basic mechanisms of inducing allergy: • • • •
Type Type Type Type
I: Anaphylactic and atopic reactions II: Cytotoxic and cytolytic reactions III: Arthus reactions induced by immunological complexes IV: Delayed cell reactions
Food allergies are most often caused by the type I mechanism, which refers to an allergic reaction occurring immediately following contact with an allergen. Protein allergens are hydrolyzed in the stomach and intestine to peptides and amino
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acids, which are recognized by the immunological system. So far no threshold value for the molecular weight decisive for the allergenic character of the formed peptides has been established. Following penetration into an antigen-presenting cell (APC), for instance, a dendritic cell, allergens undergo proteolysis. Peptides formed during hydrolysis are bound by the major histocompatibility complex (MHC) II, and are presented on the cell surface. By means of T cell receptors (TCR), T-lymphocytes recognize such complexes. Activated lymphatic T-cells initiate B-lymphocytes to produce IgE antibodies by interleukins. In case of the next contact of the organism with previously recognized allergens, immunoglobulin IgE binds to the surface of the mast cells through a high-affinity IgE receptor (FC&RI), which is followed by a release of allergic reaction mediators: leucotrienes, prostaglandins, and histamines. As a result of such reactions, itching rashes, erythema, urticaria, papular rashes, or other forms of allergic response occur.
10.4 ALLERGEN TERMINOLOGY Allergens are most frequently proteins or glycoproteins capable of inducing reactions of the organism, which depend on IgE. A system of naming newly isolated and characterized allergens has been established. The first three letters of a new allergen are given according to the taxonomic name of the species from which it has been extracted. Next, there is the first letter of the kind of a given species and Arabic number indicating the order of registering the new allergen with the Allergen Nomenclature Subcommittee of the World Health Organization/International Union of Immunological Societies (WHO/IUIS). For example, milk allergens, which originate from domestic cattle (Bos domesticus), are named Bos d 4 (α-lactalbumin), Bos d 5 (β-lactoglobulin), or Bos d 8 (caseins). In many cases it is unnecessary to specify the isoallergen or variant, and the corresponding suffixes may be deleted, such as Bet v 1 represents any Bet v 1 allergen (main allergen of birch) and Bet v 1.0101 represents a variant number 1 of isoallergen Bet v 1. When a given name is identical to that of an already existing one, it is necessary to add another letter to the first part of the name (e.g., Ves v 5—major venom allergen from Vespula vulgaris, and Ves vi 5—the allergen of Vespula vidua). Furthermore, it must be established whether the chemical compound that is to be designated as an allergen poses a threat to patients suffering from hypersensitivity. Labeling an allergen as dominating or less significant depends on the number of patients in the studied populations having IgE antibodies appropriate for the compound tested (Table 10.1). The information concerning allergens is collected and made available in databases on the Internet.
10.5 BASIC FOOD ALLERGENS 10.5.1 COW’S MILK ALLERGENS Cow’s milk contains about 30 to 35 g proteins per liter, of which caseins make 80% and whey proteins make 20%. Homologs of cow’s milk caseins occurring in the
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TABLE 10.1 Selected Food Allergens Allergen source systematic and original name Gadus callarias cod (muscle) Salmo salar Atlantic salmon (muscle) Bos domesticus cow’s (milk)
Apium graveolens celery
Daucus carota carrot Malus domestica apple
Arachis hypogaea peanut
Lycopersicon esculentum tomato
Solanum tuberosum potato
MW (kDa) SDS-PAGE
C: cDNA P: peptide sequence
Allergen M
12
C
Sal s 1
Parvalbumin
12
C
Bos d 4
α-lactalbumin
14.2
C
Bos d 5 Bos d 6 Bos d 7 Bos d 8 Api g 1
β-lactoglobulin Albumin bovine sera Immunoglobulin Casein Homologue: Bet v 1
18.3 67 160 20–30 16
C C
C
Api g 4 Api g 5 Dau c 1
Profilin 55/58 16
P C
Dau c 4 Mal d 1
Profilin homologue: Bet v 1
Mal d 2 Mal d 3 Mal d 4 Ara h 1
Thaumatin-like proteins Nonspecific lipid transfer protein Profilin Vicilin
Ara Ara Ara Ara Ara Ara Ara Lyc
Allergen name
Biochemical name
Gad c 1
Homologue: Bet v 1
C C
9 14.4 63.5
C C C C
2S Albumin Glycinin Glycinin Profilin 2S Albumin 2S Albumin Bet v 1 family Profilin
17 60 37 15 15 15 17 14
C C C C C C C C
Lyc e 2 Lyc e 3 Sola t 1
Beta-fructofuranosidase Nonspecific lipid transfer protein Patatin
50 6 43
C C P
Sola t 2 Sola t 3 Sola t 4
Cathepsin D inhibitor Cysteine protease inhibitor Aspartic protease inhibitor
21 21 16+4
P P P
h2 h3 h4 h5 h6 h7 h8 e1
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milk of other animals, such as goats, sheep, or mares, are identical in 80 to 90% of chemical structure; therefore, introducing these kinds of milk into diets of allergic patients seems pointless (Wal, 2001). European Society of Pediatric Allergy and Clinical Immunology (ESPACI) and European Society for Pediatric Gastroenterology and Nutrition (ESPGHAN) experts present a unequivocal opinion in this regard as they forbid applying both milk of a species other than cow and so-called partly hydrolyzed formulas in cases of allergic responses (Høst et al., 1999). People allergic to casein are most frequently sensitive to all four fractions of this protein. The immunological defense of the organism manifested by forming anticasein antibodies (IgE) is related to the occurrence of homologous sequences of amino acids, in particular cross-reacting epitopes. One of the epitopes with an immunoreactive character resistant to digestive processes is the phosphorylated region, present within α S1-, α S2-, and β-casein. Spuergin et al. (1997) characterized three peptide sequences (AA: 19–30, 86–103, and 141–150) of α-casein, which reacted with the serum collected from 15 allergic patients. These peptides were localized in the hydrophobic regions of the molecules and became accessible for antibodies only after casein denaturation. Among whey proteins, the β-lactoglobulin (β-lg) (Bos d 5) and α-lactalbumin (α-la) (Bos d 4) are the most potent allergens. β-lg occurs in cow’s milk in two genetic forms: A and B, whose mutations differ in positions 64 and 118. Form A contains the residues of aspartic acid and valine, while form B contains glycine and alanine. One molecule contains 2 disulfide bonds and 3 free thiol groups. Such a structure permits interaction with casein during thermal processes. β-lg is relatively resistant to acid hydrolysis and protease action; therefore, it remains largely unhydrolyzed during transport through the digestive tract, mainly the intestinal mucosa. β-lg belongs to a lypocaline family, whose characteristic features include binding and transfer of hydrophobic ligands and retinol, as well as high allergenic potential. Taking even small amounts of antigen in the form of, for example, selected hydrolyzed β-lg fractions, may activate tolerance of the organism. The α-la of cow’s milk reveals a high structural affinity to the α-la of human origin. Studies on animals (Hopp and Woods, 1982) showed that the polypeptide loop between amino acid residues (60–80):S–S:(91–96) was the strongest antigenic region. Amino acid sequences capable of binding IgE are also localized in the strongly hydrophobic part of the α-la particle, for example, between 99 and 108 amino acid residues, as well as in the region of amino acid sequences 17 to 58 and 108 to 123. The degree of affinity of these regions to human α-la is, respectively, 81 and 87%.
10.5.2 EGG ALLERGENS The majority of allergies to egg concern children aged 4 to 5 years, or up to 10 years of age. The egg white (~10%) is the most significant source of allergens, of which ovotransferrin, ovomucoid, ovalbumin, and lysozyme are the strongest. Generally, 100% of the examined patients allergic to hen’s eggs display a positive response to
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the presence of ovoalbumin (Langeland, 1983a,b). The data concerning lysozymes are not unequivocal. Allergens isolated from the hen egg yolk are apoviteline of lipoprotein fraction and α-livetin, which may cause allergic responses through the respiratory tract (Poulsen et al., 2001). Among less potent allergens are ovomucin and phosvitin. Allergy to egg may also be an occupational disease of egg processing industry workers. Therefore, environmental monitoring is recommended to check the level of egg allergens in processing plants, both in the places of direct contact in the technological lines and in the adjacent office facilities (Zanoni et al., 2002).
10.5.3 FISH ALLERGENS Allergy to fish occurs more often in countries with the highest fish consumption (Norway, Japan) than elsewhere. Allergy induced by fish consumption played a historic role in the pioneer studies on opaque organism reactions. It was the protein isolated from fish muscle tissue that was used by Prausnitz and Kustner to prove the existence of an agent in blood serum that was years later defined as immunoglobulin IgE. The protein was the first allergen whose amino acid sequence was characterized and called Cod M, thus giving rise to using new terminology for allergens. In later terminology the name was altered to Gad c 1 (Gadus is the first part of the Latin species name of cod) and classified as parvalbumin. The proteins belonging to this group control the movement of calcium ions to and from the cell. Their presence was found in the muscles of fish (0.05 to 0.1%) and reptiles. The presence of protein structurally similar to the Gad c 1 structure was confirmed in the muscle tissue of pike and carp. The molecular weight of Gad c 1 is 12.3 kDa. The allergen consists of 113 amino acid residues and one glucose molecule. At least five regions of binding IgE were demonstrated in Gad c 1 structure (Elsayed and Apold, 1983) with the arginin residue in position 75 playing the dominant role. The glucose moiety localized next to Cys-18 had no influence on the allergenicity of the whole compound. Trypsin hydrolysis made it possible to reveal a very strongly allergenic region (AA: 33 to 44) and some regions with lower allergenic potential (AA: 88 to 96). Among the less important fish allergens are Ag-17-cod, protamine sulfate and a protein with molecular weight 63 kDa (Mata et al., 1994). Also isolated was an allergen from fresh cod extract with molecular weight 41 kDa, which is a homolog of aldehyde-phosphate dehydrogenase (Das Dores et al., 2002).
10.5.4 CRUSTACEA ALLERGENS Shrimp allergens are the best characterized group. The first ones (Antigens I and II) were isolated and described by Hoffman et al. (1981). Antigen I is a dimer with a molecular weight of 45 kDa, comprising 189 amino acid residues and 0.5% saccharides. It was isolated from both raw shrimps and chitinous-calcium exoskeletons. Antigen II was extracted from boiled shrimps, as acidic thermally stable glycoprotein with a molecular weight of 38 kDa comprising 341 amino acid residues and 4% carbohydrates. In later studies, Nagpal et al. (1989) described two allergens, which
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were labelled SA-I (molecular weight 8.2 kDa) and SA-II (molecular weight 34 kDa), and in 1992 allergens Pen a 1 and Pen i 1, both with a molecular mass of 36 kDa, were isolated from boiled shrimps (Panaeus aztecus) and (Panaeus indicus), respectively. On comparing the amino acid composition of allergens and their high homology it was obvious that Pen a 1, Antigen II and SA-II were tropomyosin. It is estimated that consumption of 1 ÷ 2 medium-sized shrimps can induce an anaphylactic reaction in allergic patients. Tropomyosin has also been determined as a crayfish allergen, Pan s 1 (Panulirus stimpsoni), and a lobster allergen, Hom a 1 (Homarus americanus). Both these proteins were cloned, their sequences were studied, and it was found that they were homologous to shrimp allergen Pen a 1 (Stanley and Bannon, 1999). Hypersensitivity to crab allergens is mainly observed in populations occupationally involved in crustacea processing. IgE-dependent reactions concern mainly extracts obtained during boiling crabs, and not during contact with the raw material. Allergenic proteins with molecular weights of 37 to 42 kDa were isolated from crab broth or from boiled crabmeat extract.
10.5.5 NUT ALLERGENS This group comprises nuts growing on trees in different climatic zones, including almonds, Brazil nuts, hazelnuts, and pistachio nuts. The most common cause of allergies to nuts in Europe is the walnut (Corylus avallena). The first and main allergen, isolated by Hirschwehr and coworkers (1992), was Cor a 1 with a molecular weight of 17 kDa, which reacted with IgE present in the serum of all patients allergic to this nut species. Another recognized allergen, with a molecular weight of 14 kDa, is pollen prophyline, which induced positive responses in 16% of patients studied. Pasterello’s group studies (2002) confirmed the existence of two allergens with molecular masses of 18 kDa and 14 kDa, which turned out to be homologs of birch pollen allergens Bet v 1 and Bet v 2, respectively. Other allergens were also found: (1) with a molecular weight of 9 kDa, most likely belonging to a group of lipid-transfer proteins (LTP), (2) with a molecular weight of 32 kDa, belonging to 2S albumins, with an atomic mass of 35 kDa, described as a legumin, and (3) with a molecular weight of 47 kDa, a glycoprotein. Beyer et al. (2002) isolated a protein from walnut tree pollen with a molecular weight of 40 kDa, which was identified as Cor a 9 and classified as a globulin of the 11S type. It was the first isolated walnut allergen whose detailed amino acid sequence was described, and whose cDNA library was established based on oligonucleotides characteristics. On the basis of the current data, a recombined allergen Cor a 1.04 with a low level of IgE binding was established and proposed as a basic component of specific immunotherapy in walnut protein allergy (Luttkopf et al., 2002). Determining the structure of its epitopes made it possible to find their affinity to peanut allergen Ara h 3 and soy allergen, which can be helpful in predicting cross-reactions (Beyer et al., 2002). The main allergens of almonds are two proteins identified as albumin 2S, structurally homologous to walnut allergen Jug r 1, and gamma conglutin, 60% homologous to gamma conglutin of lupine seeds, Lupinus albus, and protein 7S of soy,
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Glycine max. Both almond allergens cross-reacted with allergens of walnut and hazelnut allergens (Poltonieri et al., 2002). The strongest allergen of Brazil nuts is an albumin protein with a molecular weight 8 kDa, that is, Ber e 1, with a high methionine content, consisting of two subunits. On analyzing the IgE of patients allergic to nuts, it was found that their serum also reacted with proteins with molecular weights of 25 and 58 kDa, both allergens of minor clinical significance (Pastorello et al., 1998). Wang et al. (2002) isolated the main allergen in cashews (Anacardium occidental), labeled Ana o 1, with a molecular weight of 50 kDa. Its structure was found to contain 11 linear epitopes, of which 3 were immunodominating. Ana o 1 is a protein from a viciline family. In 2003, the same authors discovered the next major cashew allergen, Ana o 2, which belongs to the legume family. A characteristic feature of allergens of nuts growing on trees, and of many seeds, is that their main proteins potent for inducing allergic responses are legumins and albumins 2S. This may be a valuable tip in further studies on identification of plant allergens and the occurrence of cross-reactions and it is, consequently, related to correct clinical diagnosing of allergies.
10.5.6 PEANUT (ARACHIS
HYPOGAEA)
ALLERGENS
The frequency of allergy to peanuts is 0.5 ÷ 0.7% of the total population. This kind of allergy is the most common in the United States (approximately 2 million patients), and in Europe (Great Britain, Holland, and France). Allergy to peanuts is practically not observed in Germany, although studies in this direction indicate the occurrence of allergy symptoms in patients of allergological clinics (Lepp et al., 2002). In Saudi Arabia, about 20% of allergic patients suffer from this kind of hypersensitivity. Allergy to peanuts is the most common form of this disease and accounts for numerous cases of anaphylaxis. About 7 to 10% of total peanut proteins contain substances of confirmed allergenic character. The two best-known, isolated, and characterized allergens are proteins Ara h 1 and Ara h 2. The former has a molecular weight of 65 kDa and contains 4 immunodominating epitopes and 20 determinants of lower immunological significance, and is completely resistant to high temperatures. The latter, Ara h 2, has a molecular weight of 17 kDa, while yet another, Ara h 3, is a homolog of an 11S protein. Recently isolated allergens occurring in peanuts were consecutively labeled as Ara h 4 (36 kDa), Ara h 5 (14 kDa), Ara h 6 (16 kDa), Ara h 7 (14.5 kDa), and Ara h 8 (17 kDa). Pons et al. (2002) isolated a peanut allergen belonging to an oleosine family of low-molecular proteins (18 kDa) taking part in forming peanut mastocytes. On analyzing the serum of patients allergic to peanuts using immunoblotting, the strongest reactions toward proteins were found for those with molecular weights of about 34, 50, and 68 kDa. These proteins are most likely oleosine oligomers. IgEdependent reactions were stronger in the case of extracts obtained from roasted peanuts than from raw ones. So far the threshold dose of peanut protein capable of stimulating the organism to fight the allergen has not been determined. In some patients a dose of 100 µg of
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peanut protein can provoke an allergic response, while in others this kind of reaction occurs following ingestion of more than 50 mg of peanuts (Keck-Gassenmeier et al., 1999). In practice, the only effective protection measure for patients allergic to peanuts is avoiding contact with the potential allergen. This is often difficult because various food products may be contaminated by trace amounts of peanut proteins during processing. Such contaminants are of course not specified on the product label. This also applies to beverages and cosmetics. Allergies may be triggered not only by direct consumption, but also by inhalation. Wearing a bracelet indicating peanuts as a possible cause of anaphylactic shock and carrying a first-aid kit containing epinephrine for immediate administration are presently considered the best protection measures (Dutan and Rance, 2001). Symptoms of allergy occur almost immediately from the immunological system, and the digestive and respiratory tracts. A frequent result of contact with peanut allergens is asthma.
10.5.7 SOY (GLYCINE
MAX)
ALLERGENS
Soy is a popular protein source that is introduced early to the infant diet in the form of special formulas, especially in the case of infants displaying symptoms of allergy to cow’s milk. However, it has turned out that about 30% of children allergic to milk are also allergic to soy. Therefore, studies were undertaken on the immunoreactivity of soy proteins and other components. The main soy allergens are Gly m Bd 28K, Gly m Bd 30k, and 11S glycinin. Gly m Bd 28K is a glycoprotein oligomer with a molecular weight of about 150 kDa, belonging to the vicilin family. Gly m Bd 30k also occurs as a glycoprotein with a molecular weight of more than 300 kDa. This allergen is also known as P34, a protein binding mastocyte. Gly m Bd 30k is a homolog of thiol protease from the papain family, although most likely it is not a protease because its active cysteine region has been mutated to glycine. Seed storage globulin 11S (glycinin), with molecular weights of 20 kDa and 33 kDa, occur in the form of an oligomer with a molecular weight of 322 kDa. The epitopes of this allergen have been identified. Beardslee and coworkers (2000) pointed out two immunodominating determinants A (AA: 217 to 235) and B (253 to 265). Xiang et al. (2002) characterized the shorter epitope G2A (AA: 219 to 233) as significant. The peptide consisting of section AA: 247 to 261 revealed weak binding with IgE. Helm and his group (1998, 2000) described a soy allergen as a unit of glycinin G2, with a molecular weight of 21 kDa, with 11 epitopes in six regions: 1 to 23, 57 to 111, 169 to 215, 249 to 271, 329 to 383, and 449 to 471. The epitope in regions 1 to 23 was stated as specific for soy, whereas the remaining ones were capable of binding antibodies of patients allergic to peanuts. The other allergens of smaller clinical significance were labeled as Gly m 3, Gly m 4, 2S albumin, and 7S globulin. Seed storage proteins are thermally stable, resistant to proteolysis, and usually occur as aggregates with molecular weights of more than 100,000 kDa. Denaturation of β-conglycinin requires a temperature over 75°C, and even then only partial change of the secondary and tertiary structure is noticeable (Mills et al., 2001). The capability
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of forming aggregates and gels during heating is used in food technology, but the effect of these processes on allergenicity has not yet been fully determined. Allergens occurring in soy lecithin, commonly used as an emulsifier in food processing, pharmaceutical technologies, and production of cosmetics, have also been identified. Commercial lecithin consists mainly of phospholipids, although the presence of proteins causing IgE-dependent reactions has also been confirmed with molecular weights of 7, 12, 20, 39, and 57 kDa. The 12-kDa protein has been identified as a 2S albumin with a high content of methionine, and protein 20 kDa as the Kunitz trypsin inhibitor. The 39-kDa protein has not yet been recognized; hence, it could be a new allergen. Soy lecithin may also be a source of so-called hidden allergens (Gu et al., 2001).
10.5.8 WHEAT ALLERGENS (TRITICUM
AESTIVUM)
Allergies caused by contact with wheat concern mainly patients occupationally exposed to cereal allergens, such as workers in cereal processing plants and bakeries. The main allergen that may bring about anaphylaxis is Tri a 19 (omega-5 gliadin). This protein is held responsible for immediate allergic response in children. It was suggested that omega 5-gliadin should be used during IgE tests, which would allow elimination of oral wheat provocation, which is indispensable in making diagnoses (Palosuo et al., 2001). Matsuo et al. (2004) determined 7 epitopes, of which amino acid residues in positions Gln 1, Pro 4, Gln 5, Gln 6, and Gln 7 initiated reactions with IgE. Allergy to wheat should not be mistaken for diarrhea chylosa or celiac disease, which is a gluten enteropathy.
10.6 OTHER PLANT MATERIALS AS SOURCES OF FOOD ALLERGENS Four basic allergens occurring in potato have been classified. Sol t 1 (Sola t 1 or patatin), is a storage protein that plays a protective role against parasites. It was found to be highly homologous to Hev b 7—the latex allergen. Sol t 2 is a cysteine inhibitor of proteases, mainly catepsin D. This protein has a 32% affinity to the allergen of Lolium perenne grass pollen, Lol p 11, which accounts for the commonness of cross-reactions between pollens of grass, potato, and some fruits. Sola t 3 is a cysteine inhibitor of proteases, mainly papain and bromelain, represented by two isoforms: Sola t 3.0101 and Sola t 3.0102. Sol t 4 is an aspartyl inhibitor of proteases acting specifically against trypsin and chymotrypsin (Seppälä et al., 2001). Protease inhibitors have particularly stable conformation, high resistance to heating and digestive enzymes, and are therefore capable of reaching the intestine where they can induce allergic food reactions. Allergy to tomatoes is estimated to affect 1.5 to 16% of the world’s population. The data on the tomato as a source of allergens are few. Often the issue is considered in relation to allergies to grass pollen or latex. Tomato allergen Lyc e 1 belongs to a family of proteins called profilins, which are a direct cause of cross-reactions with birch pollen, which belongs to the same family. Recently another tomato allergen,
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namely, fructofuranosidase, has been identified and labeled as Lyc e 2 (Westphal et al., 2004). IgE-dependent allergic reactions caused by the presence of Lyc e 2 affect about 17% of patients among those hypersensitive to tomato. The structure of glycane is held responsible. The most important celery allergen is Api g 1, consisting of two isoforms: Api g 1.0101 and Api g 1.0201. This allergen is a homolog of birch pollen Bet v 1. Therefore, allergies to celery are frequent in countries of northern and central Europe, where birch is a very common tree. A recombined derivative of celery allergen, Api g 4, has been identified. It is a profilin that shows a high structural affinity to Bet v 2 of birch pollen. Celery allergens cross-react not only with birch pollen antigens but also in 78% of cases with soy (Gly m 3), in 75% with barley and, to a lesser extent, with mugwort and spices. The glycoprotein with a molecular weight of about 60 kDa has been isolated and classified as Api g 5. The thermolabile chief carrot allergen, Dau c 1, is a homolog of birch pollen Bet v 1. Carrot also contains profilin corresponding to allergen Bet v 2. In central Europe about 25% of atopic patients hypersensitive to foods are allergic to carrots. In 1985 a disease syndrome called celery-carrot-birch-mugwort-spice was described for the first time. This refers to an associated kind of allergy that can be caused by consuming celery, carrot, some spices, and by birch and mugwort pollen. Simultaneously, occurrence of cross-reactions caused by allergens from the mentioned sources can be expected. In spite of a general opinion that many people are allergic to strawberries or wild strawberries, no analytical studies aimed at indicating a chemical substance responsible for hypersensitivity to these fruits were undertaken until 2004. The latest studies by Karlsson et al. (2004) showed that the most significant allergen was a homolog of protein Bet v 1, the main birch pollen allergen.
10.7 FOOD ADDITIVES So far there is no proof that chemical substances added to food can cause food allergy. They may contribute to initiating pseudoallergic reactions, so it is recommended that they be avoided by patients suffering from different kinds of hypersensitivity. Table 10.2 presents the types of clinical symptoms that may occur as a result of an organism’s reaction to substances added to foods. Among the additives initiating allergic reactions are preservatives, sweeteners, dyes, and flavorings. Benzoates, when consumed excessively, can cause hypersensitivity in patients suffering from asthma and allergy, and in those hypersensitive to aspirin disorders, in the functioning of the digestive tract. Earlier studies suggested that aspartame (E 951) could cause Quincke’s edema, swelling of the eyelids, palms, and feet, or lip reddening in hypersensitive patients. As the occurrence of such symptoms is extremely infrequent, studies are still not complete. Allergic patients should avoid food products containing artificial azo-dyes such as tartrasine (E 102), food yellow 3 (E 110), Ponceau 4R (E 124), or amaranth (E 123). Sodium glutamate (E 627) is a substance extensively used in the cuisine of the Far East and in Asian countries; therefore, the symptoms of food intolerance
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TABLE 10.2 Influence of Food Additives on Clinical Symptoms Food additives
Undesirable clinical symptoms
Dye
Azo
Preservatives and antioxidants
Nonazo Sulfuric anhydride and sulfites Benzoate
Substances that enhance taste and odor Aromas and pharmaceutical raw materials Enzymes Sweeteners
Nitrate (III), nitrate (V), sorbic acid and its salts Butylhydroxyanisole Butylhydroxytoluene Monosodium glutamate
Vanillin Cinnamic aldehyde Papain α-amylaze Aspartame
Urticaria, vessel purpura, severity on atopic changes of skin, Quincke’s edema, anaphylactic reaction, dyspnea attack Urticaria, anaphylactic shock Dyspnea attack, urticaria, edema, angioneuric edema, shock reactions Urticaria, atopic contact dermatitis, severity of atopic changes on skin Urticaria Urticaria, severity of atopic changes on skin MSG syndrome
Severity of atopic changes on skin, urticaria Severity of atopic changes on skin, atopic contact dermatitis, urticaria Anaphylactic shock, allergic rhinitis, dyspnea attack Urticaria, Quincke’s edema, anaphylactic reactions, behavioral perturbation, headache
often caused by this substance are called Chinese Restaurant Syndrome. Yet data verification does not confirm the thesis that sodium glutamate is a strong allergen. Studies point to other ingredients of Asian cooking, such as shrimps, peanuts, nuts, and some spices, which are most likely responsible for allergic reactions.
10.8 EFFECT OF PROCESSING ON ALLERGENICITY OF FOODS Allergenicity of many proteins can be lowered as a result of heating, microwave and ultrasound treatment, gamma radiation, chemical reactions (cross-linking), as well as biotechnological and enzymatic processes. The effectiveness of the treatments depends on the type and amount of allergen to be eliminated. Drastic process parameters deteriorate the sensory value of the products and may be the cause of interactions leading to the formation of new allergens. Aggregation reactions, a result of hydrophobic interactions, are an unavoidable consequence of protein denaturation. Such interactions may lead to the loss of original antigenicity of the native proteins, but also to the formation of new antigenic structures. The effect of temperature is due to the destruction of the conformation
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of the epitopes, while linear epitopes remain mostly untouched. The latter, if not digested in the digestive tract, are easily transferred to the intestine and may trigger food allergies (Davis and Wiliams, 1998). Also, chemical modifications of proteins might reduce allergenicity. The effect of acetylation and succinylation on immunoreactivity of milk whey proteins, that is, α-la and β-lg, has been confirmed. The capability of milk proteins to bind specific antibodies following their reaction with acetic and succinic anhydrides decreased by over 99% (Wróblewska, 1996). One of the most effective ways of modifying immunoreactive properties of food components is making use of milk proteins’ capability to conjugate with polyethylene glycol (PEG). PEG is a nontoxic component, approved by the U.S. Food and Drug Administration (FDA) for both internal and external medical use. Conjugation of α-la and β-lg with PEG was found useful in lowering their antigenicity. The preparations were assessed using the enzyme-linked immunosorbent assay (ELISA) method for determining immunoreactivity of particular modified proteins (0.08% for α-la and 0.05% for β-lg compared to 100% for raw milk) (Wróblewska and Jędrychowski, 2002). Studies on the influence of microorganisms on the human organism, and particularly on the immunological system, stress the significance of microflora, especially of Lactobacillus, naturally occurring in the human intestine. The Lactobacilli added to yogurts have an immunostimulating effect because they increase the production of interferon-γ and lymphokines and stimulate the production of secretory immunoglobulin A (IgA). A simultaneous growth in the activity of phagocytes and lymphocytes is observed. On analyzing fermented milk beverages obtained using meso- and thermophilic strains, a significant lowering of allergenic properties was observed in 95% of them (Wróblewska, 1996). The results demonstrating the effect of enzymes produced during the growth of Lactobacillus delbrueckii ssp. bulgaricus appear significant as this strain, along with Streptococcus salivarius ssp. thermophilus, is a component of yogurt cultures. Producing fermented beverages with lowered immunoreactive properties using the most beneficial bacteria strains seems to be the right way to obtain hypoallergenic formulas. These formulas must appear palatable, as this is a decisive element for the product’s desirability among children, which is the population group most vulnerable to allergies caused by cow’s milk. Fermented milk products have a pleasant taste with a diversified acid note. Another advantage of fermentation is lowering the content of lactose, which is a frequent cause of food intolerance. The criteria of palatability should be taken into consideration during production of hypoallergenic formulas because children (who are their main consumers), usually are very sensitive to the taste and odor of milk products. Attempts are being made to decrease the allergenicity of proteins by partial hydrolysis. Enzymatic protein hydrolysis used for producing diet formulas usually proceeds in mild conditions (pH 6 to 8, temperature 40 to 60°C), which allows minimization of noxious products. Moreover, the hydrolysates are soluble, more thermally stable, and have a higher resistance to slight acidity changes or metal ions than the proteins from which they were produced. During manufacturing, care must
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be taken in the process especially with respect to the selection of enzymes suitable for the substrate and optimization of the process parameters. The hydrolysates of whey proteins, casein and soy, have been practically put to use in the production of formulas for infants with atopy and with assimilation and digestion disorders for immunological reasons. So far, no hypoallergenic formula has been produced that is completely devoid of allergenic and immunogenic properties while being satisfactorily tasty. Only mixtures of amino acids are completely nonallergenic. Other hypoallergenic products available on the market contain proteins capable of triggering allergy mechanisms (Høst and Halken, 2004). The aim of the current research is to find less allergenic or tolerogenic fractions in modified products so as to determine the possibility of proposing a type of milk protein modification that will ensure a complete elimination of allergenicity or will be capable of stimulating the organism to produce a spontaneous immunological barrier (Calvo and Gomez, 2002). It is already known that a mixture of peptides obtained as a result of casein hydrolysis with a molecular weight below 500 kDa still has allergenic properties. Hydrolysate allergenicity can only be confirmed during clinical trials using standardized samples. The possibility of inducing allergic reactions varies depending on the patient’s physical and mental state, allergen dose, and time necessary for stimulating the organism (Høst and Halken, 2004). Allergenicity of ready products can also depend on the carbohydrates and lipids used for supplementation in special diets. Hydrolysis should be followed by suitable unit operations aimed at obtaining the most beneficial product. Such conditions are met by, for example, ultrafiltration, which permits elimination of nonhydrolyzed proteins that may still be present after hydrolysis. New products can be approved as milk-replacement preparations if they are well tolerated by 90% of the population with previously confirmed hypersensitivity to the proteins that are the basic material for the production of the hydrolysate studied. The effect of gamma radiation on the changes in food material has also been observed with respect to the antigenicity of bovine blood serum albumin (Kume and Matsuda, 1995) and β-lg (Byun et al., 2002). The authors state that antigenic structures of conformation epitopes of bovine serum albumin are destroyed as a result of radiation. However, it was also found that in the case of sequential structures antigenicity could be greater (Byun et al., 2004). In studies on animals, antigenicity of allergenic proteins was observed to decrease under the action of applied gamma radiation. This observation is significant for allergens toward which no other technological methods can be applied without losing their properties, such as egg (Lee et al., 2005).
10.9 ALLERGEN CROSS-REACTIONS Cross-reactions were defined in the IUPAC (International Union of Pure and Applied Chemistry) Compendium of Chemical Terminology in 1994 as the capability of a substance other than an analyte to bind a reacting substance, or vice versa, as the
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TABLE 10.3 Cross-Reactivity between Pollen and Food Allergens Source of allergen
Name of allergen
Origin of cross-reactive allergen
Birch
Bet v 1, Bet v 2
Mugwort
Art v 1–4
Carrot Celery
Dau c 1, Dau c 4 Api g 1, Api g 4, Api g 5
Tomato Kiwi
Lyc e 1–3 Act c 1
Peanut Tree nut
Ara h 1–7 Jug r 1, Jug r 2, Ber e 1 Hev b 1–13
Beech, oak, alder, hazel, chestnut, kernel fruits, kiwi, carrot, celery, curry, banana, lychee, mango, orange, soy, paprika, pepper, coriander, tree nuts Ragweed, chamomile, sunflower, carrot, celery, anise seed, curry, kernel fruits, kiwi, mango, fennel, paprika, caraway, pepper, latex Birch, mugwort, celery, mango, melon, cucumber Birch, grasses, mugwort, ragweed, melon, cucumber, carrot, curry, mango, paprika, pepper, caraway, coriander Birch, grasses, peanut, latex Birch, grasses, latex, banana, avocado, tree nut, sesame poppy seed Mugwort, kernel fruits, pea, soy, tomato, latex Birch, hazel, mugwort, kiwi, sesame, poppy seed
Latex
Grasses, mugwort, ragweed, kernel fruits, banana, kiwi, mango, melon, papaya, avocado, potato, tomato, peanut, chestnut, rubber plant
capability of a substance other than a reagent to bind an analyte (IUPAC Compendium, 1994). In the case of studying allergenicity in clinical diagnostics, cross-reaction determines the occurrence of clinical symptoms in a patient displaying simultaneous hypersensitivity to food, inhalation, and contact allergens. Classic cross-reactions proceed mainly between plant pollen and some fruits and vegetables, and they usually concern patients with symptoms of allergic rhinitis and bronchial asthma (Cudowska and Kaczmarski, 2003) (Table 10.3). Discovery of prolifins, carbohydrate determinants, and lipid transfer proteins (LPT) contributed to working out the pathogenic mechanisms of food allergy. Within studies on the immunoreactive and allergenic properties of milk proteins, the most significant issue is the immunological affinity of the amino acid composition of particular milk proteins, that is, alike or identical structure of epitopes. The α-la and β-lg are the best-known milk proteins with allergenic properties. α-la has been shown to have an epitope in position Val 42-Glu 49, while for β-lg it was fragment Pro 48-Glu 55. The spatial structure of these fragments is almost identical. Observations of the spatial structure of other milk proteins (lactoferrin and lactoperoxidase) revealed the occurrence of structures with similar conformations, which seem to be responsible for binding IgE antibodies. By comparing them to epitopes α-la and β-lg, amino acid loops of lactoferrin and lactoperoxidase have been defined as their most likely determinants. Experimentally determined allergenic
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regions of lactoferrin are the N-fragment (Gly 83-Thr 90) and C-fragment (Leu 572Lys 579) (Sharma et al., 2001). Explaining the immunological affinity of milk from different animal species is crucial. Special attention is focused on presenting the affinity between cow’s and goat’s milk expressed by a high level of cross-reactions, that is, a high degree of immunological affinity of particular proteins. In practice, this means a possibility of occurring health complications when goat’s milk is used as a replacement product for allergic patients (Calvani and Alessandri, 1998: Bellioni-Businco et al., 1999; Restani et al., 1999, 2002; Pessler, 2004).
10.10 ANTIBIOTIC CONTAMINATION IN FOOD Sometimes unexpected pollution or unestimated additives, such as antibiotics, present in food can cause an allergy. Sensitization can be achieved unconsciously by drinking cow’s milk or eating meat of an animal with bovine mastitis earlier treated with penicillin. Allergy caused by penicillin is relatively frequent and is estimated at 1 to 10%. Penicillin has very strong immunogenic properties. The benzyl penicillol (β-lactam and trazolidin rings) called the bigger determinant is responsible for 95% of mild allergic reactions. The most frequent symptoms of antibiotic allergies are urticaria, erythema, Quincke’s edema, and asthma. The observed 5% of anaphylactic reactions can be caused by the side chain of penicillin, which is a small determinant of antibiotics. Some groups of β-lactams antibiotics demonstrated strong cross-reactivity, such as penicillin with cephalosporins, penicillin with carbapenems, and cephalosporins with monobactams (Puchner and Zacharisen, 2002).
GLOSSARY OF BASIC TERMS USED IN IMMUNOLOGY AND ALLERGOLOGY allergen An antigen capable of inducing type I hypersensitivity with IgE involvement. allergy An organism’s hypersensitivity resulting from contact with an allergen in which the immunological system is engaged. antibody A protein from the immunoglobulin group produced by the organism and involved in regulatory reactions. Depending on the heavy chain structure, five immunoglobulin classes are distinguished: IgA, IgD, IgE, IgG, and IgM. antigen A particle that forms a specific bond/binding with an antibody or T-cell receptor. atopy The hereditary capability to excessively produce IgE antibodies. B-cell A lymphocyte maturing in secondary lymphatic organs, which on its surface has immunoglobulins acting as receptors for an antigen. cross-reaction The capability of a substance other than an allergen to react with an antibody specific to a given allergen as a result of an occurrence of the same or similar epitopes.
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epitope (antigenic determinant) Part of an antigen (fragment of an amino acid sequence) recognized by a section of an immunoglobulin chain, called a paratope, or by a cell receptor particle. immunogenicity The capability of a substance to induce defensive reactions of the organism by producing antibodies or allergic T-cells. immunological (antigenic) adjuvant A substance which, taken along with an antigen, increases the immunological response to the antigen. immunoreactivity The capability of producing a reaction between an antigen and an antibody. paratope A fragment of immunoglobulin forming a bond or binding with an epitope. T-cell A lymphocyte from the bone marrow, which transfers to the thymus as it develops, and reacts with an antigen via a specific receptor.
REFERENCES Beardslee, T.A. et al. (2000) Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h, Int. Arch. Allergy Immunol., 123, 299. Bellioni-Businco, B. et al. (1999) Allergenicity of goat’s milk in children with cow’s milk allergy, J. Allergy Clin. Immunol., 103, 1191. Beyer, K. et al. (2002) Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions, J. Allergy Clin. Immunol., 110, 517. Byun, M.W. et al. (2002) Application of gamma irradiation for inhibition of food allergy, Radiat. Phy. Chem., 63, 369. Byun, M.W. et al. (2004) Changes of the immune reactivities of antibodies produced against gamma-irradiated antigen, Radiat. Phy. Chem., 71, 127. Calvani, M., Jr. and Alessandri, C. (1998) Anaphylaxis to sheep’s milk cheese in a child unaffected by cow’s milk protein allergy, Eur. J. Pediatr., 157, 17. Calvo, M.M. and Gomez, R. (2002) Peptidic profile, molecular mass distribution and immunological properties of commercial hypoallergenic infant formulas, Milchwissenschaft, 57, 187. Cudowska, B. and Kaczmarski, M. (2003) Alergiczne reakcje krżyowe-aspekty kliniczne i diagnostyczne, Alergia, 2, 17, 41. Das Dores, S. et al. (2002) IgE-binding and cross-reactivity of a new 41 kDa allergen of codfish, Allergy, 57, 84. Davis, P.J. and Wiliams, S.C. (1998) Protein modification by thermal processing, Allergy, 53, 102. Dutan, G. and Rance, F. (2001) Peanut allergy, Revue Francaise D’Allergologie et D’Immunologie Clinique, 41, 187. Elsayed, S. and Apold, J. (1983) Immunochemical analysis of cod fish allergen M: locations of immunoglobulin binding sites as demonstrated by the native and synthetic peptides, Allergy, 38, 44. Gu, X.L. et al. (2001) Identification of IgE-binding proteins in soy lecithin, Int. Arch. Allergy Immunol., 126, 218. Helm, R. et al. (1998) Cellular and molecular characterization of a major soybean allergen, Int. Arch. Allergy Immunol., 117, 29. Helm, R.M. et al. (2000) Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K, J. Allergy Clin. Immunol., 105, 378.
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Hirschwehr, R. et al. (1992) Identification of common allergenic structures in hazel pollen and hazelnuts: a possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen, J. Allergy Clin. Immunol., 90, 927. Hoffman, D.R., Day, E.D., and Miller, J.S. (1981) The major heat stable allergen of shrimp, Ann. Allergy, 47, 17. Hopp, T.P. and Woods, K.R. (1982) Immunochemical studies on α-lactalbumin, Mol. Immunol., 19, 1453. Høst, A. and Halken, S. (2004) Hypoallergenic formulas—when, to whom and how long: after more than 15 years we know the right indication, Allergy, 59, 45. Høst, A. et al. (1999) Dietary products used in infants for treatment and prevention of food allergy, Arch. Dis. Child., 81, 80. Hozyasz, K. (2001) Choroby atopowe u dzieci (wybrane aspekty), Nowa Medycyna, 109, 1 (http://czytelnia.esculap.pl/nm_astma/04.html). IUPAC (International Union of Pure and Applied Chemistry) Compendium of Chemical Terminology (1994) 66, 2517. Jahnz-Różyk, K. (2004) Alergologia na poczętku dwudziestego pierwszego wieku, Magazyn Wojskowego Instytutu Medycznego, 2, 1. Karlsson, A.L. et al. (2004) Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens, Allergy, 59, 1277. Keck-Gassenmeier, B. et al. (1999) Determination of peanut traces in food by a commerciallyavailable ELISA test, Food Agricult. Immunol., 11, 243. Kume, T. and Matsuda, T. (1995) Changes in structural and antigenic properties of proteins by radiation, Radiat. Phy. Chem., 46, 225. Langeland, T. (1983) A clinical and immunological study of allergy to hen’s egg white. III. Allergens in hen’s egg white studied by cross radio-immunoelectrophoresis, Allergy, 37, 521. Langeland, T. (1983) A clinical and immunological study of allergy to hen’s egg white. IV. Specific IgE-antibodies to individual allergens in hens’ egg white related to clinical and immunological parameters in egg-allergic patients, Allergy, 38, 492. Lee, J.W. et al. (2005) Changes of the antigenic and allergenic properties of a hen’s egg albumin in a cake with gamma-irradiated egg white, Radiat. Phy. Chem., 72, 645. Lepp, U. et al. (2002) Peanut allergy a problem in Germany as well: results of a random sample, Allergologie, 25, 314. Luttkopf, D. et al. (2002) Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01, Mol. Immunol., 38, 515. Mata, E. et al. (1994) Surimi and native codfish contain a common allergen identified as a 63-kDa protein, Allergy, 49, 442. Matsuo, H. et al. (2004) Identification of the IgE-binding epitope in omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis, J. Biol. Chem., 279, 12135. Mills, E.N.C. et al. (2001) Formation of thermally-induced aggregates of the soy globulin beta-conglycinin, Biochim. Biophys. Acta, 1547, 339. Nagpal, S., Rajappa, L., and Metcalfe, D.D. (1989) Isolation and characterization of heatstable allergens from shrimp (Penaeus indicus), J. Allergy Clin. Immunol., 83, 26. Palosuo, K. et al. (1999) A novel wheat gliadin as a cause of exercise-induced anaphylaxis, J. Allergy Clin. Immunol., 10, 912. Passler, F. (2004) Anaphylactic reaction to goat’s milk in a cow’s milk-allergic infant, Pediatr. Allergy Immunol., 15, 183.
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Pastorello, E.A. et al. (1998) Sensitization to the major allergen of Brazil nut is correlated with the clinical expression of allergy, J. Allergy Clin. Immunol., 102, 1021. Pastorello, E.A. et al. (2002) Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results, J. Allergy Clin. Immunol., 109, 563. Poltronieri, P. et al. (2002) Identification and characterization of the IgE-binding proteins 2S albumin and conglutin gamma in almond (Prunus dulcis) seeds, Int. Arch. Allergy Immunol., 128, 97. Pons, L. et al. (2002) The 18 kDa peanut oleosin is a candidate allergen for IgE-mediated reactions to peanuts, Allergy, 57, 88. Poulsen, L.K. et al. (2001) Allergens from fish and egg, Allergy, 56, 39. Prausnitz, C. and Kustner, H. (1921) Studien über die Überempfindlichkeit. Zentrallblat für Bakteriologie, 86, 160. Puchner, T.C. and Zacharisen, M.C. (2002) A survey of antibiotic prescribing and knowledge of penicillin allergy Ann. of Allergy Asthma & Immunol., 88, 1, 24. Restani, P. et al. (1999) Cross-reactivity between milk proteins from different animal species, Clin. Exp. Allergy, 29, 997. Restani, P. et al. (2002) Cross-reactivity between mammalian proteins, Ann. Allergy Asthma Immunol., 89, 11. Seppälä, U. et al. (2001) Identification of four novel potato (Solanum tuberosum) allergens belonging to the family of soybean trypsin inhibitors, Allergy, 56, 619. Sharma, S. et al. (2001) Structure and function of proteins involved in milk allergies, J. Chromatogr. B Biomed. Sci. Appl., 756, 183. Spuergin, P. et al. (1997) Allergenicity of α-casein from cow, sheep and goat, Allergy, 52, 293. Stanley, J.S. and Bannon, G.A. (1999) Biochemical aspects of food allergens, Immunol. Allergy Clin. North Am., 19, 605. Wal, J.M. (2001) Structure and function of milk allergens, Allergy, 56, 35. Wang, F. et al. (2002) Ana o 1, a cashew (Anacardium occidental) allergen of the vicilin seed storage protein family, J. Allergy Clin. Immunol., 110, 160. Wang, F. et al. (2003) Ana o 2, a major cashew (Anacardium occidentale) nut allergen of the legumin family, Int. Arch. Allergy Immunol., 132, 27. Westphal, S. et al. ( 2004) Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency, Allergy, 59, 526. Wróblewska, B., (1996) Studia nad eliminacją alergennych właściwości białek serwatkowych mleka w wybranych procesach technologicznych i biotechnologicznych, Ph.D. Thesis, Warmia and Mazury University, Olsztyn. Wróblewska, B. and Jędrychowski, L. (2002) Effect of conjugation of cow milk whey protein with polyethylene glycol on changes in their immunoreactive and allergic properties, Food Agricult. Immunol., 14, 155. Xiang, P. et al. (2002) Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins, Arch. Biochem. Biophys., 408, 51. Zanoni, G. et al. (2002) Specific immune response to occupational antigens in asymptomatic egg processing workers, Am. J. Ind. Med., 41, 490.
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Flavor Compounds in Foods Bonnie Sun-Pan, Jen-Min Kuo, and Chung-May Wu
CONTENTS 11.1
11.2
11.3
11.4
Sources of Flavors in Foods...................................................................... 296 11.1.1 Flavors Formed Naturally in Plants............................................ 296 11.1.1.1 Spices and Herbs ......................................................... 296 11.1.1.2 Fruits ............................................................................ 296 11.1.1.3 Vegetables .................................................................... 297 11.1.2 Flavors Produced by Microbes or Enzymes............................... 297 11.1.3 Flavor Produced by Heating or Cooking.................................... 298 11.1.4 Flavors from Flavorants .............................................................. 298 Molecular Structure and Odor of Flavor Compounds .............................. 298 11.2.1 Volatility and Intensity of Aroma Compounds........................... 298 11.2.2 Flavor Compounds and Their Odors .......................................... 299 Changes in Flavor during Food Storage and Processing.......................... 300 11.3.1 Changes Due to Nature of Flavor Compounds .......................... 300 11.3.2 Changes Due to Continuing Aroma Biogenesis......................... 301 11.3.3 Changes Due to Tissue Disruption or Enzyme Reactions ......... 301 11.3.3.1 Introduction.................................................................. 301 11.3.3.2 Alliums......................................................................... 301 11.3.3.3 Brassicas ...................................................................... 303 11.3.3.4 Mushrooms .................................................................. 303 11.3.3.5 Formation of Green-Grassy Notes in Disrupted Tissues.......................................................................... 304 11.3.3.6 Glycosides as Flavor Precursors ................................. 304 11.3.4 The Maillard Reaction and Flavor.............................................. 305 11.3.5 The Role of Lipid Oxidation ...................................................... 306 11.3.6 Interaction of Lipids in the Maillard Reaction........................... 308 11.3.7 Extrusion ..................................................................................... 308 11.3.8 Concentration and Other Processes ............................................ 309 11.3.9 Changes Due to Food Storage .................................................... 309 Use of Flavors in the Food Industry ......................................................... 309 11.4.1 Functional Properties of Flavor Compounds.............................. 309 11.4.2 Collection or Production of Flavoring Materials ....................... 310 11.4.2.1 Natural Flavor Materials.............................................. 310 11.4.2.2 Organic Chemicals Used in Flavorings ...................... 312 295
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11.4.3
Flavor Manufacturing.................................................................. 312 11.4.3.1 Flavor Compounding ................................................... 312 11.4.3.2 Process Flavor.............................................................. 313 11.5 Biotechnological Production of Flavors.................................................... 314 11.5.1 Microbial Production of Flavor Compounds.............................. 314 11.5.2 Enzymatic Generation of Flavor Compounds ............................ 315 11.5.3 Recombinant DNA Technology for Flavor Formations ............. 317 11.6 Applications of Flavors ............................................................................. 318 References.............................................................................................................. 318
11.1 SOURCES OF FLAVORS IN FOODS 11.1.1 FLAVORS FORMED NATURALLY
IN
PLANTS
11.1.1.1 Spices and Herbs Since antiquity, spices, herbs, and condiments have been considered virtually indispensable in the culinary arts. They have been used to flavor foods and beverages the world over. Spices can be grouped according to the parts of the plant used: leaves (bay, laurel), fruits (allspice, anise, capsicum, caraway, coriander, cumin, dill, fennel, paprika, pepper), arils (mace), stigmas (saffron), flowers (safflower), seeds (cardamom, celery seed, fenugreek, mustard, poppy, sesame), barks (cassia, cinnamon), buds (clove, scallion), roots (horseradish, lovage), and rhizomes (ginger, turmeric). Most of the spices and herbs contain volatile oils, called essential oils, which are responsible for the characteristic aroma. Some spices (capsicum, ginger, mustard, pepper, horseradish) are pungent, while paprika, saffron, safflower, and turmeric are valued for their colors. Many spices have some antioxidant activities (Chen et al., 1999; Lee et al., 2005; Teissedre and Waterhouse, 2000). Rosemary and sage are particularly pronounced in antioxidant effects. Cloves, cinnamon, mustard seed, and garlic have antimicrobial activities (Badee et al., 2003; Firouzi et al., 1998). Some spices have physiological and medicinal effects. Spiced foods contain substances that affect the salivary glands (Pruthi, 1980). 11.1.1.2 Fruits In fruits, the ester formation is regulated by alcohol acyltransferase (AAT), alcohol dehydrogenase (ADH), and lipoxygenase (LOX). The changes in AAT activity during ripening and postharvest storage of apple show a pattern concomitant with ethylene regulation, while ADH and LOX seem to be independent of ethylene modulation. Volatile production and accumulation depend on the availability of precursors. Fatty acids and amino acids play important roles in the generation of aldelydes, alcohols, and acids for ester formation (Defilippi et al., 2005). Fruits, especially climacteric fruits such as the tomato, subject to stresses such as low or high temperatures and low oxygen levels, are generally low in volatiles. Reductions in hexanal, hexenal, hexenol, and 6-methyl-5-hepten-2-one, and increases in ethanol and acetaldehyde are stress induced. Preformed volatile compounds, such
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as isobutylthiazol and methylbutanal/ol are also subjected to changes resulting from postharvest storage conditions (Boukobza and Taylor, 2002). Volatiles of C6–C12 alkanals and alkenals emitted from plants inhibit fungal infection, for example, in corn (Zeringue et al., 1996), soybean (Boué et al., 2005), and tomato. Different cultivars of fruit can vary significantly in volatile compositions and sensory properties. For melons, the major difference lies in the content of esters, particularly sulfur-containing esters and straight 9-carbon chain compounds of aldehydes and alcohols (Kourkoutas et al., 2006). Citrus fruits contain peel oil, from which the essence oil is obtained during concentration of the juice process. Citrus oils are characterized by a high percentage of terpene hydrocarbons (limonene, C10H16), which contribute little to aroma. The unique characteristics of limonene are its relative insolubility in dilute alcohol, and its susceptibility to oxidation, causing off-flavor production. If the monoterpenes are removed, the resulting oil is called terpene-free or terpeneless oil. Aldehydes, esters, and alcohols are the main contributors to the aromas of citrus oil. These compounds are relatively polar and soluble in water; therefore, they are satisfactory for applications in foods and beverages. Citrus essence oil is a complex mixture of aroma volatiles. Compounds present in high concentration may provide little or no aroma, whereas some aroma volatiles found at trace concentrations may have intense aroma activity. The concentrations must exceed the detection threshold in order for the odorous compounds to be perceived. Using the gas chromatography-olfactometry technique, the most intense aromas in orange essence oil are produced by octanal, wine lactone, linalool, decanal, β-ionone, citronellal, and β-sinensal (Högnadöttir and Rouseff, 2003). The balance of volatiles determines the final aroma and flavor acceptance. Fruits other than citrus contain much less volatile aromatic compounds and cannot form essential oil in distillates. However, their juices, juice concentrates, extracts of dehydrated fruits, and distillates (essence) can be used as flavorants directly added to foods. 11.1.1.3 Vegetables Vegetables contain some flavor compounds, the concentrations of which are mostly too low to obtain essential oils. In the tissues of some vegetables, volatile compounds are enzymatically produced when they are disrupted. Vegetables have the function of flavoring only after their cells are disrupted or after being fried in oil. Vegetable flavors are classified as savory flavors, while fruit flavors are classified as sweet flavors. In addition to the three natural flavor categories, spices and herbs, fruits, and vegetables described above, natural products like tea, coffee beans, cocoa beans, flowers (e.g., rose, jasmine), peppermint, and balsam have flavoring properties (Arctander, 1960; Furia and Bellanca, 1975; Ka et al., 2005).
11.1.2 FLAVORS PRODUCED
BY
MICROBES
OR
ENZYMES
Fermentation has been known and commercially exploited for centuries. Products like spirits, liqueurs, wine, beer, and other alcoholic beverages; vinegar; cheese and
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yogurt; miso, soy sauce, and fermented bean curd; ham and sausage; fish sauce; curing of vanilla beans, tea, and cocoa; pickles and sauerkraut; dough, bread, and other bakery products have special flavor notes. Biotechnology for the production of flavoring materials has been developed in the past couple decades. The technology relating to the production of flavors includes cell and tissue culture; microbial fermentation; and bioconversion of substrates using whole microbial cells, plant cells, or enzymes (Baldermann et al., 2005; Harlander, 1994; Kringer and Berger, 1998; Kuo et al., 2006). For example, some important aroma compounds of fruits are formed from enzymatic degradation of carotenoids (Baldermann et al., 2005); components of bread flavors originate from fermentation and thermal reactions involving the participation of amylases, proteases, and lipoxygenase (Martinez-Anaya, 1996). In a model system, lipoxygenase (LOX) extracted from fish gill reacts with roe lipid and results in a pronounced increase in green and fresh fishlike flavor notes, but a very slight decrease in polyunsaturated fatty acids (PUFA) (Pan and Lin, 1999); fish oil and chicken fat modified with LOX yields an aroma more desirable than the untreated lipids with minor decrease in PUFA (Hu and Pan, 2000; Ma et al., 2004 ); banana leaf LOX extract reacts with 18:2 or soybean oil pretreated with bacterial lipase produces a green and melonlike aroma, while with 18:3 produces sweet, fruity, cucumberlike flavor notes (Kuo et al., 2006).
11.1.3 FLAVOR PRODUCED
BY
HEATING
OR
COOKING
After being cooked, the flavors of foods such as wheat, peanuts, and sesame are quite different from those of the raw materials. Flavor formation from flavor precursors in the processed foods are primarily via Maillard reaction, caramelization, thermal degradation, and lipid-Maillard interactions.
11.1.4 FLAVORS
FROM
FLAVORANTS
Flavorings play essential roles in the production of a wide range of food products versatile in aroma to meet consumer needs. In this regard, flavor manufacturers require expertise in flavor formulations, research, and technical services, while flavor users need a fundamental knowledge of flavor applications.
11.2 MOLECULAR STRUCTURE AND ODOR OF FLAVOR COMPOUNDS 11.2.1 VOLATILITY
AND INTENSITY OF
AROMA COMPOUNDS
The flavor of food is largely perceived as a result of the release of odorous compounds, usually present in trace amounts in foods, into the air, in the mouth, and thence to the olfactory epithelium in the nose. Therefore, an aroma compound must be volatile. The volatility is caused by the evaporation or rapid sublimation of an odoriferous substance. It is proportional to the vapor pressure of the substance, and inversely proportional to its molecular weight. The aroma in food consists of compounds of high, intermediate, and low volatility. For example, the aroma of a meatlike
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process flavoring from hydrolyzed soy protein includes: hydrogen sulfide (cookedegg odor) and methanethiol (rotten/garbage odor), which are of high volatility, while 2-methyl-3-furanthiol (meaty), 3-mercapto-2-pentanone (catty, urine), 3-methylthiolpropanal (potato) are of intermediate volatility. Maltol and furaneol both give a burnt sugar odor and are of low volatility (Wu and Cadwallader, 2002). Other characteristics relate to aroma compounds, of which flavor intensity is the most important. Threshold is used most extensively for quantification of flavor intensity. Psychophysically, a threshold can be defined as the minimum concentration of a stimulus that can be detected (absolute threshold), discriminated (just-noticeable difference), or recognized (recognition threshold). In general, the detection thresholds are lower than the recognition thresholds, if the difficulty in measuring both are comparable (Pangborn, 1981). The relationship between the molecular structure of an aroma compound and its threshold is still unclear. Volatility of a compound may not relate to its threshold. For example, the threshold of ethanol (boiling point 78°C) is much higher than octanol (boiling point 195°C) or other homologous alcohols. Ethanol has high volatility but low odor intensity. It is often used as a solvent in compounded flavors. In general, sulfur- or nitrogen-containing compounds and heterocyclic compounds have very low threshold values. Thresholds of some odorous compounds in aqueous systems can be found at (http://www.leffingwell.com/ odorthre.htm, http://www.leffingwell.com/ald1.htm). Some of those present in lipid systems have been reported in the literature (Ma et al., 2004; Meynier et al., 1998). An odor unit is defined as the ratio of concentration to threshold. This unit quantifies the contribution of a specific component or a fraction to the total odor of a mixture; however, it says nothing about the odor quality of the final mixture, nor does it imply anything about the relationship between the stimulus concentration and the intensity of sensation above the threshold (Teranishi et al., 1981).
11.2.2 FLAVOR COMPOUNDS
AND
THEIR ODORS
The relationship between the molecular structure of a chemical compound and its odor has been the subject of much research and conjecture. It is still not possible to predict the aromatic profile from the structure of a given chemical, nor is it possible to assume changes in flavor profile based on molecular structure modification. Even stereoisomers may differ in odor both qualitatively and quantitatively. Nevertheless, the relationship between structure and odor is summarized as follows. Small molecules such as ethanol, propanol, and butanol among alcohols; acetaldehyde and propionaldehyde among aldehydes; and acetic acid, propionic acid, and butyric acid among acids are highly volatile and exhibit pungent ethereal, diffusive, harsh, or chemical odor characteristics. Only in extreme dilution of these compounds will desirable odor become perceptible. Bigger molecules of alcohols, aldehydes, and acids are mild and desirable at 5°C to 10°C. Molecules containing alcohols, aldehydes, and acids reduce their volatility and odor intensities with increases in molecular size at temperatures higher than 10°C. Compounds containing functional groups, such as –OH, –CHO, –CO, and –COOH, play important roles in exporting the odors of the compound. Acid is sour,
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aldehyde yields a fresh note, and ester is fruity. However, the elongated alkyl group enhances the fatty or oily note. Ketones, having two alkyl groups attached to a carbonyl group, give a more fatty aroma than the corresponding aldehydes. Ester has a fruity note. When the initial alkyl group in alcohol or acid or both is relatively large in molecular size or with its own characteristic note, the resulting ester maintains this note in addition to the fruity note. Examples are citronellyl acetate, having the fresh rosy-fruity odor, inherits the rosy note from citronellol; bornyl acetate, having a sweet herbaceous-piney odor with a balsamic undertone, maintains the odor of borneol. The boiling point of ethyl acetate is 77°C, and its molecular weight (MW) is 88. Those of its reactant, ethyl alcohol, are 78°C and MW 46, while those of the acetic acid are 118°C and MW 60, respectively. Ester has a higher molecular weight but a lower boiling point in comparison to the precursor alcohol and acid. Many esters, such as ethyl acetate, have a fresh note. Aldehyde has a relatively low boiling point. For example, acetaldehyde has a MW of 44, while its boiling point is 21°C. Therefore, aldehydes are often used due to their fresh note. For example, decanal contributes to the fresh note in orange aroma. Lactones are cyclic compounds with an ester functional group. They have the characteristic ester notes: fruity, oily, and sweet. However, they are cyclic compounds and have relatively high boiling points. γ-Undecalactone, with a peachlike aroma, has a boiling point of 297°C. Heterocyclic compounds generally have very low thresholds. Thiazoles, thiolanes, thiophenes, furans, pyrazines, and pyridines are normally present in larger numbers and higher concentrations in cooked, fermented, processed seafoods or meat products than in fresh ones. Essential oils, oleoresin, or other natural flavoring raw materials have many valuable trace components, which play important roles in aroma. These components are not commercially available now because of their complexity and low threshold. It is not feasible to use a complicated manufacturing process for the very small amount needed. The only source available is the natural product.
11.3 CHANGES IN FLAVOR DURING FOOD STORAGE AND PROCESSING 11.3.1 CHANGES DUE
TO
NATURE
OF
FLAVOR COMPOUNDS
A volatile compound evaporates continuously even at room temperature. Higher temperature accelerates the evaporation. Some food ingredients, such as lipids and proteins, may trap flavor compounds to some extent and reduce their volatility. Different flavor compounds have different volatilities. An aged food may not only lose its total flavor, but also change the proportions of its flavor components, resulting in a changed odor. Many flavor compounds containing double bonds or aldehyde groups are susceptible to oxidation, cleavage, polymerization, or interaction among components (Sinki et al., 1997). Alcohols can be oxidized to the corresponding aldehyde and then to acid. Alcohol and acid can react and dehydrate to form esters. Esters can be
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301
hydrolyzed to alcohol and acid at neutral or alkaline pH. Aldehyde and alcohol can be dehydrated by catalysis to form hemiacetal while the reverse reaction can occur in acidic conditions or in water.
11.3.2 CHANGES DUE
TO
CONTINUING AROMA BIOGENESIS
The amount of secondary metabolites, such as aroma compounds produced by a plant during its life cycle, is a balance between formation and elimination. The two opposing functions are directly controlled by two main groups of factors. The intrinsic factors comprise all internal or hereditary properties (e.g., genotype and ontogeny), while the extrinsic factors comprise all external or environmental properties (e.g., pressure, wind, light, temperature, soil, water, nutrients). Therefore, a plant material or the essential oil may have a quite different flavor quality due to culture conditions and maturity (Nagy and Shaw, 1990; Lawrence, 1986). For example, during ripening of chili, hexanal (green aroma) and 2-isobutyl-3-methoxypyrazine (grassy aroma) decrease significantly, while compounds contributing to sweet, fruity attributes, such as 2,3-butanedione, 3-carene, trans-2-hexenal, and linalool increase noticeably (Mazida et al., 2005). Butanol, 3-methylbutanol, benzyl alcohol, and α-terpineol develop in papaya relative to fruit ripeness (Almora et al., 2004). The typical flavor of climacteric fruit such as banana, peach, pear, and cherry is not present during early fruit formation, but develops fully during a rather short period of ripening. During that time, minute quantities of lipid, carbohydrate, protein, and amino acids are enzymatically converted to volatile flavors (Reineccius, 1994a). During postharvest handling, the plant continues the biogenesis of aroma. Even pruning of tea plants has an impact on the flavor of tea. LOX activity and total fatty acids and polyenoic acids decline while hexanal, hexenal, and pentanal increase with postpruning time. Tea flavor constituents including linalool, phenylacetaldehyde, geraniol, theaflavins and sensory flavor index all elevations with time from pruning (Ravichandran, 2004).
11.3.3 CHANGES DUE REACTIONS
TO
TISSUE DISRUPTION
OR
ENZYME
11.3.3.1 Introduction Some food flavors are not present in the intact plant tissues, but are formed by enzymatic processes when the plants are cut or crushed. Under these circumstances, the cells are ruptured and the flavor precursors are released and exposed to enzymes. Unique examples of this kind of flavor formation are described below. 11.3.3.2 Alliums Garlic, onion, shallot, green onion, and chive belong to the genus Allium. Members of this genus contain volatile sulfur compounds, including thiols, sulfides, disulfides, trisulfides, and thiosulfinates (Block and Calvey, 1994; Kim et al., 2005; Yamaguchi and Wu, 1975; Yu and Wu, 1989).
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Chemical and Functional Properties of Food Components
The enzymatic flavor formation reaction of the genus Allium can be generalized as follows: 2 RSOCH2CHNH2COOH → RSSOR + 2 NH3 + CH3COCOOH where R is methyl, propyl, 1-propenyl, or allyl. The 1-propenyl compound has been identified as the lacrimator in onions. When garlic is chopped or crushed, allinase, an enzyme present in intact garlic, is activated and acts on alliin to produce allicin. Allicin (diallyl thiosulfinate) is the major thiosulfinate compound in garlic and the principal bioactive compound present in garlic extract. In common with all thiosulfinates, allicin readily forms diallyl disulfide and diallyl trisulfide at room temperature. Addition of soybean oil in the process of garlic disruption can slow down the conversion of allicin (Kim et al., 1995). The water extract of heat-treated garlic contains mainly alliin due to allinase inactivation by heat. The major compounds present in aged garlic extract are Sallylcysteine and S-allylmercaptocysteine (Kim et al., 2005). Medicinal use of garlic oil is mostly prepared by hydrodistillation of raw garlic homogenate. Garlic oil is insoluble in water, and therefore can be separated or extracted afterward. Garlic oil consists of the diallyl, allyl methyl, and dimethyl mono- to hexasulfides. Garlic oil from oil-macerated or ether extracted garlic homogenate contains the 2-vinyl[4H]-1,3-dithiin and 3-vinyl-[4H]-1,2-dithiin, allyl sulfides and ajoenes (Kimbaris et al., 2006). When alliin and deoxyllin, two important nonvolatile flavor compounds of garlic, undergo thermal reaction with an aldehyde, that is, 2,4-decadienal in a model system, volatile compounds are generated in 3 categories: (1) degradation products of alliin mainly allyl alcohols, (2) degradation products of deoxyalliin mainly allyl sulfides, allyl mercaptan, allyl thioacetic acid, and dithiacyclopentane, and (3) interaction products such as hexylthiophenes, 2-pentylpyridine, 2-pentylbenzaldehyde, and 5formyl-2-pentylthiophene (Yu et al., 1994). Aroma of fresh leek alliums is dominated by sulfur-containing volatile compounds. The formation pathway is summarized (Krest et al., 2000; Nielsen et al., 2003) as follows: nonvolatile precursors, such as S-alk(en)yl-cysteine ↓ allinase (EC 4.4.1.4) sulfenic acids (highly reactive) ↓ thiosulfinates, thiopropanal-S-oxide ↓ monosulfides/polysulfides
(pungent odor)
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Allium aroma is dominated by dipropyl disulfide and declines during frozen storage (Nielsen et al., 2004). Frozen storage of unblanched leek shows respiration, the rate of which is affected by the degree of intactness of tissue or slice thickness. Thinner slices generate higher concentrations of total sulfur compounds and total aldehydes. Extended storage periods cause reductions in total sulfur-containing volatile compounds and inductions of aldehydes later produces off-flavor. Nitrogen packaging improves the aroma profile of fresh leek in comparsion to the atmosphericair packaging (Nielsen et al., 2004 ). The lipoxygenase (LOX) pathway also participates in the aroma development in fresh alliums as follows: polyunsaturated fatty acids containing cis,cis-pentadiene structure O2 ↓ LOX (EC 1.13.11.12) hydroperoxides ↓ hydroperoxide lyase (HPL, EC 4.1.2) aldehydes ↓ alcohol dehydrogenase (EC 1.1.1.1) alcohols The alcohols contribute to fresh leek aroma, but larger amounts of alcohols produce off-flavor. 11.3.3.3 Brassicas The Brassicas of importance include turnips, rutabagas, mustards, and the cole corps: cabbage, broccoli, cauliflower, and Brussels sprouts. The production of isothiocyanates in Brassicas occurs via an enzymatic reaction on specific glycosides. Some of the isothiocyanates, especially allylthiocyanate, are highly pungent and are mainly responsible for the odor of brown mustard, horseradish, cabbage, and other crucifers. Any process that destroys or inactivates enzymes in these plants will cause decreases in aroma production, resulting in a less distinctive flavor. This is usually the case when Brassica foodstuffs are commercially preserved. 11.3.3.4 Mushrooms 1-Octen-3-ol occurs in many mushroom species (Zawirska-Wojtasiak, 2004). It contributes significantly to the flavor of edible mushrooms such as Agaricus campestris (Tressl et al., 1982), and Agaricus bisporus (Wurzenberger and Grosch, 1982; Chen and Wu, 1984). 1-Octen-3-ol is formed enzymatically from linoleic acid, the major fatty acid in A. bisporus. Enzymes involved in the pathway of formation of
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Chemical and Functional Properties of Food Components
1-octen-3-ol include lipoxygenase, hydroperoxide lyase, and allene oxide synthases (Grechkin, 1998). Shiitake (Lentinus edodes) is a kind of edible mushroom highly prized in China and Japan. Volatile flavor compounds in young, immature, mature, and old growth stages of shiitake mushrooms include 1-octen-3-ol, 3-octanol, 3-octanone, and 4octen-3-one (Cho et al., 2003). Due to the difficulties of postharvest storage, the mushroom has been traditionally preserved in dried form. The differences between fresh shiitake and dried shiitake lie in the contents of eight-carbon compounds (i.e., 3-octanone, 1-octen-3-ol, 3-octanol, n-octanol, and cis-2-octen-l-ol) and sulfurous compounds (i.e., dimethyl disulfide and dimethyl trisulfide). Fresh shiitake contains more eight-carbon compounds than dried shiitake does, while dried shiitake contains more sulfurous compounds than the fresh (Chang et al., 1991). The formation of volatile shiitake is affected greatly by the pH during blending. 1-Octen-3-ol and 2-octen-l-ol are predominantly formed at pH around 5.0 to 5.5, while the formation of sulfurous compounds, such as dimethyl disulfide and dimethyl trisulfide, is around pH 7.0. Two enzymatic systems are probably responsible for the formation of eight-carbon and sulfurous compounds. One of the two enzymes likely occurs in dried shiitake, yielding more volatile sulfur compounds than in fresh shiitake (Chen et al., 1984). 11.3.3.5 Formation of Green-Grassy Notes in Disrupted Tissues Six-carbon compounds, such as hexanal, 3Z-, and 2E-hexenal occur at high concentrations in ruptured tissue of apples, grapes, and tomatoes (Fabre and Goma., 1999; Hatanaka, 1993; Schreier and Lorenz, 1981). (Z)-3-Hexen-l-ol, (E)-2-hexenal, hexanol, (E)-2-hexen-l-ol, and hexanal are formed in bell peppers (Capsicum annuun Var. grossum, Sendt) after tissue disruption (Buttery and Ling, 1992). These compounds, having green and fruity characteristics, are biosynthetically derived from the action of 13-lipoxygenase (Fukushige and Hildebrand, 2005; Hatanaka, 1993; Hornostaj and Robinson, 1999). For example, the sensory impression of hexanol has a green flavor note; 2-hexenol gives green and fruity notes; 3-hexenol (leaf alcohol), green and fresh notes (Whitehead et al., 1995). 11.3.3.6 Glycosides as Flavor Precursors There are two forms of monoterpene derivatives in grapes: free and glycosidic conjugates. The free form consists of compounds with interesting flavor properties, such as geraniol, nerol, linalool, linalool oxides, α-terpineol, citronellol, hotrienol, and flavorless polyhydroxylated compounds (polyols) which, under mild acid hydrolysis, can yield odorous volatiles. Glycosides of volatile compounds identified in plants and fruits are mainly O-β-D-glucosides or O-diglycosides. The flavorless glycosides consist of α-D-glucopyranosides and diglycosides; 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides, 6-O-α-L-rhamnopyranosyl–β-D-glucopyranosides (rutinosides), and 6-O-β-D-apiofuranosyl-β-D-glucopyranosides of predominantly geraniol, nerol, and linalool, together with monoterpenes, are present at a higher oxidation state than the
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free forms (Osorioa et al., 2003; Sarry and Gunata, 2004). As most of the aglycones of these compounds have interesting sensory properties, their glycosides make up a potential aroma reserve more abundant than their free counterparts. Upon either acid or enzyme hydrolysis, the glycoside-bound aglycones can be released to produce flavor (Sarry and Gunata, 2004; Wu and Liou, 1986). β-Glucosidase, the most abundant glycosidase, is present with α-arabinosidase and α-rhamnosidase in grapes and berries of various cultivars. In addition, enzyme treatment of juice or wine increases the concentrations of volatile monoterpene flavorants. Prolonged aging of wine or its exposure to elevated temperatures increases the concentration of free volatile monoterpenes through hydrolysis of glycosidic precursors in wine (Gunata et al., 1992; Straus et al., 1986). Some of the liberated aglycones may already be odorous, such as linalool, geraniol, and nerol, while some give rise to potent flavor compounds, such as β-damascenone, vitispirane, and theaspirane by further enzymatic or chemical transformations during fruit juice processing or leaf products processing (Sarry and Gunata, 2004). Other plants such as papaya (Schreier and Winterhalter, 1986), nectarine (Takeoka et al., 1992), and tea leaves (Lee et al., 1984; Kobyashi et al., 1992) also contain glycosides as precursors of flavors.
11.3.4 THE MAILLARD REACTION
AND
FLAVOR
The Maillard reaction plays an important role in flavor development especially in meat and savory flavor (Aliani and Farmer, 2005; Buckholz, 1988). Products of the Maillard reaction are aldehydes, acids, sulfur compounds (e.g., hydrogen sulfide, methanethiol), nitrogen compounds (e.g., ammonia, amines), and heterocyclic compounds, such as furans, pyrazines, pyrroles, pyridines, imidazoles, oxazoles, thiazoles, thiophenes, diand trithiolanes, di- and trithianes, furanthiols, and so forth (Martins et al., 2001; Yaylayan, 2003). Higher temperatures result in production of more heterocyclic compounds, among which many have roasty, toasty, or caramel-like aromas. Sugar, ascorbic acid, amino acids, thiamine (de Roos, 1992; Aliani and Farmer, 2005; Ames and Hincelin, 1992, Guntert et al., 1992, 1994; Yoo and Ho, 1997), and peptides (Ho et al., 1992; Izzo et al., 1992; de Kok and Rosing, 1994; Lu et al., 2005) are potential reactants of the Maillard reaction. They are present in most foods, so the Maillard reaction occurs commonly when these foods are cooked. Among the reactants studied (thiamine, inosine 5′-monophosphate, ribose, ribose-5-phosphate, glucose, and glucose-6-phosphate), ribose seems to be the most important flavor precursor for chicken aroma. Using gas chromatography (GC)-odor assessment and gas chromatography-mass spectrometry (GC-MS) show that ribose increases roasted and chicken aromas. The changes in aroma due to additional ribose are probably caused by an elevated concentration of compounds such as 2-furanmethanethiol, 2methyl-3-furanthiol, and 3-methylthiopropanal (Aliani and Farmer, 2005). Aroma of meatlike process flavor can be produced by model systems containing sugar such as glucose, ribose, or xylose and a sulfur-containing amino acids, such as cysteine (Farmer et al., 1989; Hofmann and Schieberle, 1995; Ho et al., 1992). It can also be formed by thermal reaction of mixtures of enzyme-hydrolyzed vegetable protein,
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Chemical and Functional Properties of Food Components
cysteine, D-xylose, or D-ribose (Mussinan and Katz, 1973; Wu et al., 2000, Wu and Cadwallader, 2002). Cooking conditions determine the aroma of cooked foods. For example, the major volatiles of water-boiled duck meat are the common degradation products of fatty acids, while roasted duck meat contains not only the volatiles found in raw duck meat, but also pyrazines, pyridines, thiazoles, and so forth (Wu and Liou, 1992), which are Maillard reaction products. The wax gourd (Benincasa hispida, Cogn), a vegetable known as winter melon or gourd melon, is used to produce beverages, candy, or jam, which are popular in Taiwan. The flesh of the gourd melon is white in color. The major volatile compounds of fresh gourds are (E)-2-hexenal, n-hexanal, and n-hexyl formate, while those present in wax gourd beverages are 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-methyl pyrazine, and 2-ethyl-5-methyl pyrazine. The pyrazine compounds not present in the fresh wax gourd are formed from the sugar added and the endogenous amino acids during processing of the beverage (Wu et al., 1987). This is an example of changes in flavor of foods during processing where the Maillard reaction plays an important role. Hundreds of patents have been granted worldwide for processes and reaction products based on Maillard technology applied to manufacturing meat and savory flavors (Aliani and Farmer, 2005; Buckholz, 1988; Mottram and Salter, 1988; Ouweland et al., 1988). In addition, Maillard reactions may produce mutagenic components, pigments, and antioxidants, which are discussed in other sections of this book.
11.3.5 THE ROLE
OF
LIPID OXIDATION
The oxidation products of lipids are volatile aldehydes, acids, and so forth. Therefore, lipids are one of the major sources of flavors in foods. For example, much of the desirable flavors of vegetables such as tomatoes, cucumbers, mushrooms, and peas (Ho and Chen, 1994; Zawirska-Wojtasiak, 2004), fresh fish (Hsieh and Kinsella, 1989), fish oil (Hu and Pan, 2000), and cooked shrimp (Kuo and Pan, 1991; Kuo et al., 1994), as well as many deep-fat fried foods such as French-fried potatoes (Salinas et al., 1994; Sanches-Silva et al., 2005) and fried chicken (Shi and Ho, 1994) are contributed by lipid oxidation. Lipid oxidation catalyzed by lipoxygenase produces secondary derivatives, such as tetradecatrienone, which is a keynote compound of shrimp (Kuo and Pan, 1991). During storage, there is a decrease in linoleic acid proportional to the increases in volatile compounds, expecially hexanal, pentanal, and pentanol. Lipoxygenase-3 in seeds is responsible for the oxidation of linoleic acid producing stale flavor of the stored rice. In cooked rice, glutinous rice develops more of these volatiles of stale flavor than nonglutinous rice (Suzuki et al., 1999). The core flavor of mashed potatoes consists of naturally occurring and thermally generated compounds. These compounds arise mainly from the oxidation of fatty acids, especially highly unsaturated fatty acids, and from the degradation and interaction of sugar-amino acids. The extent to which these reactions affect the flavor of the final product depends on the age of the raw materials, storage conditions, and
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processing techniques. For example, both lipid oxidation and nonenzymatic browning reactions increase with age of the raw potato (Salinas et al., 1994). Garlic develops its aroma from enzymatic reactions, as described previously. When garlic slices are deep fried, microwave heated, or oven baked, the aroma changes contribute to a different kind of garlic flavor (Yu et al., 1993). A novel S-compound forms from the interaction of garlic and heated edible oil (Hsu et al., 1993; Kim et al., 2005). Alliin and deoxyalliin can react with 2,4-decadienal, which is one of the major oxidation products of polyunsaturated fatty acids, to form aroma (Yu et al., 1994). Deep-fat frying is a universal cooking method. Stir frying is common in some cuisines, especially in Chinese cooking. Volatile compounds in oils change after deep-fat frying or stir frying and subsequent storage (Wu and Chen, 1992). Soybean oil heated at 200°C for 1 h, with or without the addition of water, forms volatile aldehydes during storage at 55°C. Total volatiles increase up to 1100-fold. However, aldehyde content decreases, while volatile acid content increases significantly. The increase in hexanoic acid produces a heavy acrid, acid, fatty, and rancid odor often described as a “sweat-like” odor, which is responsible for the rancid note. Addition of water to the deep frying oil tends to retard the formation of volatile compounds during deep frying. Freshly stir-fried Chinese food has a much better flavor quality than after it has aged. The main change in volatile constituents of stir-fried bell peppers during aging is the production of volatile carbonyl compounds from autoxidative breakdown of unsaturated fatty acids (Wu et al., 1986). Generally, the undesirable flavor qualities of food are associated more closely with lipids than with proteins and carbohydrates. Lipids are responsible for the rancidity of lipid-containing foods. The term warmed-over flavor (WOF) is used to describe the rapid development of oxidized odor in cooked meat upon subsequent holding. The rancid or stale odor becomes apparent within 48 h, in contrast to the more slowly developed rancidity that becomes evident only after frozen storage for a period of months. Although WOF was first recognized in cooked meat, it also develops in raw ground meat exposed to air. Overheating of meat protects it against WOF by producing Maillard reaction products possessing antioxidant activity (Pearson and Gray, 1983). Flavor differences between meats and meat products are associated with lipid oxidation products. The major difference between the flavor of chicken broth and that of beef broth is the abundance of 2,4-decadienal and γ-dodecalactone in chicken broth (Shi and Ho, 1994). Both compounds are well-known lipid oxidation products. A total of 193 compounds have been reported in the flavor of chicken. Forty-one of them are lipid-derived aldehydes. Recently, phospholipids, such as lecithin are classified in nutraceutical foods (Colbert, 1998). The off-flavor associated with lecithin produced in fermented dairy products include compounds of 2,4-nonadienal, 2,4-decadienal, and hydrogen peroxide (Suriyaphan et al., 2001). Hexanal, 2,4-heptadienal, 2-pentenal, and 2-hexenal are major volatile compounds of oxidized pork phospholipids (Meynier et al., 1998). The flavor chemistry of lipid foods has been reviewed and compiled elsewhere in the last decade (Beltran et al., 2005; Ho and Chen, 1994; Ho and Hartman, 1994; Min and Smouse, 1989; Pan and Kuo, 1994; Shahidi and Cadwallader, 1997; Shahidi, 1998; Shahidi and Ho, 1999).
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11.3.6 INTERACTION
Chemical and Functional Properties of Food Components OF
LIPIDS
IN THE
MAILLARD REACTION
The Maillard reaction and the oxidation of lipids are two of the most important reactions for the formation of aromas in cooked foods. Interactions between lipid oxidation and the Maillard reaction have received less attention, despite the fact that lipids, sugars, and amino acids exist in close proximity in most foods. Lipids, upon exposure to heat and oxygen, decompose into secondary products, including alcohols, aldehydes, ketones, carboxylic acids, and hydrocarbons. Aldehydes and ketones produce heterocyclic flavor compounds reacting with amines and amino acids via Maillard-type reactions in cooked foods (Aliani and Farmer, 2005; Shibamoto and Yeo, 1992). Lipid degradation products, such as 2,4-decadienal and hexanal, can interact with Maillard reaction intermediates to form long-chain alkylpyrazines as well as other heterocyclic compounds (Farmer and Whitefield, 1993; Yaylayan, 2003).
11.3.7 EXTRUSION Extrusion cooking is a process whereby low-moisture (10 to 30%) foodstuffs are subjected to heat, pressure, and mechanical shearing for a short time (20 sec to 2 min). Extrusion can have a significant effect on flavor and aroma profiles of food products manufactured through this process, for example, extrusion of wheat flour products (Heinio et al., 2003; Hwang et al., 1994). Depending on the raw material composition, flavor development during processing is an important consideration for product quality. Certain mechanisms, such as nonenzymatic browning and lipid oxidation, are considered to have significant implications in the flavor characteristics of food products. Oxidation and volatility of flavor compounds are important factors during heating and extrusion cooking at different temperatures and moisture contents. Lipid oxidation products are the major compounds of aroma generation in the extrudates prepared from wheat flour at high moisture content and low die temperature. By lowering moisture content, lipid degradation compounds decrease and the Maillard reaction products dominate the flavor profile. The lipid oxidation products significantly increase during storage of the extrudate prepared at low moisture content and high die temperature (Villota and Hawkes, 1988). Formulations with starch, starch-caseinate, in biscuit mix show that when aroma compounds limonene, p-cymene, linalool, geraniol, terpenyl acetate, and β-ionone are added in water emulsion, oil solution, capsules, or inclusion complexes in β-cyclodextrin can lose free volatiles up to more than 90% during the extrusion process (Sadafian and Crouzet, 1987). Flavor retention increases through encapsulation of volatile compounds in natural or artificial inclusion complexes. Soy protein isolate (SPI) and hydrolyzed vegetable protein (HVP) are important ingredients in extruded foods. SPI is used for texturization and to increase the protein content of foods, while HVP promotes cooked and roasted aromas through Maillard reactions. The volatile aroma components from SPI and acid-hydrolyzed vegetable protein are different. Aliphatic aldehydes and ketones are mainly found in SPI, whereas pyrazines and sulfur-containing compounds are dominant in HVP (Solina et al., 2005).
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309
AND
OTHER PROCESSES
Some foods undergo special treatment in processing, which may affect the composition of volatile components. For example, in hybrid passion fruit, the presence of about 1 to 2% starch makes heat processing, such as pasteurization and concentration, impossible or impractical unless the starch is removed before processing. However, the step of removing starch and concentration causes loss of volatile compounds of fruit juice (Kuo et al., 1985).
11.3.9 CHANGES DUE
TO
FOOD STORAGE
Food preservation is designed to prevent undesirable changes in food and food products. However, flavor changes in food products during storage occur continuously, although the deterioration of flavor quality is not significant in most cases. Nonenzymatic reactions that occur during processing and storage of food products are detrimental if foods contain reducing compounds or if these compounds are produced during storage as a result of oxidation, acid hydrolysis, enzymatic reactions, or physicochemical changes. Water mediates the nonenzymatic browning reaction by controlling the liquid phase viscosity; by dissolution, concentration, and dilution of reactants; and by effects on the reaction pathways due to activation energy limitations in dehydrated foods (Saltmarch et al., 1981). Citrus juice can have off-flavor formation during processing and storage. Changes in volatile components in aseptically packaged orange juice during storage at room temperatures can be monitored. Quantities of desirable flavor components decrease during storage, while amounts of undesirable components, α-terpineol and furfural, increase progressively with prolonged storage (Moshonas and Shaw, 1987). The ultra-high temperature (UHT) processing of milk owes its commercial success to the fact that the rate of microorganism destruction increases more rapidly with temperature than do the rates of the accompanying changes in color and flavor. At very high processing temperatures, high sterility may be achieved with minimal adverse nutritional and chemical effects. However, UHT milk darkens in color during storage. This effect is noticeable after a few months of storage at 20°C. It becomes more pronounced at higher temperature and longer storage time. The milk also deteriorates in taste. The ε-amino group of lysine in milk proteins may react extensively with lactose through Maillard reaction before milk develops a marked off-flavor, discoloration, or instability (Moller, 1981). Spray-dried whey also has the problem of browning via Maillard reaction.
11.4 USE OF FLAVORS IN THE FOOD INDUSTRY 11.4.1 FUNCTIONAL PROPERTIES
OF
FLAVOR COMPOUNDS
Food flavorings are compounded from natural and synthetic aromatic substances. The compounded flavor may or may not be found in nature. The reasons for using flavors in foods are as follows (Giese, 1994a,b):
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•
• • • •
Flavors can be used to create a totally new taste. This does not happen very often, but some new flavors have been enormously successful, such as those used by Coca-Cola® or Pepsi Cola®. Flavoring ingredients may be used to enhance, extend, or increase the potency of flavors already present. Processing operations such as heating may cause loss of flavor, while some flavors already present may need supplementation or strengthening. Flavor ingredients can simulate other more expensive flavors or replace unavailable flavors. Flavors may be used to mask less desirable flavors, or to cover harsh undesired tastes naturally present in some processed foods, but not to hide spoilage.
Flavor compounds provide not only sensory quality to food, but also other functional properties including antioxidative activity, antimicrobial activity, and health-promoting functions. Volatiles of C6–C12 alkanals and alkenals emitted from plants are inhibitory to fungal infection, such as com (Zeringue et al., 1996), soybean (Boué et al., 2005), and tomato. Essential oil of clove, nutmeg (Dorman et al., 2000), Terminalia catappa leaves (Maua et al., 2003; Wang et al., 2000), and aroma extracts of dried soybean, kidney beans, mung bean, and adzuki bean (Lee and Shibamoto, 2000; Lee and Lee, 2005), in addition to extracts of several members of the Allium family (Yin and Cheng, 1998), and 23 essential oils isolated from various spices and herbs (Teissedre and Waterhouse, 2000) all have antioxidative activities. Eugenol, thymol, carvacrol, and 4-allylphenol from leaves of basil and thyme also show strong antioxidant activities (Lee et al., 2005). The aroma compounds may be used to prevent lipid oxidation in food preparation or food processing with health-related properties that are based on their antioxidative activity (Ka et al., 2005). Antimicrobial activities are found in essential oils of various herbs, spices (Badee et al., 2003; Firouzi et al., 1998), onion, garlic (Block, 1985), mustard, and horseradish (Delaquis and Mazza, 1995). Ninety-three different commercial essential oils have activity against 20 Listeria monocytogenes strains in vitro, which correlate with the chemical composition of each oil. Essential oils containing a high percentage of monoterpenes, eugenol, cinnamaldehyde, thymol (Lis-Balchin and Deans, 1997), and isothiocyanates show strong antimicrobial activity (Badee et al., 2003; Delaquis and Mazza, 1995). Flavors with antimicrobial activity can be a useful adjunct to food preservation systems. Moreover, antimutagenic, anticarcinogenic, and antiplatelet activities are found in sulfur-containing compounds of several Allium members (Chen et al., 1999; Kimbaris et al., 2006).
11.4.2 COLLECTION
OR
PRODUCTION
OF
FLAVORING MATERIALS
11.4.2.1 Natural Flavor Materials The sources, names, characteristics, and major flavor components of natural flavoring materials such as spice, herb, and so forth have been summarized in several books (Arctander, 1960; Furia and Bellanka, 1975; Reineccius, 1994b). A large number of
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constituents in natural flavor materials are not flavor compounds. These nonflavor compounds have to be removed to produce concentrated flavorants. The predominant methods for reaching this purpose are distillation and extraction. The essential oils are the distilled fraction of aromatic plants. Most often they are steam distilled. These oils, which are primarily responsible for the characteristic aroma of the plant material, are generally complex mixtures of organic compounds. For example, during the concentration of citrus juices, a layer of essential oil is formed in the condensate. This oil is called essence oil. Terpenes can be removed from both essential oil and essence oil to obtain folded oil. Some fruit or vegetable distillates containing flavor compounds are called essences. Although no essential oil layer is obtained from vegetable distillate, it is used as a flavoring raw material. Citrus peels rich in essential oils are expressed to get cold-pressed oils. Simultaneous distillation–extraction requires heating of the sample to collect volatile compounds of a wide range of volatility in an organic solvent. Sometimes it may lead to odorous artifacts. Extraction by vacuum hydrodistillation does not require heating at high temperatures. It extracts compounds at high and low boiling points. It is particularly suitable for analyses of volatile compounds of new products, such as fresh oysters (Pennarun et al., 2002) and fresh seaweed (Le Pape et al., 2002). The one disadvantage is that some volatile compounds may be lost or modified during the concentration following the vacuum hydrodistillation (Stephan et al., 2000). Dynamic headspace extraction is generally performed at temperatures close to that of vacuum hydrodistillation without heating. The extraction is only done to obtain the volatile compounds of low boiling points for analyses of materials such as honey (Radovic et al., 2001), oysters (Piveteau et al., 2000), kombu (a dried edible brown algae, Takahashi et al., 2002), and edible red algae (Le Pape et al., 2004). The nonvolatile flavoring constituents of aroma plants are recoverable by extraction. Essential oils do not contain hydrophilic flavoring components, antioxidants, or pigments. The selection of solvent is limited depending on its toxicity, regardless of whether it remains in the final product. Two kinds of solvents are used: (1) a polar solvent such as ethyl alcohol; for example, vanillin is soluble in ethyl alcohol, which is used as the solvent to prepare vanilla bean extract; (2) a nonpolar solvent such as petroleum ether. Most aroma compounds are oil soluble. Therefore, petroleum ether is used as the solvent to extract plant aroma. The extract after removal of the solvent is called concrete. Concrete may contain large portions of wax and fatty acids, and is further purified by ethyl alcohol extraction. The product is called absolute. The nonvolatile flavoring constituents of herbs and spices are recoverable by extraction. In practice, a solvent is chosen that dissolves both the essential oil and the nonvolatiles present. The resulting solvent-free product is known as oleoresin. A disadvantage of the oleoresins is that they are very viscous and thick, making them difficult to handle or to mix in processing operations. Several products have been developed into extractives, which are convenient to use and avoid handling problems. Extractives can be dispersed in salt, dextrose, or other carriers to create dry soluble spices. They may also be dispersed in fats to make fat-based soluble spices. Emulsification of extracts with starches and gums followed by spray drying
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produces encapsulated spices. Solubilization of extracts with glycerol, isopropyl alcohol, and propylene glycol produces liquid-soluble spices (Giese, 1994b). 11.4.2.2 Organic Chemicals Used in Flavorings Organic chemicals used in flavorings include hydrocarbons, such as limonene, pinene, ocimene, α-phellandrene, β-caryophyllene; alcohols, such as hexanol, cis-3-hexen-l-ol, geraniol, citronellol, eugenol, 1-menthol; aldehydes, such as acetaldehyde, hexanal, 2,4-decadienal, citral, vanillin; ketones, such as diacetyl, ionone, nootkatone; acids, such as acetic acid, butyric acid, pyroligenious acid; esters, such as ethyl acetate, linalyl acetate, ethyl phenyl acetate, methyl dihydrojasmonate; lactones, such as γ-nonalactone, δ-decalactone, γ-undecalactone; hemiacetals, such as acetaldehyde diethylacetal, citral dimethyl acetal; ethers, such as diphenyl oxide, rose oxide; nitrogen-containing compounds, such as trimethylamine; sulfur-containing compounds, such as dimethylsulfide, thiolactic acid, allyl disulfide; and heterocyclic compounds, such as furans, pyrazines, pyridines, thiazoles. The names, chemical structures, physical and sensory properties, and uses have been summarized in several books (Arctander, 1969; Furia and Bellanca, 1975; Reineccius, 1994b). Many organic chemicals used in flavorings are produced by synthetic methods and are commercially available. More and more natural compounds are used in flavorings due to the increasing demand for natural flavorings. They are produced or prepared by isolation of the compound from natural sources or by biotechnological methods. Thousands of flavoring raw materials may form a flavor. A large number of the flavoring raw materials are supplied by different manufacturers and stocked. Therefore, a strict quality control system for flavoring raw materials as well as the products is very important.
11.4.3 FLAVOR MANUFACTURING 11.4.3.1 Flavor Compounding From thousands of raw material flavors, twenty to fifty items are commonly selected and mixed in different ratios to blend a flavor as flavor compounding, which is a type of formulation. The raw material may be organic chemicals, essential oils, extracts, oleoresins, or processed flavors. Knowledge of their nature, physical and sensory properties, and applications are needed by flavorists. The professional practice of flavor compounding requires at least 3 to 5 years of training. How a flavor is formulated and modified using strawberry flavor as an example is shown in Table 11.1. The characteristic notes of strawberry are fruity, sweet, green, and a little bit oily and sour. Ethyl butyrate and methyl cinnamate have fruity notes, cis-3-hexen-l-ol, and ethyl hexanoate are green, benzaldehyde and 2,5-methyl-4-hydroxy-3 (2H) furanone are sweet, butyric acid is sour, and γ-undecalactone is oily-fruity. Formula 1 was originally designed for use in cake mix. Therefore, compounds with low boiling points, such as ethyl acetate, were not used. The solvent used was propylene glycol, which has a boiling point of 187.3°C. So the flavor compound was heat stable. However, the application of this flavor in cake resulted in a sensory characteristic that was pineapple-like, but not strawberry.
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TABLE 11.1 Formulas of Strawberry Flavors for Cake Mix Compound Ethyl butyrate Ethyl hexanoate cis-3-Hexen-1-ol Benzaldehyde Butyric acid 2,5-Dimethyl-4-hydroxy-3(2H) furanone Methyl cinnamate γ-Undecalactone Propylene glycol Total
Formula 1
Formula 2
1.5 1.0 1.0 0.3 0.4 2.0 1.3 0.9 91.6 100.0
3.0 2.0 2.0 0.2 0.8 1.0 2.0 0.5 88.5 100.0
Pineapple is oily, fruity, and sweet. Because butter, sugar, and milk were among the ingredients in the cake mix, oily or fatty and sweet notes were derived from those ingredients. Therefore, Formula 1 has to be modified by reducing the amount of γ-undecalactone, benzaldehyde, and 2,5-dimethyl-4-hydroxy-3 (2H) furanone, and increasing the amount of ethyl butyrate, ethyl hexanoate, cis-hexen-l-ol, butyric acid, and methyl cinnamate. The application test showed that Formula 2 gave the cake a strawberry flavor, although this modified flavor did not smell like strawberry before application in the cake. 11.4.3.2 Process Flavor Process flavors include reaction flavors, fat flavors, hydrolysates, autolysates, enzyme modified flavors, and so forth. Production of dairy flavor by enzyme modification of butterfat is an example (Lee et al., 1986; Manley, 1994), while meat flavor produced by enzymatic reactions has a much longer history. Raw meat has little flavor. Characteristic meat flavor varies with the species of animal, temperature, and type of cooking. Both water-soluble and lipid-soluble fractions of meat contribute to meat flavor. The water-soluble components include precursors, which upon heating are converted to volatile compounds described as “meaty.” Many desirable meat flavor volatiles are synthesized by heating water-soluble precursors, such as amino acids and carbohydrates. The Maillard reaction, including formation of Strecker degradation compounds, and interactions between aldehydes, hydrogen sulfide, and ammonia, is important in the formation of the volatile compounds of meat flavor. In addition, other kinds of flavors formed during cooking can also be obtained from heat processing. The contingency is the availability of the precursors, which may be too expensive to be isolated from natural raw materials or to be synthesized. The most practical way to characterize process flavorings is by their starting materials and processing conditions, because the resulting composition of volatiles is extremely complex comparable to the composition of cooked foods. Process
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flavorings are produced every day by housewives in kitchens, by food industries during food processing, and by the flavor industry. The International Organization of the Flavor Industry (IOFI) has guidelines for the production and labeling of process flavorings (IOFI, 1990). Some key points are that reactants are strictly appointed; flavorings, flavoring substances, flavor enhancers, and process flavor adjuncts shall be added only after processing is completed; and the processing conditions should not exceed 15 min at 180°C or proportionately longer at lower temperatures, while the pH should not exceed 8.0. Process flavors are very successful in some cases, but also unsuccessful in many cases. Natural flavor materials, such as meat extract or aromatic chemicals, may be added to process flavors to enrich some notes or to increase the overall intensity.
11.5 BIOTECHNOLOGICAL PRODUCTION OF FLAVORS 11.5.1 MICROBIAL PRODUCTION
OF
FLAVOR COMPOUNDS
Food products labeled as containing “all natural flavors” have a much higher market price than similar products containing artificial flavors. This preference has led to a strong upsurge in the requirement for natural flavor. The increasing demand for these flavor compounds now exceeds their supply from traditional sources. This has motivated research efforts toward finding alternative natural ways of obtaining natural flavor compounds. In the United States, the Code of Federal Regulations states that products produced or modified by living cells or their components, including enzymes, can be designated as “natural.” Thus, the use of biotechnological methods using microorganisms, enzymes, and recombinant DNA is a promising, economical, and environmentally friendly process for generating natural flavor compounds. Traditional fermentation using microbial activity is commonly used for the production of nonvolatile flavor compounds, such as acidulants, amino acids, and nucleotides. The formation of volatile flavor compounds via microbial fermentation on an industrial scale is still in its infancy. Although more than 100 aroma compounds may be produced microbially, only a few of them are produced on an industrial scale. The reason for that is probably due to the transformation efficiency, cost of the processes used, and our ignorance of their biosynthetic pathways. Nevertheless, the exploitation of microbial production of food flavors has proved to be successful in some cases. For example, the production of γ-decalactone by microbial biosynthetic pathways led to a price decrease from $20,000/kg to $1,200/kg (in U.S. dollars). Generally, the production of lactone can be performed from a precursor of hydroxy fatty acids, followed by β-oxidation from yeast bioconversion (Benedetti et al., 2001). Most of the hydroxy fatty acids are found in very small amounts from natural sources, and the only inexpensive natural precursor is ricinoleic acid, the major fatty acid of castor oil. Due to the few natural sources of these fatty acid precursors, the most common processes have been developed from fatty acids by microbial biotransformation (Hou, 1995). Another way to obtain hydroxy fatty acids is from the action of lipoxygenase (LOX). However, there has been only limited research on using LOX to produce lactone (Gill and Valivety, 1997).
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Isorhapontin
315
microbial stilbene dioxygenase
Eugenol Isoeugenol
Coniferaldehyde Ferulic acid
Vanillin
maize bran/sugarbeet pulp
FIGURE 11.1 Proposed pathways for the biosynthesis of vanillin.
Vanillin, perhaps the most important aroma compound, occurs in the bean of Vanilla planifolia. At present, only 0.2% of this compound is extracted from beans, and the remainder is produced synthetically in the world flavor market. Thus, the production of vanillin via microbial transformation has been the most extensively investigated. Figure 11.1 shows some of the possible routes for the production of natural vanillin by microorganisms. The transformation of the natural stilbene isorhapontin to vanillin is catalyzed by a microbial stilbene dioxygenase. This process has led to many patents (Hadegorn and Kaphammer, 1994; Walton et al., 2003). Alternative precursors are eugenol (Washisu et al., 1993) or isoeugenol. (Shimoni et al., 2000). However, the bioconversion produces relatively low yields. Direct use of ferulic acid to produce vanillin is probably the most promising approach due to the fact that this precursor is a constituent of various grasses and crops, and is a product of the microbial oxidation of lignin (Martínez-Cuesta et al., 2005). An example is the use of sugar beet pulp or maize bran as the source of ferulic acid to produce vanillin using two fungi (Bonnin et al., 2001). However, its recovery as a pure precursor is difficult, and the existence of numerous side reactions may explain the low bioconversion yields. Benzaldehyde is the second most important material after vanillin. It is used as an ingredient in cherry and other natural fruit flavors in the flavor industry. Natural benzaldehyde is generally extracted from fruit kernels such as apricots. Nowadays, the fermentation of natural substrates, such as phenylalanine, offers an alternative route for the biosynthesis of natural benzaldehyde (Moller et al., 1998). However, production via the fermentation process can become commercially acceptable only if sufficient yields can be obtained. Other applications of microorganisms used in the production of flavor compounds are listed in Table 11.2.
11.5.2 ENZYMATIC GENERATION
OF
FLAVOR COMPOUNDS
More than 3000 enzymes have been described in the literature, but only 20 are available for use in commercial processes (Armstrong and Brown, 1994). Lipases, esterases, lipoxygenases, glycosidases, proteases, and nucleases are the major enzymes used for flavor generation. Among them, lipases seem to be the most important enzyme in commercial use. The lipase-catalyzed reaction includes hydrolysis, esterification, and transesterification. The reaction can be performed not only in aqueous systems, but also in organic solvents (Jaegar et al., 1994). Based on these features, lipases are being increasingly used to synthesize “natural” flavoring materials such as esters (Abbas and Comeau, 2003; Langrand et al., 1990). The (S)-2-methylbutanoic acid
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TABLE 11.2 Aroma Compounds Produced by Microorganisms Aroma compounds
Microorganisms
Reference
Acetic acid Diacetyl Geosmin 2-Acetyl-pyrroline Lactone Linalool Benzaldehyde Vanillin 1-Octen-3-ol Jasmonate
bacteria bacteria bacteria bacteria yeast yeast fungi fungi fungi fungi
Sharpell and Stemann, 1979 Cheetham, 1996 Pollak and Berger, 1996 Romanczyk et al., 1995 Benedetti et al., 2001 Welsh, 1994 Fabre et al., 1996 Bonnin et al., 2001 Assaf et al., 1995; Zawirska-Wojtasiak, 2004 Miersch et al., 1993
methyl ester, which is known as a major flavor of apple and strawberry, is synthesized using lipase in organic media. Low-molecular-weight esters (LMWE) are flavoring agents for fruit-based products. Screening 27 commercial lipases shows that enzymes from Candida cylindracea, Pseudomonas fluorescens, and Mucor miehei (immobilized) promote synthesis of LMWE in nonaqueous systems (Rodriguez-Nogales et al., 2005; Welsh et al., 1990). LMWEs have also been produced using plant seedling lipase (Liaquat and Apenten, 2000). The catalysis efficiencies among microbial lipases are different (Kwon et al., 2000). Isoamyl acetate, one of the most employed flavor compounds in the industry, can be produced by using immobilized lipase (Krishna et al., 2001). Green note is present in a wide variety of fresh leaves, vegetables, and fruits. The characteristic aroma compounds responsible for green note include trans- and cis-2-hexenol, trans- and cis-3-hexenol (leaf alcohol), hexanol, hexanal, and cis-2hexenal (Whitehead et al., 1995). These compounds are biosynthetically produced using lipoxygenase (LOX) pathway enzymes (Fabre and Goma, 1999; Fukushige and Hildebrand, 2005; Hatanaka, 1993; Németha et al., 2004). The commercial processes for generating green odor compounds have been established using LOX pathway enzymes as shown in Figure 11.2. Four major enzymes, that is, lipolytic enzyme, LOX, hydroperoxide lyase (HPLS), and alcohol dehydrogenase (using baker yeast as a source) are involved in the formation of green odor compounds (Hatanaka, 1993). Lipids such as commercial soybean oil or vegetable oil are hydrolyzed to fatty acids by lipolytic enzymes. Fatty acid 13-hydroperoxides are formed from the action of a specific LOX, and then cleaved by HPLS into C6-green odor compounds. Among the various sources containing LOX activity, soybean (Gardner, 1989), pea (Chen and Whitaker, 1986), tomato (Riley et al., 1996), potato (Galliard and Phillips, 1971), cucumber (Hornostaj and Robinson, 1999), almond (Santino, et al., 2005), banana leaf (Kuo et al., 2006), green algae (Hu and Pan, 2000; Kuo et al., 1991), and microorganisms (Bisakowski et al., 1997) are worth mentioning. HPLS is not available in commercial sources. It is used as vegetable homogenate. Mung bean seedlings (Rehbock et al., 1998) and guava (Tijet et al., 2000) show higher HPLS
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lipid l ipolytic enzyme lipolytic linoleic acid
linolenic acid
lipoxygenase lip oxygenase 13-hydroperoxide
13-hydroperoxide
hydroperoxide lyase Hexanal alcohol dehydrogenase
isomerase
cis-3-hexenals alcohol dehydrogenase
trans-2-hexenal hexanol
cis-3-hexenol
alcohol dehydrogenase trans-2-hexenol
FIGURE 11.2 Formation of green odor compounds via lipoxygenase pathway enzymes.
activity than other sources in producing green odor. Natural 2(E)-hexenal is produced in two steps from hydrolyzed linseed oil, which contains the most linolenic acid among the available natural sources. In the first step, 13-hydroperoxy9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) is formed from linolenic acid by soybean LOX-1. In the second step, 13-HPOT is cleaved by green bell pepper HPLS (Németha et al., 2004). Continuous generation of green odor volatiles using a bioreactor immobilized with LOX and HPLS has been investigated (Cass et al., 2000).
11.5.3 RECOMBINANT DNA TECHNOLOGY FORMATIONS
FOR
FLAVOR
The application of recombinant DNA technology in the flavor industries is less advanced than in the pharmaceutical and food industries. However, this technology seems to have the most potential in future research. Nowadays, recombinant DNA technology has been increasingly used in several areas, including production of aroma chemicals, improved flavor profiles through genetic engineering, removal of off-flavor, and enzymatic formation of flavor aldehyde (Muheim, 1998). Fermentative or enzymatic processes are used to produce many flavor compounds as mentioned before. However, most of the above-mentioned techniques have only been reasonably applied so far, due to the fact that the enzymes or aroma chemicals are generally produced in very small amounts, making their recovery an expensive endeavor. Recombinant DNA technology allows efficient production of enzymes or flavor compounds. A good example is the production of green note compounds via LOX pathway enzymes. The LOX and HPLS are the determinant enzymes for the conversion of fatty acids into natural food flavor components. Due to those enzymes occurring in natural sources at low levels with instability and difficulty in purification, considerable efforts have been made to clone LOX or HPLS for commercial uses
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in the production of natural flavor components. Many plant LOX from soybean seed (Steczko et al., 1991), pea seed (Hughes et al., 1998), potato tuber (Royo et al., 1996), and HPLS from tomato (Atwal et al., 2005), guava (Tijet et al., 2000), alfalfa (Noordermeer et al., 2000), and pepper (Bourel et al., 2004; Matsui et al., 1996) have been cloned and expressed in E. coli or yeast. The green note compounds produced from such recombinant yeast cells bearing the enzyme genes are identical to those from the native enzymes. In addition, flavor compounds (such as leaf alcohol) have been produced in larger amounts using such recombinant enzymes, than using native enzymes.
11.6 APPLICATIONS OF FLAVORS Choosing the right type and dosage of flavor and adding the flavor at the right step in food processing are important to flavor applications. A flavor can be admired only after suitable application. Due to different application conditions, flavors are made to have different characteristics, such as solubility in water or oil, and heat stability. There is no general rule for flavor applications. Flavor users need some basic knowledge of flavor, food chemistry, and processing technology, in order to handle flavor applications properly. For example, citral is the key compound of lemon flavor. If the flavor has undergone thermal treatment severe enough to let it be oxidized, then the hemiacetal formed causes changes in flavor. Limonene is a major constituent in citrus oils. It has to be removed to prevent off-flavor production in food processing and storage. The extent of evaporation loss of each flavor ingredient is different in food processing. Some food components, such as starch, lipids, and proteins, can trap flavor compounds and reduce their volatilities. Some foods have their own flavors or flavor production. Therefore, modification of flavor formulas is needed to accommodate the flavor identity of different processed foods. Studies on flavor application for each food product are required to find the right strength, right form, and right step. Technical support for flavor users are standard services provided by flavor manufacturers. Flavors can be used to develop new food products. At the 1995 IFT Food EXPO, flavor manufacturers created unique berry flavors that do not exist naturally and are more exciting than natural aroma products. Mayberry, pepperberry, juneberry, mountainberry, bugleberry, and bellberry were all fabricated by creative flavor manufacturers (Sloan, 1995). Creating new flavors for innovative flavor applications is a challenge to the flavor industry, thus leading to the development of newer and highquality food products for quality living.
REFERENCES Abbas, H. and Comeau, L., Aroma synthesis by immobilized lipase from Mucor sp., Enzyme & Microbial Technol., 32, 589, 2003. Aliani, M. and Farmer, L.J., Precursors of chicken flavor. I. Determination of some flavor precursors in chicken muscle, J. Agric. Food Chem., 53, 6067, 2005.
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Salinas, J.P, Hartman, T.G., Karmas, K., Lech, J., and Rosem, R.T., Lipid derived aroma compounds in cooked potatoes and reconstituted dehydrated potato granules, Lipids in Food Flavors, Ho, C.T. and Hartman, T.G., Eds., Am. Chem. Soc., Washington, DC, 1994, p. 108. Saltmarch, M., Vagnini-Ferrari, M., and Labuza, T.P., Theoretical basis and application of kinetics to browning in spray-dried whey food systems, Maillard Reactions in Food, Eriksson, C., Ed., Pergamon Press, Oxford, U.K., 1981, p. 331. Sanches-Silva, A., Lopez-Hernandez, J., and Paseiro-Losada, P., Profiling flavor compounds of potato crisps during storage using solid-phase microextraction, J. Chromatogr., 1064, 239, 2005. Santino, A., Iannaconeb, R., Hughes, R., Casey, R., and Mita, G., Cloning and characterisation of an almond 9-lipoxygenase expressed early during seed development, Plant Science, 168, 699, 2005. Sarry, J.E. and Gunata, Z., Plant and microbial glycoside hydrolases: volatiles release from glycosidic aroma precursors, Food Chem., 87, 509, 2004. Schreier, P. and Lorenz, G., Formation of “green-grassy”-notes in disrupted plant tissues: characterization of the tomato enzyme systems, Flavour '81, Schreier, P., Ed., Walter de Gruyter, Berlin, 1981, p. 495. Schreier, P. and Winterhalter, P., Precursors of papaya (Carica papaya, L.) fruit volatiles, Biogeneration of Aromas, Parliment, T.H. and Croteau, R., Eds., Am. Chem. Soc., Washington, DC, 1986, p. 85. Shahidi, F., Flavor of Meat, Meat Products and Seafoods, 2nd ed., Blackie Acad. and Prof., London, 1998. Shahidi, F. and Cadwallader, K.R., Flavor and Lipid Chemistry of Seafoods, ACS Symposium Series 674, Am. Chem. Soc., Washington, DC, 1997. Shahidi, F. and Ho, C.T., Flavor Chemistry of Ethnic Food, Kluwer Academic/Plenum Publishers, New York, 1999. Sharpell, F. and Stemann, C., Development of fermentation media for the production of butyric acid, Advance in Biotechnology, Moo-Young, M., Ed., vol. II, Pergamon, Toronto, 1979, p. 71. Shi, H. and Ho, C.T., The flavor of poultry meat, Flavor of Meat and Meat Products, Shahidi, R., Ed., Blackie Acad. and Prof., London, 1994, p. 52. Shibamoto, T. and Yeo, H., Flavor compounds formed from lipids by heat treatment, Flavor Precursors Thermal and Enzymatic Conversions, Teranishi, R., Takeoka, G.R., and Guntert, M., Eds., Am. Chem. Soc., Washington, DC, 1992, p. 175. Shimoni, E., Ravid, U., and Shoham, Y., Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin, J. Biotechnol., 78, 1, 2000. Sinki, S., Assaf, R., and Lombarco, J., Flavor changes: a review of the principal causes and reactions, Perfumer Flavorist., 22, 23, 1997. Sloan, A.E., Ingredients add more fun, flavor, freshness & nutrition, Food Technology, August 1995, 102. Solina, M., Baumgartner, P., Johnson, R.L., and Whitfield, F.B., Volatile aroma components of soy protein isolate and acid-hydrolysed vegetable protein, Food Chem., 90, 861, 2005. Steczko, J., Donoho, G.A., Dixon, J.E., Sugimoto, T., and Axelrod, B., Effect of ethanol and low temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli, Prot. Express. Purif., 2, 221, 1991. Stephan, A., Bücking, M., and Steinhart, H., Novel analytical tools for food flavours, Food Res. Int., 33, 199, 2000.
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Straus, C.R., Wilson, B., Gooley, P.R., and Williams, P.J., Role of monoterpenes in grape and wine flavor, Biogeneration of Aromas, Parliment, T.H. and Croteau, R., Eds., Am. Chem. Soc., Washington, DC, 1986, p. 222. Suriyaphan, O., Cadwallader K.R., and Drake, M.A., Lecithin associated off-aromas in fermented milk, J. Food Sci., 66, 517, 2001. Suzuki, Y., Ise, K., Li, C., Honda, I., Iwai, Y., and Matsukura, U., Volatile components in stored rice [Oryza sativa (L.)] of varieties with and without lipoxygenase-3 in seeds, J. Agric. Food Chem., 47, 1119, 1999. Takahashi, H., Sumitani, H., Inada, Y., and Mori, D., Identification of volatile compounds of Kombu (Laminaria spp.) and their odor description, Nippon Kagaku Kaishi., 49, 228, 2002. Takeoka, G.R., Flath, R.A., Buttery, R.G., Winterhalter, P., Guntert, M., Ramming, D.W., and Teranishi, R., Free and bound flavor constituents of white-fleshed nectarines, Flavor Precursors Thermal and Enzymatic Conversions, Teranishi, R., Takeoka, G.R., and Guntert, M., Eds., Am. Chem. Soc., Washington, DC, 1992, p. 116. Teissedre, P.L. and Waterhouse, A.L., Inhibition of oxidation of human low-density lipoproteins by phenolic substances in different essential oils varieties, J. Agri. Food Chem., 48, 3801, 2000. Teranishi, R., Buttery, R.G., and Guadagni, D.G., Some properties of odoriferous molecules, Flavor '81. Schreier, P., Ed., Walter de Gruyter, Berlin, 1981, p. 133. Tijet, N., Waspi, U., Gaskin, D.J.H., Hunziker, P., Muller, B.l., Vulfson, E.N., Slusarenko, A., Brash, A.R., and Whitehead, I.M., Purification, molecular cloning and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava), Lipids, 35, 709, 2000. Tressl, R., Bahri, D., and Engel, K.H., Formation of eight-carbon and ten-carbon components in mushrooms (Agaricus campestris), J. Agric. Food Chem., 30, 89, 1982. Villota, R. and Hawkes, J.G., Flavoring in extrusion, an overview, Thermally Generated Flavors: Maillard, Microwave, and Extrusion Processes, Parliment, T.H., Ed., Am. Chem. Soc., Washington, DC, 1988, p. 280. Walton, N.J., Mayer, M.J., and Narbad, A., Vanillin, Phytochem, 63, 505, 2003. Wang, H.F., Ko, P.T., Chyau, C.C., Mau, J.L., and Kao, M.D., Composition and antioxidative activity of essential oil from Terminalia catappa L. leaves, Taiwanese J. Agric. Chem. Food Sci., 38, 27, 2000. Washisu, Y., Tetsushi, A., Hashimoto, N., and Kanisawa, T., Manufacture of vanillin and related compounds with Pseudomonas, Japan, Patent, 5,227,980, 1993. Welsh, F.W., Overview of bioprocess flavor and fragrance production, Bioprocess Production of Flavor, Fragrance, and Color Ingredients, Gabelman, A., Ed., Wiley, New York, 1994, p. 1. Welsh, F.W., Williams, R.E., and Dawson, K.H., Lipase mediated synthesis of low molecular weight flavor esters, J. Food Sci., 55, 1679, 1990. Whitehead, I.M., Muller, B.L., and Dean, C., Industrial use of soybean lipoxygenase for the production of natural green note flavor compounds, Cereal Foods World, 40, 193, 1995. Wu, C.M. and Chen, S.Y., Volatile compounds in oils after deep frying or stir frying and subsequent storage, JAOCS, 69, 858, 1992. Wu, C.M. and Liou, S.E., Volatile components of water-boiled duck meat and Cantonese style roasted duck, J. Agric. Food Chem., 40, 838, 1992. Wu, C.M. and Liou, S.E., Effect of tissue disruption on volatile constituents of bell peppers, J. Agric. Food Chem., 34, 770, 1986.
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Wu, C.M., Liou, S.E., and Chiang, W., Volatile compounds of the wax gourd (Benincasa hispida, Cogn) and a wax gourd beverage, J. Food Sci. 52, 132, 1987. Wu, C.M., Liou, S.E., and Wang, M.C., Changes in volatile constituents of bell peppers immediately and 30 minutes after stir frying, JAOCS, 63, 1172, 1986. Wu, Y.F., Baek, H.H., and Cadwallader, K.R., Development of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein (E-HVP), J. Food Sci., 65, 1220, 2000. Wu, Y.F. and Cadwallader, K.R., Characterization of the aroma of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein, J. Agric. Food Chem., 50, 2900, 2002. Wurzenberger, M. and Grosch, W., The enzymic oxidative breakdown of linoleic acid in mushrooms (Psalliota bispora), Z. Lebensm. Unters Forsch., 175, 186, 1982. Yamaguchi, M. and Wu, C.M., Composition and nutritive value of vegetables for processing, Commercial Vegetable Processing, Luh, B.S. and Woodroof, J.G., Eds., The AVI Publishing Company, Inc., Westport, CT, 1975, p. 652. Yaylayan, V.A., Recent advances in the chemistry of Strecker degradation and Amadori rearrangement: implications to aroma and color formation, Food Sci. Technol. Res., 9, 1, 2003. Yin, M.C. and Cheng, W.S., Antioxidant activity of several Allium members, J. Agric. Food Chem., 46, 4097, 1998. Yoo, S.S. and Ho, C.T., Pyrazine generation from the Malliard reaction of mixed amino acids in model systems, Perfumer Flavorist., 22, 49, 1997. Yu, T.H., Lee, M.H., Wu, C.M., and Ho, C.T., Volatile compounds generated from thermal interaction of 2,4-decadienal and the flavor precursors of garlic, Lipids in Food Flavors, Ho, C.T. and Hartman, T.G., Eds., Am. Chem. Soc., Washington, DC, 1994, p. 61. Yu, T.H. and Wu, C.M., Stability of allicin in garlic juice, J. Food Sci. 54, 977, 1989. Yu, T.H., Wu, C.M., and Ho, C.T., Volatile compounds of deep-oil fried, microwave-heated, and oven-baked garlic slices, J. Agric. Food Chem., 41, 800, 1993. Yu, T.H., Wu, C.M., and Ho, C.T., Meat-like flavor generated from thermal interactions of glucose and alliin or deoxyalliin, J. Agric. Food Chem., 42, 1005, 1994. Zawirska-Wojtasiak, R., Optical purity of (R)-(-)-1-octen-3-ol in the aroma of various species of edible mushrooms, Food Chem., 86, 113, 2004. Zeringue, H.J., Jr., Brown, R.L., Neucere, J.N., and Cleveland, T.E., Relationships between C6-C12 alkanal and alkenal volatile contents and resistance of maize genotypes to Aspergillus flavus and aflatoxin production, J. Agric. Food Chem., 44, 403, 1996.
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Interactions of Food Components Zdzisław E. Sikorski and Norman F. Haard
CONTENTS 12.1 12.2
Introduction................................................................................................ 330 Water–Protein Interactions ........................................................................ 331 12.2.1 Water in Proteinaceous Structures .............................................. 331 12.2.2 Antifreeze Proteins—Ice Crystal Interactions ............................ 333 12.2.3 Protein–Protein and Protein–Polysaccharide Interactions.......... 334 12.3 Water–Lipid and Protein–Lipid Interactions............................................. 335 12.4 Polysaccharide Interactions in Food Systems........................................... 336 12.4.1 Polysaccharide–Water Ions–Polysaccharide Interactions........... 336 12.4.2 Polysaccharide–Lipid Interactions .............................................. 337 12.5 The Effect of Interactions on the Color of Foods .................................... 338 12.5.1 Changes in Meat Pigments ......................................................... 338 12.5.2 Changes of Chlorophylls............................................................. 339 12.5.3 Interactions of Carotenoids ......................................................... 340 12.5.4 Interactions of Other Pigments ................................................... 341 12.5.5 Browning and Formation of Black Spots ................................... 341 12.6 Interactions Affecting Food Flavor ........................................................... 342 12.6.1 Introduction ................................................................................. 342 12.6.2 Hydrolytic, Oxidative, and Pyrolytic Reactions......................... 343 12.7 Changes in Texture of Foods .................................................................... 344 12.7.1 Introduction ................................................................................. 344 12.7.2 Rigor Mortis and Tenderization of Meat.................................... 344 12.7.3 Texture Changes in Frozen Fish ................................................. 346 12.7.4 Cross-Linking in Gels and Films................................................ 346 12.7.5 Interactions in the Dough............................................................ 347 12.7.6 Lipids and the Texture of Foods................................................. 348 12.8 Interactions and the Nutritive Value of Foods .......................................... 349 12.8.1 Introduction ................................................................................. 349 12.8.2 Changes in Digestibility.............................................................. 349 12.8.3 Changes in Biological Value....................................................... 350 12.9 The Effect of Interactions on the Safety of Foods ................................... 351 12.10 Final Remarks............................................................................................ 352 References.............................................................................................................. 353 329
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12.1 INTRODUCTION Foods contain a multitude of various compounds. The components of foods form the structural elements of different tissues in living plants and animals, serve as reserve material and sources of energy, and perform numerous other biochemical functions. In natural or processed foods they are present in solutions or as a variety of structures, stabilized by covalent bonds, hydrogen bridges, ionic forces, and hydrophobic interactions. Foods can be classified as (1) intact tissue systems, such as fruits and vegetables, cereals, pulses, eggs, meats; (2) disrupted tissue systems, such as ground meat, purees, flour, tomato catsup; (3) noncellular or disrupted cellular systems, such as milk, fruit juices, honey, oils; or (4) combinations thereof (Haard, 1995). The interactions of components are of a physicochemical, biochemical, or chemical nature. The physicochemical interactions are responsible for the formation of emulsions, foams, and also partially gels. Thus they have great impact on the sensory quality of various food commodities and on the resistance to shearing, as well as on the flow of the material in the processing equipment. Chemical and biochemical reactions in fresh and processed foodstuffs may affect all quality attributes including the color, appearance, flavor, aroma, texture, biological value, nutritional properties, safety, and processing suitability of foods. Thus they may improve the edible characteristics or in other cases delimit storage life. It is not always clear whether interactions or reactions occurring in foodstuffs are nonenzymic, enzyme catalyzed, or result from microbial metabolism. For example, the development of bad odor in coleslaw prepared with mayonnaise and sour cream was first attributed to microbial spoilage. However, study of the problem revealed that endogenous enzymes in the cabbage were activated by the anaerobic conditions in the package and were responsible for the formation of offensively smelling volatiles. In general, enzyme-catalyzed reactions are of primary importance in untreated and intact plant and animal tissues, while nonenzymic reactions predominate in properly processed foodstuffs. There are, however, some exceptions to this general rule. In addition, enzymatic and nonenzymatic reactions may act in concert. For example, a color change of the surface of red meats is caused by nonenzymatic reactions of desirable oxygenation and subsequent undesirable oxidation of myoglobin (see Section 12.5.1). However, enzymatic reactions involved with respiration at the tissue surface can lower oxygen concentration and indirectly promote oxidation of myoglobin (MbFe(II)). Some tissues also contain the enzyme metmyoglobin (MbFe(III)) reductase, which can reverse the nonenzymic oxidation of MbFe(II). Likewise, texture deterioration that occurs during frozen storage of fish of certain species is primarily caused by the nonenzymic denaturation and aggregation of the major muscle protein myosin. However, protein denaturation and cross-linking may be accelerated by the action of trimethylamine oxide demethylase, which liberates reactive formaldehyde or by phospholipase, which forms interactive free fatty acids (see Section 12.7.3). The chemical and biochemical reactivity of the components in food systems is controlled by their chemical character (Table 12.1), compartmentalization in the tissues, the activity of enzymes and different enzyme inhibitors, and predominantly by environmental conditions of storage and processing. The most chemically reactive
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TABLE 12.1 Reactive Groups of Food Components –SH –S–S– –NH2 –NH–C(=NH)NH2 –CONH2 –OH –CHO R2C=O 1O •O – •OH H2O2 RO• ROO• ROOH ArO• ArOO• 2 2 –COOH –O–SO3H –O–PO3H2 –CH=CH– –CH=CH–CH2–CH=CH– CH3–(CH2)n–CH2– and other hydrophobic groups Ca2+ Mg2+ Fe2+ Fe3+ Cu2+ Co2+ Zn2+ and other cations Additives: NO• NO2• O=N–OOH O=N–OO–
food components are reducing sugars and other carbonyl compounds, thiol groups in proteins, ionizable and heterocyclic amino acid (AA) residues, polyenoic lipids and pigments that are readily oxidizable, phenolics, terpenes, and radicals. The concentration of some of these compounds changes during storage of foods due to the activity of endogenous enzymes. Some reactive groups are hidden in the structure of food polymers and thereby are not always accessible to interactions; other potential substrates are separated by cellular compartmentalization in the intact tissues. Initially masked or protected food components may later interact as a consequence of food storage and processing conditions. Maillard reactions, some enzyme-catalyzed reactions, and various oxidation processes are examples of important interactions that are facilitated by tissue disruption or processing. The interactions in many foods may be affected by physical changes associated with handling of the raw material that leads to damage of the structure, like rupturing of the cell membranes in fish muscle, fruits, and vegetables by large ice crystals formed in the intracellular spaces during slow freezing, selective cell breakage under controlled conditions of milling, which results in disruption of the endosperm cells of the wheat grain, or disintegration of the muscle architecture during comminuting of meat in sausage manufacturing. Different interactions of food components may also be enhanced by high hydrostatic pressure of the order of 100 to 600 Mpa. Biochemical reactions in living tissues can also be a function of physiological processes related to ontogeny (e.g., fruit ripening and plant senescence) as well as stress and biological strain (e.g., chilling injury of plants or pale, soft, exudative condition in myosystems). Moreover, the chemical reactivity and quantity of tissue components that interact postharvest may be influenced by preharvest factors such as the age or nutrition of the plant or animal and the plant cultivar or animal breed.
12.2 WATER–PROTEIN INTERACTIONS 12.2.1 WATER
IN
PROTEINACEOUS STRUCTURES
Water in foods serves as the solvent of numerous solutes and is normally the continuous, dispersing phase of solid components. In dehydrated foods, with lowered water activity, the mobile and dispersing phase may be lipid. Water is not only the
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solvent in which reactions occur. It participates directly in many reactions such as protein folding, protein–protein interactions, protein condensation, and hydrolysis. Proteins may occur in foods in solution or as fibrils, filaments, sheets, and particles. Fibrils, filaments, and sheets build the structure of cross-striated muscle (see Chapter 2). Wheat gluten forms fibrils and strands, with starch granules adhering to the protein matrix. The caseins of milk form polydisperse, spherical particles, from about 25 nm to about 600 nm in diameter. Almost all seeds contain protein granules, also called protein bodies or aleurone grains. These bodies, generally spherical, differ in size, depending on the species and variety of the cereal or legume. In corn, the average diameter is about 2 µm, in sorghum it ranges from 0.5 to 3 µm; in broad beans 1 to 5 µm, in peanuts 2 to 10 µm, and in soybeans 2 to 20 µm. The protein bodies of soybeans are nestled within a spongelike cytoplasmic protein network and are accompanied there by spherical lipid droplets (Heertje 1993). Interactions with water are crucial for the conformation of proteins in moist food systems due to hydrogen bonding, hydrophobic and van der Waals interactions, and the influence on ionic bridges. Water participates in hydrogen bonds with polar side chains of proteins (Figure 12.1) and may form highly ordered water molecules as cages around hydrophobic residues. Because the latter is energetically unfavorable due to the entropy decrease, the formation of amphipathic proteins into stable structures, such as micelles or protein bodies, is closely related to water–protein interactions. They are thus responsible for the stability of proteinaceous structures in many foods, such as the functional properties of proteins in meat and fish products and the casein micelles. Water is also an important reactant in which it is eliminated (condensation) or added (hydrolysis). Water is the most abundant substance in living systems, making up more than 70% of the weight of most tissues. Its amount in the tissues of slaughter animals, fish, shellfish, and mollusks is from about 3 to 7 times higher than that of proteins. Water can be held in meat mainly due to entrapment within the microstructure of tissues and because of various direct interactions with protein molecules. This property of muscle foods is known as water-holding capacity or water-binding potential. Any treatments, additives, and biochemical processes that result in loosening of the
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FIGURE 12.1 Water molecules forming hydrogen bonds with amino acid residues.
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myofibrillar structure by enhancing the mutual repelling of the fibrils, strands, and sheets increase the water-binding potential. Thus the factors affecting the retention of water in meat systems, and thereby the juiciness of the products, include the characteristics of the proteins, the pH in the meat, the concentration of postmortem metabolites, which accumulate in the process of tenderization, the presence of additives, mainly saccharides, salt, and phosphates, as well as changes in proteins due to freezing, frozen storage, mincing, and cooking. The effect of the state of the proteins on the water-holding capacity can be observed in pale, soft, exudative pork (PSE) and similar disorders in other food myosystems such as poultry and burned tuna meat. The PSE condition occurs due to denaturation of muscle proteins at comparatively high temperatures and low pH in the carcass after slaughter. Burned tuna meat, instead of being red, translucent, and firm as prime quality tuna, is pale, exudative, soft, and slightly sour. It occurs especially frequently in hand-line-hooked fish that fought only a few minutes during the catch, but rarely in specimens that were subjected to destruction of the spinal column or brain immediately after capture. According to the hypothesis of Watson et al. (1988), high stress of the animal during hooking and fierce fighting causes a large increase in concentration of the hormones norepinephrine and epinephrine in the blood. These catecholamines potentiate muscle proteolysis by calpains in postmortem muscle. Because the catecholamines in tuna are rapidly metabolized and have a half-life of about 30 minutes, their concentration in the blood of long-linecaught fish after several hours in water is not high enough to affect the proteolysis of muscle proteins. The rapid lowering of pH in muscles is due to the decompartmentalization of Ca+2 and its interaction with Ca+2-ATPase. The hydrolysis of ATP results in H+ formation and a lowering of pH. Brain destruction decreases the rate of postmortem dephosphorylation of ATP in the muscles, and thus prevents rapid leaking of Ca2+ from the sarcoplasmic reticulum into the sarcoplasm. At a low concentration of Ca2+ there is no activation of calpains or myosin ATPase.
12.2.2 ANTIFREEZE PROTEINS—ICE CRYSTAL INTERACTIONS In frozen foods most of the water, about 80% at a temperature of about –30°C, is present as ice crystals. The size and distribution of the crystals depend, for example, in ice cream, primarily on the rate of freezing and on shearing forces during processing. Some proteins interact with the water molecules in ice crystals. The blood of fishes inhabiting polar oceans, as well as the blood of some insects, contains specific proteins, known as antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP). The AFP are responsible for the noncolligative depression of the freezing point of the blood serum of these animals. Both the AFP and AFGP are preferentially bound to the ice lattice and retard the growth of the crystals. Different mechanisms of the specific binding of AFP to the ice lattice have been proposed, whereby the contribution of the hydrogen bonds, entropic effects, and van der Waals interactions has been postulated (Chao et al. 1997). The winter flounder AFP contains 4 ice-binding regions grouped along the polypeptide chain so that they are compatible with the ice surface topology (Cheng and Merz 1997). Therefore the
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AA residues in all regions simultaneously form 5 to 6 hydrogen bonds each with the water molecules in the crystals. The proper sequence of the AA residues is also necessary for the van der Waals interactions and the hydrophobic effect to participate in the binding of the protein.
12.2.3 PROTEIN–PROTEIN INTERACTIONS
AND
PROTEIN–POLYSACCHARIDE
Most proteins with a molecular weight greater than 100 KDa have multiple subunits, identical or different. In nature, multimeric proteins can have from two to hundreds of subunits. Some protein assemblages, such as collagen, serve primarily a structural role in living systems. In other cases multimeric proteins have enhanced binding or catalytic properties, such as the binding of oxygen to multimeric hemoglobin. In living systems, protein folding and protein–protein interactions may be spontaneous or may require the assistance of specialized proteins called molecular chaperones. As well, disulfide bond formation and the cis-trans isomerization of proline peptide bonds are catalyzed by specific proteins. In food systems, protein–protein interactions are normally facilitated by changes in environmental conditions, notably temperature and pH, by chemical cross-linking agents like formaldehyde, or by the activity of enzymes, such as chymosin or transglutaminase. There are numerous proteins in particular food commodities. Differing in AA composition and sequence and in molecular weight they behave differently in food systems. In some proteins the distribution of hydrophilic and hydrophobic segments of the polypeptide chains is such that it favors hydrogen bonds, ionic interactions, and hydrophobic associations of the molecules after their deconformation (Table 12.2). Interactions of various reactive groups and hydrophobic patches exposed on the surface of the molecules lead to formation of various intermolecular links, which result in changes in protein hydration, solubility, viscosity of solutions, film formation, gelling, and adsorption on the interface between aqueous and lipid phases. Examples of important food protein aggregations and interactions include the formation of cheese curds (aggregated, destabilized casein micelles) from acidification
TABLE 12.2 Characteristic Sequences in Some Food Proteins Protein
Characteristic sequences
αs1-casein β-casein Collagen α-Gliadin Phosvitin
–SerP–Ile–SerP–SerP–SerP–Glu– –SerP–Leu–SerP–SerP–SerP–Glu– –Gly–Pro–Hyp– –(His)3–(Gln)6–Pro–Ser2–Gln–Val–Ser–Tyr–(Gln)2– –Asp–(SerP)6–Arg–Asp– –Ser–Asn–Ser–Gly–(SerP)8–Arg–Ser–
Winter flounder antifreeze glycoprotein
–Asp–Thr–Ala–Ser–Asp–Ala6–Leu–Thr–Ala2–Asn–Ala–Lys–Ala3–Glu– Leu–Thr–Ala2–Asn–Ala7–Thr–Ala–Arg–
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of milk or chymosin catalyzed hydrolysis of κ-casein, interaction and cross-linking of myosin causing loss of succulence of texture in fish of some species, aggregation of albumin proteins in heated egg, and development of gluten from the wheat proteins gliadin and glutenin. Many foods contain substantial amounts of glycoproteins, that is, proteins linked to various monosaccharide or oligosaccharide moieties via O-glycosidic or N-glycosidic bonds. The saccharides are bound to the residues of hydroxyamino acids or asparagine. The best known food glycoproteins include κ-casein, ovoglycoprotein, ovomucoid, ovomucin, avidin, ovoinhibitor, ovalbumin, and some collagens, especially fish collagens. The saccharide moieties may participate in covalent interchain cross-links and affect many functional properties of the glycoproteins, such as their tensile strength, solubility, viscosity in solutions, ability of interactions with other food components, and biological activity. Some proteins participate in interactions with polysaccharides. This may cause precipitation or stabilization of the proteins even in the presence of precipitating agents. The neutral polysaccharides like locust bean gum and guar gum, as well as most saccharide polyanions, including gum Arabic, carboxymethylcellulose, and pectins do not prevent precipitation of casein micelles and αS1-casein by Ca2+ at pH 6.8, while carrageenans, especially κ-carrageenan, form stable complexes with this protein (Phillips and Williams 1995). From NMR studies it can be deduced that both hydrogen bonds and van der Waals contacts confer stability on the interaction of wheat germ agglutinin with oligosaccharides (Espinosa et al. 2000). Under appropriate conditions, polysaccharides may interact with proteins via Maillard reactions and Strecker degradation forming products with reduced nutritional value, changed color and flavor, and altered texture.
12.3 WATER–LIPID AND PROTEIN–LIPID INTERACTIONS In water-in-oil emulsion-type products, such as butter, margarine, and fat spreads, water occurs as droplets. In butter, the diameter of the droplets is below 10 µm, in spreads below 5 µm. Water droplets have a structure-forming role in such foods. Depending on their size and distribution they affect the rheological and sensory properties, as well as bacterial stability of the products. The stabilization of the droplets is due to interactions with solid fat particles, monoacylglycerols and phospholipids, or proteins that accumulate at the water–lipid interface. The coalescence of the droplets is prevented by interactions with fat lamellae. In milk and dairy commodities, in mayonnaise, salad dressings, ice cream, and comminuted meat and fish products, the lipids form globules dispersed in the aqueous phase. In fresh and in ultrafiltered milk the globules, 0.5 to 10 µm and 1 to 18 µm in diameter, respectively, are separated from each other and from the aqueous medium by phospholipid-proteinaceous membranes, about 10 nm thick. On the surface of these membranes are adsorbed lipoprotein particles. In whipped cream and ice cream the fat globules are assembled on the interface of the aqueous dispersing phase/air bubbles. In conventionally produced ice cream the fat droplets are
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about 1 µm in diameter. High-pressure homogenization, at about 2000 bar, makes it possible to reduce the size of the droplets to 0.3 µm and thus increase the perception of creaminess. In fresh cheese the globules are surrounded by a protein shell and distributed in a protein network. In butter the globules are in a matrix of free fat. The core of these globules is formed by liquid fat that is covered by fat crystals. The softening temperature of butter depends on the proportion of fluid fat to fat crystals. In mayonnaise the oil droplets form a honeycomb structure stabilized within the dispersing aqueous phase by the egg yolk phospholipids, and probably by lipid crystals at the surface. Amphiphilic lipids form micelles or lamellar phases, when present in the aqueous environment in concentration above the critical value. Different types of micelles have various shapes, depending on the surface area of the polar groups and the volume and length of the hydrophobic chains. The lipids may assemble into hexagonal micelles. In one type of micelle, the polar heads of the amphiphilic lipids protrude to the surface, while the hydrocarbon chains are directed axially toward the center of the cylinder. In another type of structure, the polar groups point to the axis forming a hydrophilic channel inside, with the hydrophobic tails stretching outside. Lipidous lamellar liquid crystalline phases self-assemble by stacking of stretched lipid bilayers separated by water lamellae. In wheat flour lipids, a transition from the micellar to the lamellar phase takes place on hydration (Marion et al. 1998). Noncovalent lipid protein complexes in foodstuffs are known to alter texture, for example, in the interaction of fatty acids and protein in frozen fish and in wheat dough gluten development. Wheat flour contains some low-molecular-weight proteins rich in cysteine and basic AA residues, which in the native state spontaneously bind lipids or lipid aggregates. The thionins, ligoline, lipid transfer proteins, and puroindolines belong here. They facilitate the spreading of monolayers of lipids at the air–water interfaces, thus acting as very efficient foam stabilizers. Interactions of puroindoline-a with lysophosphatidylcholine have a synergistic effect on foam stability (Marion et al. 1998). Moreover, degradation products of fatty acid hydroperoxides (aldehydes, alcohols, acids, epoxides, ketones, cyclic fatty acid monomers, dimers, polymers) can interact with proteins in the Maillard reaction. Fiber formation during high-moisture extrusion can be enhanced by the addition of some polysaccharides, such as starch, soy arabinogalactans, or maltodextrins in the food mix before extrusion. During extrusion, polysaccharides form a separate phase, which appears to enhance protein aggregation in the direction of flow and reduce it in the direction perpendicular to extrusion (Akdogan 1999).
12.4 POLYSACCHARIDE INTERACTIONS IN FOOD SYSTEMS 12.4.1 POLYSACCHARIDE–WATER IONS–POLYSACCHARIDE INTERACTIONS Interactions of polysaccharides with each other, ions, proteins, and lipids affect the water holding, gelling, film forming, viscosity, crystal growth, and stabilization of foams and lipid emulsions. Thus they contribute to the sensory properties of meat
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and fish products, milk gels, custards, creams, ice cream, salad dressings, cakes, bakery fillings, jam, marmalade, jelly, fruit drinks, and instant beverages, as well as to the tensile strength and barrier properties of biodegradable films and coatings. Starch granules are abundant in many raw plant tissues and in processed foods. In most food products the granules occur in different modified forms due to various stages of gelatinization and swelling, depending on the botanical variety of the starchy food material, the water availability, and conditions of heating during processing. The hydration of neutral polysaccharide networks depends on their affinity to the solvent. The immobilization of water molecules is, to a large extent, due to hydrogen bonding with the hydrophilic groups of the hydrocolloids. In a polyelectrolyte gel, like that of pectins, the swelling is increased by the effect of the counterions that accumulate within the network to neutralize the electrical charge of the polymer. However, cross-linking of the pectins via Ca2+-mediated ionic bonds may decrease swelling by counteracting expansion of the network. Polyanionic hydrocolloids interact with cationic counterions. Depending on the kind of anionic group of the polysaccharide—CO2–, PO32–, or SO4–—and the properties of the cations the counterions are bound together electrostatically with their coordinated water dipoles or after displacement of the hydration shell. Thus the effects of binding of various cations to different anionic hydrocolloids, such as carboxymethylcellulose or carrageenan, must be considered in selecting gum additives in food systems. The interactions of water with many plant hydrocolloids (gums) generally do not lead to gelling. Instead, very viscous, pseudoplastic solutions are formed, even at a low concentration of polysaccharides, due to the large molecular size of the polymers. The rheological behavior of solutions of neutral gums is pH-stable, while the viscosity of solutions of polymers with ionizable groups depends significantly on the pH of the system (Ramsden 2004). Cryoprotective agents have been studied for use in food processing. A series of mono-, oligo-, and polysaccharides; di- and polyalcohols; hydroxymonocarboxylic acids; and di- and tricarboxylic acids and highly branched oligosaccharides have been used as cryoprotectants (Auh et al. 2003). The physical mechanisms of cryoprotection by saccharides are not fully understood. It has been suggested that cryoprotectant activity is related to the replacement of the main hydration shell of polar head groups of membrane phospholipids by hydroxyl groups of sugars. In other words, sugars serve as water substitutes when the hydration shell of a protein is removed. All sugars interact strongly with water and participate in the water lattice through hydrogen bonds. This presumably leads to the formation of extended regions of hydrogen-bonded structured water in the vicinity of the cryoprotective molecule, which in turn may stabilize the surface hydration of proteins and protect them from freeze injury. The interaction of saccharide molecules with water is an essential process in the cryopreservation mechanism, but no real evidence of this hydration process has yet been produced.
12.4.2 POLYSACCHARIDE–LIPID INTERACTIONS In aqueous systems these interactions are due to hydrophobic effects, like in binding of a monoacylglycerol molecule by amylose. Accommodating the hydrophobic
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saturated fatty acid residue inside the amylose helix forms an amylose–lipid complex. Heating increases the degree of binding of lipids by amylose, as in dough during bread baking. Amphiphilic lipid–amylose interactions decrease the rate of bread staling. The amylose–lipid complexes may also form gels. The rheological properties of these gels depend on the concentration and type of lipids, and on the crystalline form of the complexes (Eliasson 1998). Interactions with lipids are responsible for the emulsifying properties of some hydrocolloids, such as gum Arabic. This polysaccharide, although having large molecules, forms water solutions of comparatively low viscosity. This is because gum Arabic also contains among its components a high molecular mass arabinogalactan–protein complex, in which the polypeptide chain is probably located at the periphery of the macromolecule. The surface hydrophobicity of the polypeptide enables the adsorption of the gum on the oil–water interface. Denaturation of the proteinaceous moiety by heating leads to loss in the emulsifying efficiency of the hydrocolloid. Because the polysaccharide–lipid interactions affect the rheological properties of the system, they may be utilized in formulating low-calorie emulsion-type foods. An emulsion of 20% soybean oil in a water solution containing 1 to 1.5% microcrystalline cellulose behaves like a 65% pure oil emulsion with respect to viscosity, flow properties, and stability (Phillips and Williams 1995).
12.5 THE EFFECT OF INTERACTIONS ON THE COLOR OF FOODS 12.5.1 CHANGES
IN
MEAT PIGMENTS
The color of meat of slaughter animals, fish, mollusks, and crustaceans is affected mainly by the contents and the chemical state of hemoproteins, predominantly MbFe(II) and to a lesser degree hemoglobin, cytochromes, and hemocyanin; the pink to red color of the flesh of salmonid fishes is due to carotenoids. The content of hemoproteins depends on the species, age, sex, nutrition, and living conditions of the animal, the type of muscle and muscle fibers, and on the efficiency of blood removal during slaughtering. The natural, cherry-red color of beef meat on the freshcut surface is due to reduced forms of the muscle hemoproteins. Oxygenation of MbFe(II), hemoglobin, and cytochromes at high partial pressure of oxygen leads to the formation of light cherry-red pigments. Oxymyoglobin (MbFe(II)02) oxidizes slowly to brown MbFe(III), which undergoes further oxidation by H2O2, which may be generated due to bacterial metabolism, to the strong oxidant ferrylmyoglobin (MbFe(IV)=O). Oxidation of thiol groups in meat proteins may lead to cross-linking. MbFe(IV)=O decomposes slowly or reacts with another molecule of MbFe(II) to yield MbFe(III): MbFe(IV)=O → MbFe(III) MbFe(IV)=O + MbFe(II) → 2 MbFe(III)
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A green discoloration in meat is due to sulfmyoglobin MbFe(III)SH, but other factors may also influence the color. In order to stabilize the desirable light-red color of fresh meat, appropriate packaging of the cuts or various antioxidants may be used. Different carotenoids are able to decrease the rate of oxidation of MbFe(II)02 and to reduce MbFe(IV)=O, thus increasing the color stability of meat (Mortensen and Skibsted 2000). Also α-tocopherol is known to stabilize MbFe(II)02 in beef. It acts most probably by delaying the generation of primary lipid peroxides and the release of their watersoluble free-radical breakdown products, predominantly unsaturated aldehydes or HO⋅ radicals. It may also increase the potential of reduction of MbFe(III) in meat, thereby stabilizing the desirable color (Faustman and Wang 2000). Dietary supplementation of livestock with vitamin E is effective in delaying lipid oxidation and discoloration of fresh beef. The desirable color of fresh meat can also be obtained by the formation of the bright cherry-red carboxymyoglobin. Storing of the meat cuts in modified atmosphere packages in the presence of about 0.5% CO has been approved in various forms in different countries. In order to prevent browning due to cooking, as well as to develop the desirable cured-meat flavor and inhibit the outgrowth of Clostridium botulinum spores in pasteurized products, NaNO3 and NaNO2 are used for curing. NaNO3 in the curing brine and in the meat undergoes reduction by bacterial enzymes to NaNO2, which is reduced by the endogenous cytochrome oxidase further to NO. MbFe(II) can also reduce NO2 to NO. The interaction of NO with MbFe(III) in the presence of ascorbate, thiol compounds, and NADH-flavins results in the formation of light-red nitrosyl myoglobin (MbFe(II)=NO). In heated cured meat, the color is due to the thermally stable nitrosyl hemochromogen (see Chapter 6). In cooked poultry, the pinking of white meat is regarded as a defect that suggests an undercooked and thus unsafe product. The possible causes of this defect may be the presence of nitrosyl hemochrome formed due to an incidental nitrate or nitrite contamination, residues of undenatured MbFe(II) or MbFe(II)02, the occurrence of reduced globin hemochromes in well-cooked products, and the formation of carbon monoxide myoglobin. As little as 1 µg of NaNO2 in 1 g of meat is enough to develop a pink color in cooked chicken and turkey. Other factors affecting the occurrence of the defect include the initial color of the raw material, as well as pH and the oxidation-reduction potential in the meat (Holownia et al. 2004).
12.5.2 CHANGES
OF
CHLOROPHYLLS
Chlorophylls are rather unstable pigments. Depending on the conditions of cooking and blanching of green vegetables, and particularly pH, temperature, cations, oxidizing agents, and enzyme activity, they turn into various green, olive-green, olivebrown, grayish-brown, or colorless compounds. Pigment synthesis and degradation in detached fruits and vegetables can be influenced by storage conditions, such as light, temperature, and relative humidity, and by ethylene. Ethylene is formed in plant tissue during ripening, following wounding, or by exposure of the plant to air pollutants. By virtue of its hormone action, ethylene initiates the degradation of
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chlorophyll in mature plant tissues like fruits and leafy vegetables. Normally procedures are established to retard the process in vegetables (via low temperatures and modified atmosphere storage) and promote it in ripening fruit (by ethylene exposure—degreening). The exact mechanism by which ethylene action leads to chlorophyll degradation is not completely known. Chlorophyllase (EC 3.1.1.14) is a thylakoid membrane glycoprotein that catalyzes hydrolysis of the phytol side chain of chlorophyll (no change in color), although it appears that hydrolysis is followed by oxidative degradation of the tetrapyrrole involving a peroxidase (chlorophyll:H2O2 oxidoreductase). The participation of different lipoxygenase isoenzymes has also been implicated in chlorophyll and carotenoid degradation in growing plants and seeds. Conditions of low pH and high temperature favor the conversion of chlorophylls to their corresponding pheophytins, which results in olive-brown discoloration of canned green vegetables (see Chapter 9).
12.5.3 INTERACTIONS
OF
CAROTENOIDS
Carotenoid pigments in vegetables and fruits are mixtures of up to several dozen compounds. They easily undergo oxidation in the presence of air and light, but if properly protected they resist oxidation even at cooking temperatures. Oxidation of carotenoids during storage and processing of foods causes loss of the characteristic color of many commodities. A typical example is the appearance of brown discoloration on stored red peppers due to oxidation of capsanthin. Oxidation of carotenoids may also lead to the development of aroma compounds. Heat, light, or dilute acid conditions cause isomerization of all-trans-carotenoids to various cis isomers. The reaction results in loss of vitamin A activity and changes in color. The direction of color change (red or blue shift) differs with the carotenoid. In some cases, such as canning of pineapple, the orange hue is intensified. The skin of many fish and the shell of marine crustaceans may have vivid yellow, orange, red, purple, blue, silver, or green colors. The major components of the pigments are various carotenoids and the products of their interactions with proteins. In marine organisms the complexes are predominantly formed due to noncovalent interactions of astaxanthin and canthaxanthin or their derivatives with proteins, glycoproteins, phosphorylated glycoproteins, glycolipoproteins, and lipoproteins. Numerous structures and colors of carotenoproteins are known due to the diversity of interacting molecules (Zagalsky et al. 1990). The colors fade during storage of the caught fish, especially in direct, bright light, because the carotenoprotein complexes dissociate. The major carotenoid component in the carapace of lobster is the red astaxanthin, but in the live animal it is present in the form of a blue carotenoprotein crustacyanin. However, boiling the blue or blue-gray crustacean denatures the protein and releases free astaxanthin, whereby the color of the carapace turns bright red. Carotenoids are also responsible for the pink flesh pigmentation of salmonid fish, mainly astaxanthin and canthaxanthin. Oxidation of these pigments, especially in the presence of oxidized lipids, is responsible for the loss of the appealing color of the flesh. Bleaching of the surface of cold smoked salmon fillets may also be due to leaking of the pigments in processing, in the course of salting and rinsing. Dietary
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supplementation with carotenoids is effective in improving the pigmentation of the meat of reared salmonid fishes. In marine animals, other pigments also contribute to color and iridescence, such as the brown-black melanins and melanoproteins and blue-purple indigoins.
12.5.4 INTERACTIONS
OF
OTHER PIGMENTS
The red, violet, or blue color of anthocyanins present in fruits and flowers depends on their structure and on the pH in aqueous media. Hydrophobic interactions and hydrogen bonds between different anthocyanins and with some phenolic acids and alkaloids affect the color and its intensity in various plants. The stability of anthocyanins in food products is rather low and depends on their structure. They are susceptible to enzyme-catalyzed degradation, to light, heat, oxygen, pH, and ascorbic acid. Various anthocyanins differ significantly in stability. In stored, heat-treated fruits and juices the color may gradually diminish or turn brown due to polymerization reactions. In the presence of Al, Fe, and Sn, purplish-blue or slate-gray pigments may be formed in processed fruits (see Chapter 9). The addition of SO2 to the 4-position of anthocyanins forms a bisulfite derivative and causes decolorization. In processed foods (e.g., during storage of frozen tissue that has not been blanched) anthocyanins can be co-oxidized by degradation products resulting from polyphenol oxidase or peroxidase-catalyzed oxidation of phenolic compounds. Decolorization of anthocyanins normally occurs following deglycosylation, and this is catalyzed by endogenous or fungal glycosidases called anthocyanases. Betalains, occurring predominantly in red beets and in some mushrooms as redviolet betacyanins and yellow betaxanthins, are sensitive to heat degradation in acid foods and to oxidation. The major pigment of red beets, betanin, turns into colorless and yellow compounds due to hydrolysis. The rate of degradation of betalains increases in the presence of metal ions.
12.5.5 BROWNING
AND
FORMATION
OF
BLACK SPOTS
Browning reactions are important because they affect the color, flavor, safety, and nutritional value of processed fruits and vegetables. Maillard browning is promoted by a high concentration of reactants (reducing sugars, amino acids/proteins), high temperature, and alkaline pH. Reducing sugars differ in their reactivity in this reaction, e.g., glycoaldehyde > glyceraldehyde >> xylose >> glucose > fructose. Phosphorylated sugars, such as those that sometimes accumulate in postmortem myosystems, are generally much more reactive than their nonphosphorylated counterparts. An example of quality loss due to the participation of phosphorylated sugars in Maillard browning is orange discoloration of canned tuna. The compounds formed in the early stages of Maillard reactions are essentially colorless or slightly yellowish. They are called premelanoidins—precursors of brown-colored melanoidins. The colorless products of molecular mass below 1000 Da are produced mainly by reactions with free AA. Brown-colored melanoidins are generated predominantly by polymerization of intermediary products and by their reactions with proteins. Another possible way is the formation of covalent bonds by
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POO + O2
tyrosine
POO + O2
amino acids
DOPA o-quinone colored colorless reducing additive
brown polymers
FIGURE 12.2 Black spot formation.
reaction of low-molecular-weight chromophor structures with side chains of proteins, especially lysine, arginine, or cysteine residues. The lysine residue can react with furan-2-carboxaldehyde. The reactions of oxidized lipids with amino acids and proteins also result in the formation of brown compounds—so-called protein-lipid browning. The browning rate is particularly high in reactions with cysteine, methionine, and tryptophan. The discoloration is objectionable especially in white poultry or fish muscle, where browning appears even under refrigeration or frozen storage. The shells and surface layers of marine crustaceans, predominantly lobsters, shrimps, and crabs, lose their attractive color shortly after they are caught and develop very dark or black spots. The melanosis starts with enzymatic changes catalyzed by the endogenous polyphenoloxidase (PPO) complex and is followed by nonenzymatic polymerization. In the first step, tyrosine is oxidized in the presence of monophenol oxidase to dihydroxyphenylalanine (DOPA), while diphenoloxidase catalyzes the oxidation of DOPA. Polymerization of the DOPA chinone leads to high-molecularweight black pigments (Figure 12.2). In the polymerization reactions of DOPA, cysteine, tyrosine, and lysine residues in proteins are also involved. Very extensive blackening is regarded as a drastic quality deterioration of the crustaceans. The formation of black spots can be prevented by reducing the quinone to the colorless DOPA by different sulfiting agents in the form of dips shortly after catch, usually on the fishing boats. Bisulfite also acts as a competitive inhibitor of PPO. A number of other reducing compounds, antioxidants, and enzyme inhibitors have been proposed (Kim, Marshall, and Wei 2000). Similar reactions also occur in potatoes, bananas, figs, apples, nuts, or cereal products.
12.6 INTERACTIONS AFFECTING FOOD FLAVOR 12.6.1 INTRODUCTION The flavor of a food is related predominantly to the concentration of the volatile flavor compounds in the vapor phase above the product, which is affected by the concentration of the aroma compounds in foodstuffs, their volatility at the given temperature, and on their affinity to other components. Proteins, lipids, and polysaccharides have the ability of binding flavors due to hydrophobic interaction and hydrogen, ionic, or covalent bonds, depending on the structure of the compounds. This binding decreases the volatility of the molecules, thus affecting the rate of loss of flavor during storage of the products. The stability of the system depends on the pH and temperature of food. Binding of alcohols, aldehydes, ketones, and other volatile compounds by various proteins have been described (Bakker 1995).
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Hydrophobic aroma compounds accumulate predominantly in the lipid phase of foods, thus decreasing their partitioning into the aqueous and vapor phases. The air–lipid partition coefficient for any hydrophobic flavor compound is affected at a given temperature by the fatty acid composition of the lipid and the degree of fat dispersion. Hydrocolloids decrease the perceived intensity of various flavors in food systems, not only by binding of the volatile compounds, but also due to lowering the diffusion rate due to their high viscosity. Another important factor is complexation. Formation of cyclodextrin molecular-inclusion complexes with aroma compounds may protect flavors against evaporation and inhibit their undesirable chemical degradation during processing and storage due to interactions with other food components. The formation of inclusion complexes is controlled by the concentration of the guest molecules and the dimensional compatibility of the flavor compounds and the cyclodextrin cavity. Complexation improves the stability of various flavors, but it may also decrease their release from the cyclodextrin trap during consumption of the products (Reineccius et al. 2004).
12.6.2 HYDROLYTIC, OXIDATIVE,
AND
PYROLYTIC REACTIONS
Hydrolytic reactions in foods are broad ranging. They are catalyzed by enzymes and also by acid or alkaline conditions. Hydrolysis of triacyglycerols, so-called hydrolytic rancidity, leads to free fatty acids, which sometimes have a soapy flavor (e.g., lauric acid). Heating starch or other saccharides under mild acidic conditions leads to hydrolysis, for example, formation of invert sugar from sucrose. Phenolic glycosides are hydrolyzed under mild alkaline conditions. Such reactions can influence the texture and flavor of fruits and vegetables. Phenolic glycosides may also be hydrolyzed by hydrolases such as anthocyanases. Aging changes in bovine meat lead to the liberation of peptides and AAs and thus enhancement of flavor formation in the roasted meat. The increase in the concentration of free AAs in tenderized meats is caused mainly by endogenous proteolytic enzymes from the lysosomes and by calpains. Electrical stimulation of beef carcasses may increase the rate of releasing of the enzymes from the lysosomes, and thus the formation of free AAs. The secondary products of lipid oxidation, especially carbonyl compounds, react with AAs and proteins forming rancid off-flavors. Oxidation of polyenoic fatty acids leads to 2,4-alkabienals and conjugated alkatrienals, which reacting with AAs generate a fishy off-flavor. Terpenes present in the majority of foods of plant origin are also easily oxidized on storage and heating. Oxidized terpenes and products of the decomposition of the intermediary hydroperoxides, including radicals, react with AAs and proteins forming numerous off-flavor compounds, often resembling, in their flavor, rotten or spoiled food. Oxidation of ascorbate in the presence of molecular O2 results in the loss of vitamin C activity, and may be coupled to other reactions, such as disulfide bond formation during gluten development, or conversion of ethanol to acetaldehyde in wine aging. Pyrolytic reactions, especially at temperatures higher than 150°C, mainly between AA and saccharides, lead to the development of numerous flavor compounds
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(see Chapter 11). Pyrrolidone carboxylic acid formed from glutamic acid during thermoprocessing of canned vegetables is believed to contribute to acid-catalyzed pheophytin formation and off-flavor.
12.7 CHANGES IN TEXTURE OF FOODS 12.7.1 INTRODUCTION These interactions occur in the original, intact raw materials of food, for example, in the muscles of slaughter animals and fish postmortem or in frozen fish, stored vegetables and fruits, as well as in processed systems, like comminuted sausage batters, fish gels, dairy products, extrudates, polysaccharide gels, dough, and bread crumbs. They comprise predominantly the formation of various cross-links, due to covalent bonds, hydrogen bonding, electrostatic forces, and hydrophobic interactions. These cross-links may decrease the quality of proteinaceous foods by inducing toughness, for example, in some frozen stored fish, or are necessary for developing the desirable, rheological properties of many products, such as the ability of a protein to form disulfide bridges in extrudates when heated to high temperatures at low moisture content, which is a prerequisite to yielding high-quality products. The crosslinking may be induced by endogenous or added enzymes, different metabolites of enzymatic reactions, chemical additives, or various processing factors, including shearing and heating. On the other hand, endogenous hydrolases may decrease the effect of cross-linking by degrading the polymers. In stored apples, enzymatic degradation of the pectin structural material is responsible for a gradual decrease in the firmness of the tissue due to the loss of binding between the adjacent cell walls. In fresh apples, the shear forces cause cutting of the tissue across the cells, which results in juicy texture, while apples after prolonged storage have a floury texture. In various food gels, a high content of uniform starch particles, 1 to 2 µm in diameter, Ca-pectinate, about 40 µm in diameter, or microcrystalline cellulose particles, is responsible for fat mimetic properties. Other interactions affecting food texture are those that facilitate the formation and stabilize different emulsions and foams. Air and other gases occur in many foods, such as the interstitial space of fruits and vegetables (e.g., apple tissue may contain more than 25% air by volume), and as bubbles in ice cream, whipped cream, bread, and beer foam. In bread or cake the gas bubbles are dispersed in a solid matrix, for example, of starch and protein, while in a fluid such as whipped cream, stabilization is due to a protein film and fat globules. Thus, the texture of the products depends significantly on various interactions of food components and additives and is affected by the processing parameters, predominantly time and temperature of heating, pH, shearing forces, and the presence of metal ions.
12.7.2 RIGOR MORTIS
AND
TENDERIZATION
OF
MEAT
The postmortem changes in the rheological properties of muscle tissues, that is, the development of rigor mortis, gradual diminishing of the stiffness, and tenderization,
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although triggered by biochemical processes, involve physical and chemical interactions of the muscle proteins. The stiffness of the fish or meat is a physical manifestation of interactions between the myosin heads in the thick microfibrils in the sarcomeres and the active centers of the actin molecules in the thin microfibrils. These chemical interactions are the consequences of earlier biochemical events. The concentration of Ca2+ in the sarcoplasm increases about 10 times with the time postmortem until the onset of stiffening. This is due to depletion of the reserves of phosphocreatine and ATP, and in consequence, lack of energy needed for pumping the ions against the concentration gradient back into the sarcoplasmic reticulum. At a high enough concentration of Ca2+ in the sarcoplasm, conformational changes in the regulatory proteins occur, which unblock the reaction sites on the actin molecules for cross-linking. In the flesh of fish belonging to several temperate and tropical species, a phenomenon known as cold shortening occurs. In these fish the prerigor state after death is about twice shorter at 0°C than at 10°C, which is rather unusual. This is accompanied by a correspondingly faster release of Ca2+ from the sarcoplasmic reticulum and a higher rate of degradation of ATP at 0°C than at 10°C. According to Ushio et al. (1991) this phenomenon is caused by the fact that at 0°C, the Ca2+ uptake ability of the reticulum is decreased, which leads to an increase in the concentration of Ca2+ in the sarcoplasm and thus activation of the myofibrillar ATPase. During aging of the carcass, rigor mortis gradually disappears and the meat becomes less tough or overly tender in the case of some fish myosystems. At a temperature of about 3°C to 5°C, the highest tenderness in beef appears after 2 to 4 weeks, and in pork from 6 to 10 days—the toughness decreases to 50 to 60% of the initial value. In current models, formed on the basis of biochemical research, it has been suggested that tenderization during postslaughter conditioning of meat is due to a gradual degradation of the proteins of the myofibrils and of the cytoskeletal proteins. Changes in the collagens and of the proteoglycan component of the extracellular matrix may also be involved (Purslow et al. 2001). These changes result from a concerted action of different endogenous enzymes—large-scale disassembly of the original structural elements of the muscle fiber, followed by further degradation of the smaller components. The cleavage of several proteins should lead to weakening of the cytoskeletal network and thus of the sarcomere structure, resulting in decreased integrity and tensile strength of the muscle fibers. The rate of these processes is affected by temperature and other conditions in the meat, which are both the cause and effect of different enzymatic responses to the activity of endogenous enzyme inhibitors, changes in compartmentalization, concentration of Ca2+ in the sarcoplasm, and pH. These factors are not constant during the aging of meat. In the carcasses of conventionally slaughtered animals, the temperature in the center of the thickest part drops during cooling in industrial conditions to 5°C after about 2 days. After 15 hours the pH in such beef carcasses is about 6. These data and the properties of the endogenous proteolytic enzymes, as well as the fact that most chemically evidenced proteolysis occurs only after the development of meat tenderness, may indicate that the endogenous cathepsins, m-calpain, and µ-calpain, cannot contribute significantly to meat tenderization in practical conditions of carcass handling. The weakening of the structures of the myofibrils
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and the desmin intermediate filaments may be due to Ca2+ that accumulates in the sarcoplasm postmortem. Ca2+ ions at a concentration of 0.1 mM disrupt the filamentous structure of titin, nebulin, and desmin. They also weaken the Z-disks in the sarcomeres by binding to the phospholipids, which are the main components of the matrix reinforcing the Z-disk structure (Kanawa et al. 2002, Ahn et al. 2003).
12.7.3 TEXTURE CHANGES
IN
FROZEN FISH
Prolonged storage of frozen fish, mainly belonging to the Gadidae family, may lead to increased toughness of the meat, loss in functional properties, and decreased ATPase activity of myosin. This is the result of cross-linking of myofibrillar proteins caused by interactions of AA residues with different reactive components, predominantly with formaldehyde generated by endogenous trimethylamine oxide demethylase, and with several products of lipid oxidation or hydrolysis. Oxidizing lipids may contribute to protein cross-linking via radical polymerization and due to reactions of secondary oxidation products. Natural antioxidants—mixtures of plant extracts—are effective in inhibiting undesirable changes in frozen fatty fish. The loss in functional properties is especially severe in minced fish due to increased contact of the interacting components of the mince. By removing from the minced fish the water-soluble proteins and nonprotein nitrogenous components, prooxidative compounds and ions, as well as most of the lipids, a concentrate of myofibrillar proteins, known as surimi, is produced. Surimi is more resistant to undesirable changes in functional properties than the original fish mince. However, extracting of the water-soluble components results in about a 30% loss of crude protein from the fish meat. The changes in myofibrillar proteins in frozen fish can also be limited by adding different cryoprotective compounds, decreasing the storage temperature, and preventing lipid oxidation in the mince. Among the most common cryoprotectors are sucrose, sorbitol, mannitol, alginates, polyphosphates, citrates, ascorbate, and sodium chloride in different, proprietary mixtures. Several AAs, hydroxy carboxylic acids, branched oligosaccharides, and adenosine nucleotides are effective in model systems and in surimi (Matsumoto and Noguchi 1992). AAs with a high negative net charge are most effective in preventing freezing denaturation of fish myofibrillar proteins. Generally, the low molecular cryoprotectors contain more than one reactive group suitably spaced in the molecule.
12.7.4 CROSS-LINKING
IN
GELS
AND
FILMS
Food gels may be made from proteins and polysaccharides due to chemical or enzymatic interactions of the polymers, and in some cases involvement of endogenous or added low molecular compounds. After deconformation of the native macromolecules a three-dimensional structure is formed, which is capable of immobilizing large quantities of solvent, various solutes, and inert filling material. The concentration of the polymers may be as low as a few percent of the total mass of some gels. The cross-links buttressing the structure result from hydrophobic interactions between the exposed nonpolar fragments of the chains in the aqueous environment, hydrogen bonds, ionic forces, and various covalent bonds.
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A typical polysaccharide gel is that formed by starch. Native granular starch absorbs in aqueous media up to 30% of water, by weight. The swollen granules heated in water lose their crystalline structure and turn into gelatinized starch, which gradually forms a paste of high viscosity. Salt and some low-molecular-mass hydrophilic components, competing for available water molecules, increase the gelatinization temperature. Cooling of the paste leads to further increase in viscosity due to the development of a network of the hydrated starch molecules and the mass turns into a gel. During prolonged storage of the paste or gel, the crystalline structure of starch gradually develops again in a process called retrogradation and some water is exuded. The rate of retrogradation depends predominantly on the proportions of amylose and amylopectin in the granules and the contents of lipids, as well as on the temperature (Jane 2004). The mutual interactions of different polysaccharides may lead to increased viscosity of solutions and gelling. The extent of synergism is affected by the chemical composition of the polymers. The gelling of agarose, carrageenan, and furcellaran can be enhanced in the presence of other, nongelling polysaccharides. κ-Carrageenan increases the gelling of mixtures of locust bean gum and other galacto- and glucomannans in aqueous media. The mechanism of this synergism may involve hydrogen bonding between the two interacting polysaccharides, self-aggregation of the galactomannan chains in the presence of the carrageenan network, or mutual exclusion of incompatible polymers. In some cases, even a very small addition of a specific polysaccharide, about 0.5% of the total polymer concentration, can have a significant synergistic effect on gelling of the system. Interchain associations of alginates and pectins enhance the gelling of these polymer mixtures (Phillips and Williams 1995). Reactions influencing the intracellular cement of fruits and vegetables may also have a profound effect on texture. Phytic or citric acids can sequester Ca2+ from intracellular pectate and thereby cause softening of canned fruit. Likewise, the presence of added Ca2+ or the enzyme-catalyzed deesterification of pectin leads to the firming of cooked vegetables like green beans and potatoes. Cross-linking is also involved in the preparation of edible films, which are used for foods because of their barrier properties, as carriers of ingredients and antimicrobial agents, and as enzyme supports. Such films can be made of various proteins or of proteins in mixtures with saccharides or lipids. Their properties depend on the proportions and characteristics of the components and on the interactions between the polymers, cross-linking agents, and plasticizers. The procedures applied for preparing films generally comprise in the first step deconformation of the polymer molecules by heating, shearing, or exposing thin layers of the interface with air. At appropriate concentrations of the denatured polymer in the dope, cross-linking is induced by evaporation of the solvent or shearing in a spinneret and immersing in a coagulating bath.
12.7.5 INTERACTIONS
IN THE
DOUGH
Protein cross-links and protein–lipid–polysaccharide interactions are necessary for the development of the desirable texture of cereal-based baked goods. During mixing with water and salt, the flour particles hydrate and the glutenin polymers disaggregate
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and form a membrane network. During preparation of bread dough, after addition of water, the wheat endosperm particles very rapidly exude proteinaceous fibrils. These fibrils are formed from the gluten protein film at the air–water interface, which is created due to interfacial forces. Interactions of the strands with each other lead to cross-linking of the flour particles and cohesion in the dough (Bushuk 1998). The dough can be viewed as a viscoelastic, hydrated matrix of gluten proteins with inserted starch granules and cell wall debris. Thus it may be regarded as a filled gel system. Intra- and interpeptide disulfide bonds play a key role in building the required structure of the dough. In the presence of soluble compounds containing thiol groups, some of the –S–S– bridges originally present in the gluten proteins are mobile due to disulfide interchange reactions, and interchain cross-links can be formed. Oxidizing agents added to the dough improve the texture of bread by promoting the formation of the cross-links. Baking increases the number of disulfide bridges, adding to the stability of the loaf structure. Other covalent bonds also contribute to the properties of the dough. Cross-linking may be caused by dehydroascorbic acid and by the products of its thermal degradation, like methyl glyoxal, glyoxal, diacetyl, and threose, which interact with the lysine residue of proteins (Fayle et al. 2000). Other covalent cross-links can probably result from enzymatic formation of γ-butyraldehyde catalyzed by endogenous diamine oxidase and subsequent addition to it of nucleophilic AA residues in the presence of Cu(II) ions (Chiarello et al. 1996). Hydrolysis of a number of peptide bonds in the gluten proteins by endogenous or added enzymes leads to weakening of the structure. The interactions between proteins and starch are also important (Friedman 1995). The structure of the dough is significantly affected by hydrogen bridges. According to Levebvre et al. (2003) gluten forms a network due to aggregation of particles primarily under the effect of hydrogen bonds and hydrophobic interactions, with the disulfide bridges probably not involved directly in the network formation. Although hydrogen bonds are much weaker than covalent bonds, they may be formed in large numbers due to a high content of glutamine residues in cereal proteins and numerous hydroxyl groups in starch and pentosans. Interchange reactions of the hydrogen bonds under mechanical stress facilitate stress relaxation of molded dough. Hydrophobic interactions in aqueous systems between the apolar AA residues of the flour proteins also contribute to buttressing the dough structure, especially during baking of the loaf. Ionic repulsion–attraction forces, although not numerous because of a relatively small number of ionizable residues in cereal proteins, also play a role in forming the desirable, porous structure of bread crumbs (Wrigley et al. 1998).
12.7.6 LIPIDS
AND THE
TEXTURE
OF
FOODS
Lipid droplets have a structure-forming role in oil-in-water emulsions like milk, cream, ice cream, cheese, dressing, mayonnaise, and comminuted meat products. These droplets are stabilized by interactions with proteins, lecithins, or various synthetic surfactants. The rheological properties of milk gels depend on the size of the fat droplets and the nature of the stabilizing agent. Low-molecular-weight surfactants make the surface of the droplets smooth and noninteractive and the gels weak. In gels stabilized by whey proteins, the fat globules are cross-linked due to
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protein interactions. On standing of chilled, natural milk, the lipoprotein membranes of fat globules interact with a serum protein, thereby forming clusters of globules. Clustering of the fat globules around the air bubbles contributes to the desirable characteristics of ice cream. In the cream-churning process, concentration of the fat globules and spreading of the oil at the air–water interface occur, leading to suppression of the foam and formation of clumps of the water-in-oil emulsion of butter. In various dairy products, margarine, shortenings, and chocolate, the fats occur in the form of crystals. Triacylglycerols crystallize in at least four forms: sub-α, α, β′, and β, differing in stability and melting temperatures, as well as in their effect on the creaming and rheological properties of the products. Transformations of the polymorphic forms of cocoa butter are thought to be responsible for the development of the undesirable appearance of white or gray powdery surface deposits and loss of gloss of stored chocolate, known as bloom. Spherulites with random arrangement of fat crystals can be responsible for the sandy texture of various foods. As discussed in Section 12.7.3, lipids and lipid oxidation and hydrolysis products also influence texture by interacting with proteins.
12.8 INTERACTIONS AND THE NUTRITIVE VALUE OF FOODS 12.8.1 INTRODUCTION The nutritive value of foods may be affected primarily by interactions among proteins, proteins and saccharides, proteins and lipids, and with oxidizing agents. The rate of these interactions increases with the temperature applied in food processing and cookery. However, their effects also occur during prolonged storage even in refrigerated and frozen products. The nutritive value may decrease due to loss in digestibility or chemical modifications making the nutrients unavailable for the human organism.
12.8.2 CHANGES
IN
DIGESTIBILITY
The digestibility of a protein depends, inter alia, on the structure of the protein. Tightening of the protein structure by additional cross-links that prevent unfolding of the polypeptide chains may decrease the digestibility by making some peptide bonds, buried inside of the molecule, inaccessible to the enzymes. Heating of foods may lead to cross-linking in proteins by isopeptide bonds between the ε-NH2 group of lysine and the β- and γ-carboxyl groups of aspartic and glutamic acid residues, respectively, or their amides. This may decrease the rate of protein hydrolysis. Protein cross-linking may also be caused by interactions of amino groups with reducing saccharides or other compounds containing carbonyl groups. In foods these are especially the secondary products of lipid oxidation and aldehydes contained in smoked commodities. As a result of a number of further reactions, different unsaturated compounds are generated, which also interact with amines and AAs. Various cross-linking reactions may be involved, including the formation of α-amino acid
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amides and α-hydroxyacid amides (Büttner, Gerum, and Severin 1997). Crosslinking also occurs in food systems due to alkaline treatment (see Chapter 6).
12.8.3 CHANGES
IN
BIOLOGICAL VALUE
While the early products of the Maillard reaction can be utilized in the human organism, further changes gradually make the lysine residue nutritionally unavailable. This happens mainly in the outer parts of baked, grilled, or fried products. In the center of a baked bread loaf, turkey, or chicken, the temperature is below 100°C and the loss of lysine is negligible. However, sterilization leads to a significant decrease of available lysine in products rich in reducing sugars and high-value proteins. In various commercially produced enteral proteinaceous food formulas, the lysine availability is only about 75% (Castillo et al. 2002). Significant loss of lysine has also been found in commercial infant formulas (Gonzales et al. 2003). The Maillard products are formed at a high rate, mainly in the outer parts of heated foods. However, in condensed milk, the reaction also proceeds at ambient temperature. Although the rate of reaction is much slower, the content of available lysine may drop by several percent due to months of storage. The decrease in availability of lysine can be followed by determining the unchanged AA residue, or by assaying some early Maillard reaction products, such as furosine, Nε-carboxymethyllysine or pyrraline (Hartkopf and Erbersdobler 1995; Belitz, Grosch, and Schieberle 2001). Thiamine and its decomposition products formed in heated foods may also participate in the Maillard reaction. In acid foods thiamine is stable, but decomposes readily at alkaline pH. The average loss of thiamine due to boiling of eggs is about 15%, curing of meat 20%, baking of bread 15 to 20%, cooking of vegetables 25 to 40%; in different cooked, canned, roasted, and fried meat and fish products the loss ranges from about 15 to 75%. Interactions of dehydroalanine formed during heating in alkaline conditions (see Chapter 6) with the residues of other AAs lead to cross-linking, and in hydrolysates of the heated protein a mixture of unnatural and D-forms of AAs can be found. However, the D-forms of AAs may be present in various foods not only due to heating in alkaline conditions. In the edible parts of some aquatic crustaceans and mollusks, D-alanine is synthesized by alanine racemase. Furthermore, fish sauces and fermented dairy products contain substantial amounts of D-alanine and Daspartic acid of microbial origin. The interactions due to prolonged heating at alkaline conditions may decrease the nutritional value of proteins due to the loss of essential AAs and lower absorption of the D-forms. Some products of the reactions inactivate metalloenzymes, induce nephrocytomegaly in rats and kidney damage (Pearce and Friedman 1988; Friedman and Pearce 1989). A beneficial effect of heating under alkaline conditions is the release of nutritionally available niacin from grains. The Hopi Indians’ traditional procedure of cooking mature corn grain with alkaline wood ashes hydrolyzed the ester linkages binding niacin to saccharides and turned the nutritionally unavailable polymer into free niacin (Wall and Carpenter 1988). High-acid foods like lemon juice undergo nonoxidative degradation of ascorbic acid to 3-deoxypentulose 2-furaldehyde. The reaction appears to contribute to ascorbate browning of these products as well as loss of vitamin C.
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Several components of wood smoke, mainly phenols, aldehydes, and nitric oxide, may react with some AA residues in meat proteins during smoking and storage of the products. The results of various model experiments indicate that the contents of SH and NH2 groups, as well as lysine availability decreases by from several up to about 90% in cysteine solution in beef meat and fish due to smoking in various conditions. Recent investigations have shown that in mild hot smoked fish, the loss of available lysine is negligible (Kołodziejska et al. 2004). Oxidation may damage AAs, decrease the digestibility of the protein by stable cross-linking, and lead to antinutritional degradation products. The rate of changes in proteins is controlled by the concentration of various endogenous and added oxidizing agents, prooxidants, and sensitizers, such as chlorophyll, methylene blue, erythrosine, and riboflavin, antioxidants, temperature, and the sensitivity of various AA residues. The effect also depends on the distribution of the reactive AA residues in the polypeptide chains and on their availability for reaction with the oxidizing agent at the actual conformation of the protein. The oxidation of thiol groups in native proteins is less efficient than in denatured proteins. The number of surface reactive SH groups in proteins increases due to heating in the temperature range of 20 to 50°C (Sano et al. 1994). Oxidation of thiol groups in proteins by oxidized lipids proceeds predominantly via the free radical mechanism. Tryptophan undergoes autoxidation in acidic and alkaline solutions when heated to 100°C or higher temperatures, in a free-radical reaction, whereby different degradation products appear (Friedman and Cuq, 1988). The destruction of tryptophan residues in various proteins under conditions similar to those prevailing in food cookery and sterilization is not higher than a few percent. Oxidation of ascorbic acid with molecular O2 to form diketogulonic acid results in loss of vitamin C activity. Heating foods with thiamin at pH less than 6 results in cleavage of the methylene bridge of vitamin B1 to form pyrimidine and thiazole and loss of vitamin activity. Likewise, the isomerization of all-trans carotenoids with provitamin A activity favored by light, heat, and dilute acid conditions causes loss of vitamin A activity. Increased lipid oxidation from conversion of Fe2+ to Fe3+ and the resulting reduced iron absorption or bioavailability is the consequence that can occur when iron is oxidized.
12.9 THE EFFECT OF INTERACTIONS ON THE SAFETY OF FOODS Heating may lead to the formation of N-nitroso compounds (NNC) in foods containing high amounts of nitrates or nitrites. About 90% of 300 tested NNCs have been reported to be carcinogenic in animal experiments. Although NNCs are found mainly in heated foods, they are also formed in frozen stored products, albeit at a low rate. Additives capable of binding the nitrosating agents, such as ascorbate and α-tocopherol, can effectively retard the formation of NNCs. Several other compounds have also been found to be efficient inhibitors of N-nitrosation, such as long-chain acetals of ascorbic and erythorbic acids. Foods low in nitrates and amines contain usually up to 10 ng NNCs/g, while cured and heavily smoked meat and fish up to several hundred ng/g. In smoked foods, C-nitroso- and C-nitrophenols have also been found.
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In potato chips, French fries, and processed cereals, fried beetroot, and fried spinach, different amounts of acrylamide may be found. The content of acrylamide in various foods increases with the time and temperature of heating, and may be above the actual detection limit of about 10 ng/g (Food and Agriculture Organization/World Health Organization [FAO/WHO] 2002). It may be formed in Maillardtype reactions between glucose and asparagine, as well as by changes of AAs, acrolein, acrylic acid, and lactic acid at about 120°C. The contents of acrylamide in French fries can be significantly decreased by extracting the reducing sugars and asparagine from the surface of the cut potato (Biedermann-Brem et al. 2003). About 20 different mutagenic and carcinogenic heterocyclic aromatic amines were found in different heated foods. Their content in foods depends on the concentration of substrates, enhancers and inhibitors, time and temperature of heating, water activity, and pH. Some of them are formed even at about 37 to 60°C, however, only after several weeks. In the presence of lipids, Fe2+, and Fe3+, the rate of reaction increases, probably due to oxidation and the generation of radicals (Jägerstad et al. 1998). Antioxidant spices—rosemary, thyme, sage, and garlic—as well as curing brine, reduce the formation of several heterocyclic amines in fried meat (Murkovic, Steinberger, and Pfannhauser 1998). The total contents of these compounds in broiled, fried, and grilled protein-rich products are generally on the order of several ng/g wet weight; however, in some commodities it may even reach 85ng/g (Tai, Lee, and Chen 2001). Interactions leading to the formation of these compounds are presented in detail in Chapter 19. Interactions of lipids and NaCl in heated foods may cause the formation of carcinogenic 3-monochloropropanediol. This compound has been detected in concentrations on the order of 50 ng/g in bacon, salami, cured fish, bread, cakes, pastries, and cheeses, and up to 10 times more in malts. The rate of formation of 3-monochloropropanediol increases with the temperature of food processing.
12.10 FINAL REMARKS Interaction is the key word with respect to many aspects of the quality of industrially processed and home-prepared foods. For the food technologist, it is not enough to know the physical and chemical properties of individual food constituents. In order to control the quality of various commodities, it is necessary to understand the effect of processing variables and storage conditions on the interactions of the different components in whole or disintegrated food materials. Although the results of numerous experiments carried out in model systems have already given insight into the mechanisms of the interactions of many food constituents, more information must be obtained on these reactions in composite food systems. While studying physical and chemical interactions in foods, it must be considered that ongoing biochemical processes often rapidly modify the system and new components may emerge, which can play the role of reactive substrates or inhibitors that can change the reaction conditions and outcome.
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REFERENCES Ahn, D.H., Shimada. K., and Takahashi, K., 2003, Relationship between weakening of Zdisks and liberation of phospholipids during postmortem aging of pork and beef, J. Food Sci., 68(1), 94–98. Akdogan, H., 1999, High moisture food extrusion, Intl. J. Food Sci. Technol., 34(3), 195–207. Auh, J.H., Kim, Y.R., Cornillon, P., Yoon, J., Yoo, S.H., and Park, K.H., 2003, Cryoprotection of protein by highly concentrated branched oligosaccharides, Intl. J. Food Sci. Technol., 38(5), 553–563. Bakker, J., 1995, Flavor interaction with the food matrix and their effects on perception, in ingredient interactions, in Effects on Food Quality, Gaonkar, A.G., Ed., Marcel Dekker, New York, Basel, Hong Kong, pp. 411–438. Belitz, H.-D., Grosch, W., and Schieberle, P., 2001, Lehrbuch der Lebensmittelchemie. Fünfte, vollständig überarbeitete Auflage, Springer-Verlag, Berlin. Biedermann-Brem, S., Noti, A., Grob, K., Imhof, D., Bazzacco, D., and Pfefferle, A., 2003, How much reducing sugar may potatoes contain to avoid excessive acrylamide formation during roasting and baking?, Eur. Food Res. Technol., 271(3), 185–194. Bushuk, W., 1998, Interactions in wheat doughs, in Interactions: the Keys to Cereal Quality, Hammer, R.J. and Hoseney, R.C., Eds., American Association of Cereal Chemists, Inc., St. Paul, MN, pp. 1–16. Büttner, U., Gerum, F., and Severin, T., 1997, Formation of α-amino-acid amides and α-hydroxy-acid amides by degradation of sugars with primary amines, Carbohyd. Res., 300, 265–269. Castillo, G., Ángeles Sanz, M., Ángeles Serrano, M., and Hernández, A., 2002, Influence of protein source, type, and concentration, and product form on the protein quality of commercial enteral formulas, J. Food Sci., 67(1), 328–334. Chao, H., Houston, M.E., Hodges, R.S., Kay, C.M., Sykes, B.D., Loewen, M.C., Davies, P.L., and Sönnichsen, F.D., 1997, A diminished role for hydrogen bonds in antifreeze protein binding to ice, Biochemistry, 36(48), 14652–14660. Cheng, A. and Merz, K.M., 1997, Ice-binding mechanism of winter flounder antifreeze proteins, Biophys. J., 73, 2851–2873. Chiarello, M.D., Larre, C., Kedzior, Z.M., and Gueguen, J., 1996, Pea seedling extracts catalyze protein amine binding and protein cross-linking. 2. Contribution of diamine oxidase to these reactions. J. Agric. Food Chem., 44, 3723–3726. Eliasson, A.Ch., 1998, Lipid-carbohydrate interactions, in Interactions: the Keys to Cereal Quality, Hammer, R.J. and Hoseney, R.C., Eds., American Association of Cereal Chemists, Inc., St. Paul, MN, pp. 47–80. Espinosa, J.F, Juan Luis Asensio, J.L., García, J.L., Laynez, J., Bruix, M., Wright, Ch., Siebert, H-Ch., Gabius, H.-J., Cañada, F.J., and Jiménez-Barbero, J., 2000, NMR investigations of protein carbohydrate interactions. Binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N′, N″-triacetylchitotriose, Eur. J. Biochem., 267(13), 3965. FAO/WHO (Food and Agriculture Organization/World Health Organization), 2002, Health implications of acrylamide in food, Report of a Joint FAO/WHO Consultation, Geneva. Fayle, S.E., Gerrard, J.A., Simmons, L., Meade, S.J., Reid, E.A., and Johnston, A.C., 2000, Crosslinkage of proteins by dehydroascorbic acid and its degradation products, Food Chem., 70, 193–198.
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Faustman, C. and Wang, K.W., 2000, Potential mechanisms by which vitamin E improves oxidative stability of myoglobin, in Antioxidants in Muscle Foods. Nutritional Strategies to Improve Quality, Decker, E., Faustman, C., and Lopez-Bote, C.J., Eds., WileyInterscience, New York, pp. 135–152. Friedman, M. and Cuq, J.L., 1988, Chemistry, analysis, nutritional value, and toxicology of tryptophan in food, A review, J. Agric. Food Chem., 36, 1079–1093. Friedman, R.B., 1995, Interactions of starches in foods. Interaction of water with food components, in Ingredient Interactions. Effects on Food Quality, Gaonkar, A.G., Ed., Marcel Dekker, New York, Basel, Hong Kong, 171–195. Friedman, M. and Pearce, K.N., 1989, Copper(II) and cobalt(II) affinities of LL- and LDlysinoalanine diastereomers: implications for food safety and nutrition, J. Agric. Food Chem. 37, 123–127. Gonzáles, A.S.P., Naranjo, G.B., Malec, L.S., and Vigo, M.S., 2003, Available lysine, protein digestibility and lactulose in commercial infant formulas, Intl. Dairy J., 13, 95–99. Haard, N.F., 1995, Foods as cellular systems: impact on quality and preservation. A review, J. Food Biochem., 19, 191–238. Hartkopf, J. and Erbersdobler, H.F., 1995, Model experiments with sausage meat on the formation of Nε-carboxymethyllysine, Z. Lebensm. Unters. Forsch., 201, 27–29. Heertje, I., 1993, Structure and function of food products: a review, Food Structure, 12, 343–364. Holownia, K., Chinnan, M.S., and Reynolds, A.E., 2004, Cooked chicken breast meat conditions related to simulated pink defect, J. Food Sci., 69, FCT194–FCT199. Jane, J., 2004, Starch: structure and properties, in Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, pp. 81–101. Jägerstad, M., Skog, K., Arvidsson, P., and Solyakov, A., 1998, Chemistry, formation and occurrence of genotoxic heterocyclic amines identified in model systems and cooked foods, Z. Lebensm. Unters. Forsch A, 207, 419–427. Kanawa, B., Ji, J.R., and Takahashi, K., 2002, Inactivity of µ-calpain throughout postmortem aging of meat. J. Food Sci., 67(2), 635–638. Kim, J., Marshall, M.R., and Wie, Ch., 2000, Polyphenoloxidase, in Seafood Enzymes. Utilization and Influence on Postharvest Seafood Quality, Haard, N.F. and Simpson, B.K., Eds., Marcel Dekker, New York, pp. 271–315. Kołodziejska, I., Niecikowska, C., Sikorski, Z. E., and Kołakowska A., 2004, Lipid oxidation and lysine availability in Atlantic mackerel hot smoked in mild conditions, Bulletin of the Sea Fisheries Institute, 1(161), 15–27. Lefebvre, J., Pruska-Kędzior, A., Kędzior, Z., and Lavenant, L., 2003, A phenomenological analysis of wheat gluten viscoelastic response in retardation and in dynamic experiments over a large time scale, J. Cereal Sci., 38, 257–267. Marion, D., Dubreil, L., Wilde, P.J., and Clark, D.C., 1998, Lipids, lipid-protein interactions and the quality of baked cereal products, in Interactions: the Keys to Cereal Quality. Hammer, R.J. and Hoseney, R.C., Eds., American Association of Cereal Chemists, Inc., St. Paul, MN, pp. 131–158. Matsumoto, J.J. and Noguchi, S.F., 1992, Cryostabilization of protein in surimi, in Surimi Technology, Lanier, T.C. and Lee, C.M., Eds., Marcel Dekker, New York, Basel, Hong Kong, pp. 357–388. Mortensen, A. and Skibsted, L.H., 2000, Antioxidant activity of carotenoids in muscle foods, in Antioxidants in Muscle Foods. Nutritional Strategies to Improve Quality, Decker, E., Faustman, C., and Lopez-Bote, C.J., Eds., Wiley-Interscience, New York, pp. 61–82.
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Murkovic, M., Steinberger, D, and Pfannhauser, W., 1998, Antioxidant spices reduce the formation of heterocyclic amines in fried meat, Z. Lebensm. Unters. Forsch., A. 207, 477–480. Pearce, K.N. and Friedman, M., 1988, Binding of copper(II) and other metal ions by lysinoalanine and related compounds and its significance for food safety, J. Agric. Food Chem., 36, 707–717. Phillips, G.O. and Williams, P.A., 1995, Interaction of hydrocolloids in food systems, in Ingredient Interactions. Effect on Food Quality. Gaonkar, A.G., Ed., Marcel Dekker, New York, pp. 131–169. Purslow, P.P., Ertbjerg, P., Baron, C.P., Christensen, M., and Lawson, M.A., 2001, Patterns of variation in enzyme activity and cytoskeletal proteolysis in muscle. 47th International Congress of Meat Science and Technology, Kraków, August 26–31, Congress Proceedings, Vol. 1, pp. 38–43. Ramsden, L., 2004, Plant and algal gums and mucilages, in Chemical and Functional Properties of Food Saccharides, Tomasik, P., Ed., CRC Press, Boca Raton, FL, pp. 231–254. Reineccius, T.A., Reineccius, G.A., and Peppard, T.L., 2004, Utilization of β-cyclodextrin for improved flavor retention in thermally processed foods, J. Food Sci., 69, FCT58–FCT62. Sano, T., Ohno, T., Otsuka-Fuchino, H., Matsumoto, J.J., and Tsuchiya, T., 1994, Carp natural actomyosin: thermal denaturation mechanism, J. Food Sci., 59, 1002–1008. Tai, C.-Y., Lee, K.H., and Chen, B.H., 2001, Effects of various additives on the formation of heterocyclic amines in fried fish fibre, Food Chem., 75, 309–316. Ushio, H., Watabe, S., Iwamoto, M., and Hashimoto, K., 1991, Ultrastructural evidence for temperature-dependent Ca2+ release from fish sarcoplasmic reticulum during rigor mortis, Food Structure, 10, 267–275. Wall, J.S. and Carpenter, K.J., 1988, Variation in availability of niacin in grain products, Food Technol., (10), 198–204. Watson, C., Bourke, R.E., and Brill, R.W., 1988, A comprehensive theory on the etiology of burned tuna, Fishery Bull., 86(2), 367–372. Wrigley, C.W., Andrews, J.L., Bekes, F., Gras, P.W., Gupta, R.B., MacRitchie, F., and Skerritt, J.H., 1998, Protein–protein interactions—essential to dough rheology, in Interactions: The Keys to Cereal Quality, Hammer, R.J. and Hoseney, R.C., Eds., American Association of Cereal Chemists, Inc., St. Paul, MN, pp. 17–46. Zagalsky, P.F., Eliopaulos, E.E., and Findlay, J.B.C., 1990, The architecture of invertebrate carotenoproteins, Comp. Biochem. Physiol., 97B(1), 1–18.
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13
Main Food Additives Adriaan Ruiter and Alphons G.J. Voragen
CONTENTS 13.1 13.2 13.3
Introduction................................................................................................ 357 Classification.............................................................................................. 358 Preservatives .............................................................................................. 359 13.3.1 Introduction ................................................................................. 359 13.3.2 Sulfite........................................................................................... 360 13.3.3 Nitrite........................................................................................... 361 13.3.4 Sorbic Acid.................................................................................. 361 13.3.5 Benzoic Acid ............................................................................... 362 13.4 Antioxidants............................................................................................... 362 13.5 Flavorings, Colorants, and Sweeteners ..................................................... 363 13.6 Stabilizers, Emulsifiers, and Thickening Agents ...................................... 365 13.7 Clarifying Agents and Film Formers ........................................................ 367 13.8 Acidulants .................................................................................................. 367 13.9 Fat Substitutes and Fat Mimetics .............................................................. 368 13.10 Prebiotics ................................................................................................... 369 References.............................................................................................................. 371
13.1 INTRODUCTION The addition of certain substances to foodstuffs was practiced in ancient times, mostly for improving preservation. Salt was added to perishable foodstuffs, such as meat and fish, from prehistoric ages on. Smoke curing can also be considered as the fortuitous addition of constituents to food, as wood smoke contains a number of compounds that are absorbed by the food during the smoke-curing process or are deposited onto the surface. These treatments not only prolong the shelf life of the food, but also add to the flavor. The preparation of any food product includes the addition of a number of ingredients that are not considered to be additives, but that clearly improve some properties of the food, such as maintenance of quality, and are originally intended as such. Preparation of a marinade of sour wine or vinegar, for example, is a technique for preserving fish, which was known to the Romans, but acetic acid is not an additive in the strict sense of the word. In some cases, it is not so easy to determine whether the substance under consideration is an additive. It is helpful, however, to keep in mind that an additive is intended as an aid, for some purpose or another, and not as an ingredient. 357
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In 1955, the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Nutrition defined food additives as non-nutritive substances which are intentionally added mostly in small quantities, with the aim of improving the appearance, flavor, taste, composition, or shelf-life of foods. In a more recent wording, these food additives are described as generally not intended as a foodstuff or as a characteristic ingredient which, irrespective of any nutritional value, are added, for any technological or sensoric reason, to a foodstuff during manufacturing, preparation, packaging, transport or storage, and from which it is expected that either the substance itself or reaction or decomposition products become a permanent component of the foodstuff or the raw material (van Dokkum, 1985; Kamsteeg and Baas, 1985). In the latter definition, the term improvement is not included, and the remaining presence of the compound, or reaction products from that compound, are included in the definition. This may be the result of a shift in the attitude toward additives. The ranking, by the public, of actual food hazards, was almost inverse to that given by Wodicka (1977), in which microbiological and nutritional risks were listed at the top and food additives in the last position of the ranking. To some extent, this inversion exists even today (Hall, 1999). The preparation of any food product includes the addition of a number of ingredients that are not considered to be additives, but that clearly improve some properties of the food, such as maintaining quality, taste, flavor, or texture, and are originally intended as such. The origin of food additives remains a point of discussion. There is a continuing demand, from the consumer’s side, for “natural” additives. No additive, however, is completely free of impurities. Products of chemical synthesis should be purified, eliminating starting materials and compounds resulting from side reactions. It must be stated that enzyme-catalyzed synthesis or modification more and more frequently replaces purely chemical synthesis of modification. “Natural” compounds should be purified as well in order to remove accompanying substances that have no significance in the final product. Generally speaking, purification is more difficult and more complicated for natural additives, as it is also much more problematic to characterize the raw material, which may contain a great many ill-defined compounds whose toxicity is largely unknown (Ruiter, 1989). Feberwee (1989) points out that official legislation does not discriminate between safe natural and safe artificial food additives. The main difference in safety evaluation between these two categories is our long experience with natural additives (Lüthy, 1989).
13.2 CLASSIFICATION Additives are most often listed and classified in the following categories: • • • •
preservatives to extend the shelf life of foodstuffs antioxidants to protect lipids in food from attack by oxygen flavor enhancers to improve the perception of taste and flavors sweeteners to replace sugars to provide a sweet taste to the product
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colorants to improve the appearance of a foodstuff emulsifying agents to enable and maintain a fine partition between oils or fats in water (or a partition of water between oils or fats), gas in liquid (foam) thickening and gel-forming agents clarifying agents film formers glazing agents acidulants fat substitutes and fat replacers substances improving the nutritional value pro- and prebiotics many other substances such as anticlotting agents, moisteners, antifoaming agents, flour improvers, leavening agents and baking powders, melting salts, stiffening agents, complexing agents, fillers, enzymes, and so on
With respect to the reactivity of additives, it is preferable to add another classification, in which three groups can be distinguished: •
•
•
Substances that, simply by their presence, lead to the desired improvement. Most colorants, sweeteners, and some preservatives belong to this class. Many of these additives do not display a strong reactivity toward other constituents during preparation and storage of the foodstuffs to which they are added. Substances that are added because of their reactivity toward undesirable components already present or arising during manufacturing or storage, and that may be bound by these added substances. The reaction may be directed toward these components themselves or toward their precursors. Antioxidants are an example of this category, as well as some peculiar substances reacting with matrix components to make desirable components such as flavor compounds. Food additives that participate in fortuitous reactions which, in some cases, may be undesirable.
This classification, however, also has its limits. First, there is hardly any additive that does not take part in some chemical reaction. Furthermore, some food additives may also participate in unintentional reactions. Therefore, in this presentation, some additives are discussed in an individual way, with emphasis on their reactivity with respect to matrix compounds.
13.3 PRESERVATIVES 13.3.1 INTRODUCTION Preservatives are added in order to protect a variety of foodstuffs against microbial spoilage. This protection is possible, in many cases, because of a chemical reaction between the preservative and the microorganisms. It may therefore be expected that these compounds show some reactivity toward food components as well.
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Many preservatives are comparatively reactive compounds, such as sulfite, nitrite, and sorbic acid, but some of these show a moderate reactivity only (e.g., benzoic acid). Sulfite, nitrite, sorbic acid, and benzoic acid are discussed below. 13.3.2
Sulfite
Disinfection by the vapors of burning sulfur is an old technique that was frequently used to decontaminate wine casks. Some sulfur dioxide was left in these vessels, preventing the wine from unwanted microbial infections. At present, sulfites are used both as preservatives and as agents that stop browning reactions. In food, the HSO3– species predominates, while in dehydrated food, it is expected that S(IV) mainly exists as metabisulfite (S2O52 – ), which is in equilibrium with HSO3– and SO32 – (Wedzicha et al., 1991). Because of the nucleophilicity of the sulfite ion, many reactions with food components are possible (Wedzicha, 1991), one of which is a reversible addition to carbonyl compounds. It is suggested that the sulfite rather than the bisulfite ion acts as the nucleophilic agent (Wedzicha et al., 1991). This reaction has many implications for foodstuffs. Aroma components possessing a carbonyl group, for example, become involatile and do not contribute any longer to the overall flavor. Other nucleophilic reactions include the cleavage of S–S bonds in proteins and addition to C=C bonds of α,β-unsaturated carbonyl compounds. Control of nonenzymatic browning is based on this latter reaction (McWeeny et al., 1974). A key intermediate of the Maillard reaction, that is, 3,4-deoxyhexulos3-ene, is efficiently blocked by a fast reaction with sulfite, leading to the formation of 3,4-dideoxy-4-sulphohexosulose, which is much less reactive and in which sulfite is irreversibly bound. Ascorbic acid browning is also inhibited by the addition of sulfite (Wedzicha and McWeeny, 1974). The same holds for polyphenol oxidase-catalyzed oxidation of natural phenols in fruit. The mechanism of the inhibition is by reaction of o-quinone intermediates with sulfite, which leads to nonreactive sulfocatechols (Wedzicha, 1995). An undesirable reaction of sulfite in food is the cleavage of thiamin by means of an attack on the pyrimidin moiety (Zoltewicz et al., 1984). This was one of the reasons for a ban, in many countries, on the use of sulfite in meat. Another reason is the preserving effect on the meat color, which makes stale meat look as if it were fresh. Sulfite, however, is unable to reduce metmyoglobin back to myoglobin (Wedzicha and Mountfort, 1991). An important reaction, in a quantitative respect, is the cleavage of disulfide bonds in meat proteins, in particular in lean meat. The reducing capacities of sulfite should be emphasized as well. In fact, cleavage of S–S bonds by sulfite can be considered as a reduction. This property of sulfite makes it useful as an additive to flour for biscuit making (Wedzicha, 1995). The cleavage of disulfide bonds in wheat proteins speeds up and facilitates the production of a satisfactory dough. A quite different type of reaction, which also may occur in food, is that of reduction of azo dyes to colorless hydrazo compounds. As in the reaction with carbon compounds, the reactive species is SO32 – and not HSO3 – (Wedzicha and Rumbelow, 1981).
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Nitrite
lt has been known for a long time that small amounts of saltpeter (KNO3) are able to cause a reddish discoloration of meat, which is characteristic for many meat products. In about 1900 it was established that it was not nitrate but its reduction product, nitrite, that was responsible for this color development. Some decades later, nitrite was recognized as a potent inhibitor of microorganisms, including pathogens, in many meat products. In particular, the inhibition of Clostridium botulinum, with accompanying toxin formation, was established. The role of nitrite in the characteristic cured meat flavor was not noticed until later. The inhibitive action of nitrite is pH-dependent, which led to the assumption that undissociated nitrous acid was the active substance. This, however, is a hypothetical acid from which equimolar parts of H2O, NO and NO2 are formed. In the 1960s it became clear that nitric oxide (NO) is the active species. Nitric oxide, however, can also be generated through the reduction of nitrite, for example, by ascorbate or isoascorbate (Wirth, 1985). Nitric oxide shows a strong binding to iron and is able to block iron atoms in biologically active compounds important in cell metabolism, in particular in the outgrowth of germinated spores (Woods, Wood, and Gibbs, 1989). In meat products preserved with nitrite, NO binds to heme iron, thus forming nitrosomyoglobin (Giddings, 1977). There is some evidence that inhibition of Cl. botulinum outgrowth in nitrite-cured meat products is mainly due to the binding of iron in such a way that it is no longer available for outgrowth of Clostridium spores. This strong binding also explains the antioxidative properties of nitrite in these products (Grever and Ruiter, 2001). Fat also is able to bind some nitric oxide. The amount incorporated in fat is considerably higher in unsaturated than in saturated lipids. Furthermore, nitrite is able to react with intermediates of the Maillard reaction, such as 3-deoxyosulose (Wedzicha and Wei Tian, 1989). The reaction of nitrite with secondary or tertiary amines, though unimportant in quantitative respect, leads to N-nitroso compounds, which for a considerable part, are potent carcinogens. These compounds may rearrange to form highly electrophilic diazonium ions that react with cellular nucleophiles, such as water, proteins, and nucleic acids. Nitrate ingested with food or drinking water is partially reduced to nitrite in the body, and contributes more than nitrite in meat products to the possible endogenous formation of N-nitroso compounds. A ban on the use of low amounts of nitrite as an additive is therefore not very rational, and deprives the consumer of a very effective guard against a number of pathogenic microorganisms, in particular Cl. botulinum, but also Cl. perfringens and Staphylococcus aureus. Finally, nitrite may react in physiological concentrations and under gastric pH conditions with naturally occurring, as well as synthetic, antioxidants (Kalus et al., 1990). There are no indications for the formation of hazardous reaction conditions from a mutagenicity viewpoint. 13.3.4
Sorbic Acid
The preserving properties of sorbic acid were recognized around 1940. During the late 1940s and the 1950s, sorbic acid became available on a commercial scale,
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resulting in its extensive use as a food preservative throughout the world (Sofos and Busta, 1983). As a straight-chain trans-trans dienoic fatty acid (FA) (CH3–CH=CH–CH=CH–COOH), it is susceptible to nucleophilic attack (Khandelwal and Wedzicha, 1990b). The lowest electron density is associated with position 3. Nucleophilic groups, e.g., the thiol group, however, may bind to the carbon atom at the 5 position as the terminal methyl group delocalizes the charge on that (Khandelwal and Wedzicha, 1990a; Wedzicha, 1995). Sorbic acid inactivates several intracellular enzymes by this mechanism. It passes the cell wall in its undissociated form only, which explains its low activity at higher pH values. The pKa value at 25°C amounts to 4.76. It is mainly active against yeasts, molds, and strictly aerobic bacteria. The toxicity toward mammals is low. Sorbic acid is easily oxidized. This oxidation is accompanied by the development of a glyoxal-like flavor in sorbic acid preparations, and a brown color in a wide variety of model foods in which sorbic acid is included. Amino acids accelerate color development (Wedzicha et al., 1991). 13.3.5
Benzoic Acid
This compound is relatively stable during food processing and in food products. It was proposed in 1875 by H. Fleck as a replacement for salicylic acid, and can be considered as one of the first safe food preservatives. Like sorbic acid, it is mainly active against yeast and molds, but the growth of micrococci, E. coli, and many other bacteria is retarded as well. As is the case with sorbic acid, benzoic acid penetrates the cell wall in the undissociated form. As a consequence, it is active at lower pH values only (pKa at 25°C = 4.19), and therefore serves as a preservative for sour products such as fruit juices and jams. In shrimp preservation it is applied as a powder that is spread over the shrimps, passes cell walls, and then ionizes in the intracellular fluid to yield protons that acidify the alkaline interior of the cell. The main cause of its activity, however, are biochemical effects (Eklund, 1980) such as inhibition of oxidative phosphorylation and of enzymes from the citric acid cycle (Chipley, 1983). In mayonnaise preserved by benzoic acid, the undissociated acid is mainly present in the lipid phase, which can be considered as a reservoir for the aqueous phase. Benzoic acid is often combined with sorbic acid in order to reduce its peculiar flavor. It must be stated, however, that these are at least partly caused by impurities in the benzoic acid preparation used.
13.4 ANTIOXIDANTS Antioxidants can be defined as “substances that, when present in low concentrations compared to those of an oxidizable substrate, significantly delay or inhibit oxidation of that substrate” (Halliwell and Gutteridge, 1989). Antioxidants are frequently added to unsaturated fats and oils in order to protect them against oxidative deterioration. For this reason, they are also added to a variety of food products containing unsaturated lipids. Antioxidants frequently applied are esters of gallic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene
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OH
OH
OH
OH
HO
C(CH3)3
C(CH3)3 OCH3
COOR OH
OCH3 OH C(CH3)3
C(CH3)3
(CH3)3C
CH3
OH
FIGURE 13.1 Antioxodants. Top: from left to right, alkyl gallates, the two isomers of BHA. Bottom: from left to right, BHT and TBHQ.
(BHT), and tertiary butylhydroxyquinone (TBHQ). Of these, TBHQ is by far the most potent antioxidant. In both BHA and BHT, the butyl groups are also of a tertiary structure (Figure 13.1). Antioxidants are naturally present in many foodstuffs and are of great importance as inactivators of radical formation. Some antioxidative enzyme systems are produced in the human body and are supposed to play an important role in the cellular defense against oxidative damage (Langseth, 1995). Many of the antioxidants present in food function as terminators of chain reactions. A variety of compounds, such as phenols, aromatic amines, and conjugates, can function as chain-breaking antioxidants. They react with the chain-propagating radical species, which results in the formation of radical species incapable of extracting hydrogen atoms from unsaturated lipids. These radicals may rapidly combine with other radicals, or if a polyphenolic structure is present as in gallic acid esters, disproportionate into their original state and a quinoid form. Because there are synergistic effects among antioxidants, commercial preparations usually contain mixtures of them. As oxidative rancidity is strongly catalyzed by some heavy metal ions, in particular Cu++, antioxidant mixtures often contain sequestrants (e.g., citric acid, EDTA) in order to complex these ions. Reductants such as ascorbic acid, which decrease the local concentration of oxygen, are also able to decrease the formation of peroxy radicals. Fat oxidation by bacteria can be suppressed by the addition of preservatives such as benzoic acid or sorbic acid.
13.5 FLAVORINGS, COLORANTS, AND SWEETENERS Artificial flavorings are frequently added to a variety of foodstuffs. These preparations mostly consist of a large number of different compounds, of which some show
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a considerable reactivity. The way in which flavor components interact with the food matrix, and how this influences flavor perception, has been recently reviewed by Bakker (1995). Many interactions are of a purely chemical nature and may result from the presence of aldehydes and their reactivity toward amino and thiol groups of proteins. Another frequently occurring type of interaction is the formation of hydrogen bonds between food compounds and polar flavor components such as alcohols. Starch and starch-derived maltodextrins and β-cyclodextrin are able to form inclusion complexes with many flavor components. Many other interactions, although of great influence on flavor perception, are of a physical nature and therefore are not mentioned in this chapter. Food additives such as dyes and sweeteners are not intended to react with matrix compounds or to undergo other reactions. Some reactions may occur, however, and a few examples are given here. As for food dyes, many of these are azo compounds, which implies the possibility of reduction, for example, by the action of certain bacteria. The loss of color, in these cases, is an indication of spoilage. Bisulfites are also able to reduce azo dyes (Wedzicha and Rumbelow, 1981). Sweeteners have also been applied to foodstuffs for many years. Compounds such as saccharin, sodium cyclamate, aspartame, and several others are well known and hardly need to be discussed here. In more recent times, sweeteners such as sucralose and thaumatin have appeared on the scene. Sucralose is a sucrose derivative obtained by chlorination by which the D-glucopyranosyl unit is converted to a galactopyranosyl unit chlorinated at position 4, while the fructofuranosyl unit is chlorinated at position 6. This results in an intense sweetness (600 times that of sucrose) and a greater stability toward acids (Figure 13.2).
CH2OH Cl
O OH
OH O CH2Cl O HO CH2Cl OH FIGURE 13.2 Sucralose.
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Thaumatin is a protein from katemfe fruit (Thaumatococcus danielli) with the strongest sweetening properties hitherto known (2500× as sweet as sucrose). The protein is freely soluble in water, consists of 207 amino acids, shows a molecular weight of 22 kDa, and an isoelectric point (I.E.P.) of 11.5. The electrical charge in the molecule is thought to be a major factor in its interaction with taste receptors (Boy, 1994). It synergizes with aspartame and with flavor enhancers such as 5'nucleotides and sodium glutamate. Apart from this, it masks the aftertaste of saccharin. It also masks metallic and bitter taste components. Taste interactions between sweeteners are not uncommon. Another example is the synergism between aspartame and acesulfame K. A 70%/30% blend, in some cases, yields a taste that cannot be distinguished from sugar. Acesulfam K also shortens the long, sweet aftertaste of sucralose (Meyer, 2001). Apart from taste interactions, some sweeteners also show synergism with flavors. Some sweeteners may undergo chemical reactions, thereby losing their sweet properties. The instability of saccharin at higher temperatures is well known. Apart from this, saccharin is subject to some degradation under acid conditions, yielding sulfobenzoic acid, which has a disagreeable phenolic flavor. Another example is aspartame, which is the methyl ester of N-L-α-aspartyl-L-phenylalanine. Because of its nature, its stability in aqueous systems is limited. The maximum stability is between pH 3 and 5 and decreases at higher temperatures with a concomitant loss of sweetening power. The main degradation product is 3,6-dioxo-5-(phenylmethyl)2-piperazinoacetic acid (Furda et al., 1975). Other decomposition products were listed by Stamp and Labuza (l989), who added some novel components to this group. These all have in common a loss of sweet taste. Apart from this, aspartame shows remarkable reactivity toward a number of aldehydes that may be present in foodstuffs and contribute to flavor (Hussein et al., 1984; Cha and Ho, 1988), and thaumatin reacts with carrageenans if these are present (Ohashi et al., 1990). Many unintended and sometimes unwanted reactions of artificial dyes, sweeteners, and other additives with the food matrix are imaginable and should always be taken into consideration when the consequences of such additions to food are discussed.
13.6 STABILIZERS, EMULSIFIERS, AND THICKENING AGENTS The most important representatives of these compounds are polysaccharides, such as starch and starch derivatives (α-1,4 D-glucans), cellulose and cellulose derivatives (β-1,4 D-glucans), plant extracts (pectins: α-1,4 D-galacturonans), seaweed extracts (carrageenan, agar, alginates), seed flour (guar and locust bean galactomannans, tamarind xyloglucans, konjac glucomannans), exudate gums (arabic, karaya, tragacanth), and microbial gums (xanthan, gellan, curdlan). Polymers are built of one or more types of sugar residues, covalently attached in linear, linearly branched, and branched structures. Their anomeric form (α/β), types of linkages (1,2; 1,3; 1,4; 1,4,6; 1,3,6; etc.), presence of functional groups (carboxyl, phosphate, sulfate, esters, ethers), and molecular weight distribution determine their conformation in aqueous
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systems (stiff/rodlike, random coil, helices), the intra- and intermolecular interactions between molecules (dimerization, association, ionic interactions, hydrophobic interactions), and the interactions with other molecules (other polysaccharides, proteins, lipids). These interactions are the basis for viscous behavior, gelling, water binding, film forming, bulking, stabilizing, and emulsifying properties. Some polysaccharides act synergistically in imparting these functions, such as locust bean gum and carrageenan, locust bean gum and xanthan, and pectin and alginate. Important parameters for applications are pH, heat and shear stability, syneresis properties, shelf life, and compatibility with other food constituents. Derivatives of these polysaccharides with improved functional properties are also used. Emulsifiers are amphophilic compounds that concentrate at oil–water interfaces, causing a significant lowering of interfacial tension and a reduction in the energy needed to form emulsions. They can be anionic, cationic, and nonionic compounds that have one or more of the following characteristics: surface active, viscosity enhancer, solid absorbent, or hydrophilic–lipophilic balance (HLB). They are added to food emulsions to increase emulsion stability and to attain an acceptable shelf life. Polysaccharides are not surface-active agents, but rather macromolecular stabilizers that generally function through enhancement of viscosity and enveloping oil droplets in oil-in-water emulsions. The emulsifiers being used in food manufacturing were categorized by Artz (1990) (see Table 13.1). Only lecithin is of natural origin. Its main source is the soybean, but it is also present in corn, sunflower, cottonseed, rapeseed, and eggs. Emulsifiers stabilize emulsions in various ways. They reduce interfacial tension and may form an interfacial film that prevents coalescence of droplets. In addition, ionic emulsifiers provide charged groups on the surface of the emulsion droplets, and thus increase repulsive forces between droplets. Emulsifiers can also form liquid crystalline microstructures, such as micelles, at the interface of emulsion droplets. These are formed only at emulsifier concentrations larger than the critical micelleforming concentration. These microstructures have a stabilizing effect.
TABLE 13.1 Food Emulsifier Categories Category
Typical application
Lecithin (naturally occurring) and lecithin derivatives Glycerol FA esters Hydroxycarboxylic acid and FA esters Lactylate FA esters Polyglycerol FA esters Polyethylene and propylene glycol FA esters Ethoxylated derivatives of monoglycerides Sorbitan FA esters
— — Baking goods, margarine — Baked goods O/W emulsions — Antistaling
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Proteins also have a major influence on emulsion stability, although various types of instability may occur: instability caused by coalescence or flocculation, and cream layer formation caused by differences in density between oil droplets and the aqueous phase. In practice the latter problem is the most difficult to solve. Creaming can be retarded by increasing the viscosity of the aqueous phase and by reducing the diameter of oil droplets or the density difference. The density difference between oil droplet and serum can be reduced by loading as much protein as possible on the oil droplet surface. An open, random coil protein, such as sodium caseinate, is expected to yield less protein loaded on the oil surface than compact proteins do. It has been shown that open structure proteins, indeed, show a lower equilibrium surface load than do compact structure proteins. (Zwijgers, 1992). The higher the equilibrium surface load, the better the emulsion-stabilizing protein. In line with the foregoing, milk protein hydrolysates are useful foamers and emulgators. The foam- and emulsion-forming properties of milk peptides are even superior to those of intact milk proteins, as long as these show both charged and hydrophobic areas (Caessens, 1999). The selection of emulsifiers for food preparations is mainly based on their HLB numbers. This index expresses the hydrophile–lipophile balance, and is based on the relative percentage of hydrophilic to lipophilic groups within the emulsifier molecule. Lower HLB numbers indicate a more lipophilic emulsifier, while higher numbers indicate a more hydrophilic emulsifier. Emulsifiers with HLB numbers between 3 and 6 are best for water-in-oil emulsions and emulsifiers with numbers between 8 and 18 are best for oil-in-water emulsions.
13.7 CLARIFYING AGENTS AND FILM FORMERS Clarifying agents or flocculants are used to eliminate turbidity or suspended particles from liquids, such as chill haze in beer, precipitates in fruit juices and wines, or haze in oils. Often, they provide a nucleation site for suspended fine particles. Examples of clarifying agents are lime in sugar juice clarification, pectic enzymes to break down pectins in fruit juices, and gelatin for clarification of fruit juices. Film formers are used to coat a food by providing it with a protective layer and so make it more attractive in appearance or to increase its palatability. Film formers may not impart flavor or mouth-feel of their own to the food. Examples are starches to coat proteins to prevent Maillard reactions, mineral oils to seal pores of eggs, or sodium caseinate to encapsulate fat in whiteners.
13.8 ACIDULANTS Food acidulants find their application, for the most part, in beverages and in fruit and vegetable processing. Apart from pH lowering, acidulants provide buffer capacity, impair sourness and tartness, enhance the effect of preservatives, and for some acidulants such as citric acid, prevent discoloration caused by trace metals (Seifert, 1992).
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CH2OH O OH
O
OH OH FIGURE 13.3 D-Gluconolactone.
Besides acids, other components that produce or release acids are applied as acidulants, in particular when a slow release of acid is of importance. A well-known, slow-release acidulant is glucono-delta-lactone (GDL), which is used in bakery products, dairy products, and in particular, meat products (Watine, 1995) (Figure 13.3). The use of GDL in the maturation of dry sausages is well known. During preparation of these sausages, GDL standardizes acidification, strongly reduces the risk of contamination, and improves the quality. GDL gradually lowers the pH to 5.4, and after filling the sausage casings, the temperature is lowered to 0 to 4°C for some hours. During this period the growth of starters and undesirable bacteria is inhibited. Then the temperature is increased so that fermentation can take place normally.
13.9 FAT SUBSTITUTES AND FAT MIMETICS Dietary fat contributes to the combined perception of mouth-feel, taste, and aroma. Fat also contributes to creaminess, appearance, palatability, texture, lubrication properties of foods, and increases the feeling of satiety during meals. Apart from this, it is able to carry lipophilic flavor compounds and can act as a precursor for flavor development. A very important characteristic of fat is its suitability for use in frying. Due to their high caloric value, there is an increasing tendency to replace fats and oils with components that are not calorific, but which can impart the same technological and sensory functionalities. Two types of fat replacers can be distinguished—fat substitutes and fat mimetics. Fat substitutes are lipid- or fat-based macromolecules that physically and chemically resemble triacylglycerols (e.g., sucrose polyesters or alkyl glycoside polyesters). Fat mimetics are protein- or carbohydrate-based compounds that imitate organoleptic or physical proteins or triacylglycerols (Voragen, 1998). Fat substitutes physically and chemically resemble fats and oils, as they are esters of polyols. They are stable at cooking and frying temperatures. Many carbohydrate-based fat substitutes are mixtures of sucrose esters formed by chemical transesterification or interesterification of sucrose with one to eight fatty acids (FA). The class with six to eight FAs are called sucrose fatty acid polyesters. These molecules are too large to be broken down by intestinal lipase enzymes, and for that reason, do not have any caloric value (Voragen, 1998).
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Olestra, in this respect, is a promising compound. The stability of Olestra is comparable to natural fats and even better. Olestra can be used as a frying medium, and appears to undergo slower oxidative and hydrolytic degradation (Lindley, 1996). The functionality and potential application of the sucrose FA polyesters is governed by the type of fatty esters used in the manufacturing process. The melting character depends on the length and the degree of saturation of the FA in a similar way. In theory, this melting character may be adjusted by the right choice of FA (Lindley, 1996). Esters containing one to three fatty acids are called sucrose FA esters (SFEs). Unlike sucrose FA polyesters, the SFEs are easily hydrolyzed and absorbed by digestive lipases, and are thus caloric. SFEs containing five to seven free hydroxyl groups with one to three FA esters show both hydrophilic and lipophilic properties, and therefore has excellent emulsifying and surface-active properties. In addition, they are effective lubricants, anticaking agents, thinning agents, and antimicrobials (Voragen, 1998). Other carbohydrates modified to fatty acid esters are sorbitol, trehalose, raffinose, and stachyose. Saccharide-based fat mimetics differ strongly from fats and oils. Generally they absorb a substantial amount of water and are therefore not suitable for frying. As they can only carry water-soluble flavors, they lack the flavor of fats and oils. Inulin and starch hydrolysates (dextrose equivalent ~2) are striking examples of fat mimetics. The fat substitution is based upon its ability to stabilize water into a creamy structure that has a fatlike mouth-feel (Blomsma, 1997). Most carbohydrate-based fat replacers are extracted from by-products rich in cell wall polysaccharides. Polydextrose, which can be applied both as a carbohydrate and as a fat replacer, is obtained by vacuum thermal polymerization of glucose, using citric acid as a catalyst and sorbitol as a plasticizer (Voragen, 1998).
13.10 PREBIOTICS In Chapter 12, it is stated that the use of prebiotics in association with useful probiotics may be a worthwhile approach, as prebiotics may stimulate some probiotic strains. The term prebiotic is derived from the Greek and can be translated as “prior to life.” Generally, prebiotics comprise food additives that are barely or not digested in the small intestine and end up in the colon where they, as already stated, may stimulate beneficial bacteria in the colon by serving as fermentation substrates. Promotion and regulation of health is the aim of these bioactive products, which present an important trend in the present-day production of foods. Nondigestible carbohydrates are the main representatives of this class of food additives. There are three main types of carbohydrates that are indigestible in the human small intestine, that is, nonstarch polysaccharides, resistant starch, and nondigestible oligosaccharides (NDOs). As the average daily ingestion of the latter group is lower than the level considered as safe (not over 15 g/day), supplementation of NDOs could be beneficial (Voragen, 1998). These can be used in a wide range of processed foods, including dairy products, confectionery, bakery products, ready meals, breakfast cereals, and drinks. Other significant beneficial effects are (Van Haastrecht, 1995):
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• • • • •
replacement of sugar and fat reduction of the number of unwanted bacteria in the colon fiber enrichment of foods hitherto poor in fiber, such as white bread, dairy products, and transparent drinks prevention of tooth decay regulation of lipid metabolism
Two specific groups of NDOs, which are commercially available, must be mentioned, that is, fructo-oligosaccharides (FOS), obtained by transfructosylation of sucrose using a β-D-fructosyltransferase or by hydrolysis of inulin by endo-inulinase; and galacto-oligosacharides (GOS) by transgalactosylation of lactose using β-galactosidase. Inulin is a virtually linear fructose polymer in which the fructose molecules are linked by β-(2-1) bonds with an average degree of polymerization ranging from 2 to 60 (average length of 10). Most oligosaccharides have a moderate reducing power by which they are still subject to Maillard reactions when used in food to be heat processed. Fructo-oligosaccharides of the GFn type, composed of fructofuranosyl residues and one terminal, nonreducing glucosyl residue as obtained by transfructosylation; lactosucrose and glycosylsucrose have no reducing power. Fructo-oligosaccharides obtained by hydrolysis of inulin can be of the GFn type or of the Fm type, the latter having a reducing fructofuranosyl residue (Figure 13.4). CH2OH
CH2OH
O
O OH
O
OH
OH
OH
OH
OH
OH OH
OH
OH O
CH2OH O
O
O CH2OH O
CH2OH O HO
HO CH2OH
OH
CH2
HO CH2
CH2 OH
O
n-1
OH
O
CH2OH O
CH2OH O
HO
HO
CH2OH
CH2OH OH
OH
Sucrose
m-2
GFn
Fm
FIGURE 13.4 Two types of FOS (sucrose of comparison). (After van Haastrecht, J., Int. Food Ingredients, 1995.)
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At a pH less than 4 and treatments at elevated temperatures or prolonged storage at ambient conditions, oligosaccharides present in a food can be hydrolyzed resulting in the loss of nutritional and physicochemical properties. For fructo-oligosaccharides it is reported that in a 10% solution of pH 3.5, less than 10% is hydrolyzed after heat treatments of 10 s at 145°C, 5 min at 45°C, or 60 min at 70°C. After two days at 30°C, less than 5% is hydrolyzed. The stability can greatly differ for the various classes of oligosaccharides depending on the sugar residues present, their ring form and anomeric configuration, and linkage types. Generally β-linkages are stronger than α-linkages, hexoses are more strongly linked than pentoses and deoxysugars, and pyranoses are more strongly linked than furanoses. Finally, resistant starches are dietary carbohydrates that have attracted increased interest on the part of food manufacturers due to the beneficial effects these might have on human health. It escapes digestion and absorption in the small intestine of man and reaches the large bowel, where fermentation takes place. As a result, the pH in the colon is lowered and short-chain FAs are formed. They further increase fecal bulk and may protect against colon cancer, improve glucose tolerance, and lower blood lipid levels. The most important forms of resistant starch in the diet are botanically encapsulated starch present in intact foods, starches with a B-type crystalline structure present in unheated foods, starch retrogradated as a result of full gelatinization and dispersion by processing, and thermally or chemically modified starches (Voragen, 1998).
REFERENCES Artz, W. Emulsifiers, in Food Additives, Bramen, A.L., Davidson, R.M., and Salminen S., Eds., Marcel Dekker, New York, 1990, p. 347. Bakker, J., Flavor interactions with the food matrix and their effects on perception, in Ingredient interactions—Effects on food quality, Goankar, A.G., Ed., Marcel Dekker, New York, 1995, p. 411. Blomsma, C.A., Ingenious inulin, International Food Ingredients, 2, 22, 1997. Boy, C., Thaumatin: a taste-modifying protein, International Food Ingredients, 6, 23, 1994. Caessens, P.J.W.R., Vissers, S., Gruppen, H., and Voragen, A.G.J. β-Lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V9 protease, J. Agric. Food Chem., 47, 2973, 1999. Cha, A.S. and Ho, C.T., Studies of the interaction between aspartame and flavor vanillin by high-performance liquid chromatography, J. Food Science, 53, 562, 1988. Chipley, J.R., Sodium benzoate and benzoic acid, in Antimicrobials in Foods, Branen, A.L. and Davidson, P.M., Eds., Marcel Dekker, New York, 1983, p. 11. Eklund, T., Inhibition of growth and uptake processes in bacteria by some food preservatives, J. Appl. Bacteriol., 48, 423, 1980. Feberwee, A., Legal aspects of food additives of natural origin, in Proceedings of the International Symposium Food Additives of Natural Origin, Plovdiv, Bulgaria, 31 May–2 June 1989, p. 22. Furda, I., Malizia, P.D., Kolor, M.G., and Vernieri, P.J., Decomposition products of L-aspartylL-phenylalanine methyl ester in various food products and formulations, J. Agr. Food Chem., 23, 340, 1975.
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Giddings, G.G., The basis of color in muscle foods, J. Food Science, 42, 288, 1977. Grever, A.B.G., Ruiter, A., Prevention of Clostridium botulinum outgrowth in heated and hermetically sealed meat products by nitrite—A review, Eur. Food Res. Technol., 213, 165, 2001. Hall, R.L., 1999, Food safety: elusive goal and essential quest, IUFoST Founders Lecture, Sydney, October, Food Australia, 51, 12, 601, 1999. Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, 2nd ed, Clarendon Press, Oxford, U.K., 1989. Hussein, M.M., D’Amelia, R.P., Manz, A.L., Jacin, H., and Chen, W.-T.C., Determination of reactivity of aspartame with flavor aldehydes by gas chromatography, HPLC and GPC, J. Food Science, 49, 520, 1984. Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Nutrition, 4th report. WHO Technical Report Series, No. 97. World Health Organization, Geneva, 1955, p. 29. Kalus, W.H., Münzner, R., and Filby, W.G., Isolation and characterization of some products of the BHA-nitrite reaction: examination of their mutagenicity, Food Additives and Contaminants, 7, 223, 1990. Kamsteeg, J. and Baas, M.I.A., E = Eetbaar (E = Edible), H.J.W. Becht, Amsterdam, 1985. Khandelwal, G.D. and Wedzicha, B.L., Derivatives of sorbic acid-thiol adducts, Food Chemistry, 37, 159, 1990a. Khandelwal, G.D. and Wedzicha, B.L., Nucleophilic reactions of sorbic acid, Food Additives and Contaminants, 7, 685, 1990b. Ladikos, D. and Lougovois, V., Lipid oxidation in muscle foods: a review, Food Chemistry, 35, 295, 1990. Langseth, L., Oxidants, Antioxidants, and Disease Prevention, ILSI Europe Concise Monograph series, ILSI Europe, Brussels, 1995. Lindley, M.G., Olestra: the ultimate in fat substitution? International Food Ingredients, 3, 35, 1996. Lüthy, J., Safety evaluation of natural food additives, in Proceedings of the International Symposium Food Additives of Natural Origin, Plovdiv, Bulgaria, 31 May–2 June 1989, pp. 35–40. McWeeny, D.J., Knowles, M.E., and Hearne, J.F., The chemistry of non-enzymic browning in foods and its control by sulphur, J. Sci. Food Agric., 25, 735, 1974. Meyer, S., Taste interactions of acesulfame potassium and other high intensity sweeteners with fruit flavours in different food proteins, Abstracts, 2nd IUPAC International Symposium on Sweeteners (2nd IUPAC-ISS), November 13–17, 2001, Hiroshima, Japan, pp. 55–56. Möhler, K., Formation of curing pigments by chemical, biochemical or enzymatic reactions, in Proc. Int. Symp. Nitrite in Meat Products, Zeist, The Netherlands, Krol, B. and Tinbergen, B.J., Eds., Pudoc, Wageningen, 1973, p. 13. Ohashi, S., Ura, F., Takeuchi, M., Iida, H., Sakaue, K., Ochi, T., Ukai, S., and Hiramatsu, K., The decrease of thaumatin’s sweetness intensity upon interaction with carrageenan, Food Hydrocolloids, 4, 323, 1990. Ruiter, A., Safety of food: the vision of the chemical food hygienist, in Food Science: Basic Research for Technological Progress. Proceedings of the Symposium in Honour of Professor W. Pilnik, Wageningen, The Netherlands, 25 November 1988, Roozen, J.P., Rombouts, F.M., and Voragen, A.G.J., Eds., 1989, p. 19. Seifert, D., Functionality of food acidulants, International Food Ingredients, 3, 4, 1992. Sofos, J.N. and Busta, F.F., in Antimicrobials in foods, Branen, A.M. and Davidson, R.M., Eds., Marcel Dekker, New York, Basel, 1983, p. 141.
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Stamp, J.A. and Labuza, T.R., Mass spectrometric determination of aspartame decomposition products: Evidence for β-isomer formation in solution, Food Additives and Contaminants, 6, 397, 1989. Van Dokkum, W., Additieven en contaminanten (additives and contaminants), Voeding in de praktijk, 6, 1, 1985. Van Haastrecht, J., Oligosaccharides: promising performers in new product development, International Food Ingredients, (1), 23, 1995. Voragen, A.G.J., Technological aspects of functional food-related carbohydrates, Trends in Food Science and Technology, 9, 328, 1998. Watine, Ph., Glucono-delta-lactone: functional properties and applications, International Food Ingredients, 3, 39, 1995. Wedzicha, B.L., Sulphur dioxide—the most versatile food additive? Chemistry in Britain, 1030–1032, 1991. Wedzicha, B.L., Interactions involving sulfites, sorbic acid, and benzoic acid, in Ingredient Interactions—Effects on Food Quality, Goankar, A.G., Ed., Marcel Dekker, New York, Basel, 1995, p. 529. Wedzicha, B.L., Bellion, I., and Goddard, S.J., Inhibition of browning by sulfites, in Nutritional and Toxicological Consequences of Food Processing, Friesman, M., Ed., Plenum Press, New York, 1991, p. 217. Wedzicha, B.L. and McWeeny, D.J., Non-enzymic browning of ascorbic acid and their inhibition. The production of 3-deoxy-4-sulphopentosulose in mixtures of ascorbic acid, glycine and bisulphite ion, J. Sci. Food Agric., 25, 577, 1974. Wedzicha, B.L. and Mountfort, K.A., Reactivity of sulphur dioxide in comminuted meal, Food Chemistry, 39, 281, 1991. Wedzicha, B.L., Rimmer, Y.L., and Khandelwal, G.D., Catalysis of Maillard browning by sorbic acid, Lebensmittel-Wiss. u.-Technol., 24, 278, 1991. Wedzicha, B.L. and Rumbelow, S.J., The reaction of an azo food dye with hydrogen sulphite ions, J. Sci. Food Agric., 32, 699, 1981. Wedzicha, B.L. and Wei Tian, Kinetics of the reaction between 3-deoxyhexulose and nitrite ion, Food Chemistry, 31, 189, 1989. Wirth, F., Pökeln: Farbbildung und Farbhaltung by Brühwurst (Curing: formation and maintaining of colour in fermented sausages), Fleischwirtschaft, 65, 423, 1985. Wodicka, V.O., Food safety—rationalizing the ground rules for safety evaluation, Food Technology, 9, 31, 1977. Woods, L.F.J., Wood, J.M., and Gibbs, P.A., Nitrite, in Mechanisms of Action of Food Preservation Procedures, Gould, G.W., Ed., Elsevier, London, 1989, p. 225. Zoltewicz, J.A., Kauffman, G.M., and Uray, G., A mechanism for sulphite ion reacting with vitamin B2 and its analogues, Food Chemistry, 15, 75, 1984. Zwijgers, A., Outline of milk protein concentrate, International Food Ingredients, 3, 18, 1992.
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14
Food Safety Julie Miller Jones
CONTENTS 14.1 14.2 14.3 14.4 14.5 14.6 14.7
Introduction................................................................................................ 375 Consumer Attitudes toward the Food Safety Problem ............................. 376 Tests to Determine Food Safety ................................................................ 378 Food Safety Concerns ............................................................................... 380 Microbial Contamination of Food............................................................. 381 Risk/Benefit as It Applies to Food ............................................................ 383 Nutritional Evaluation of Food Processing............................................... 384 14.7.1 Beneficial and Detrimental Effects ............................................. 384 14.7.2 Effects on Vitamins ..................................................................... 385 14.7.3 Effects on Minerals ..................................................................... 387 14.8 Newer and Novel Technologies................................................................. 387 14.8.1 Irradiation .................................................................................... 387 14.8.2 Biotechnology ............................................................................. 387 14.9 Additives .................................................................................................... 388 14.10 Summary.................................................................................................... 389 References.............................................................................................................. 389
14.1 INTRODUCTION Safe food—it is what every individual expects in every mouthful and every government strives to provide for its populous. Because food is the object of our earliest preferences and the subject of our strongest prejudices, food safety is a gut issue. Yet what seems on the surface to be both basic and imperative is not at all simple and, in fact, is not achievable in the absolute sense. It is extremely hard for many to accept even the idea that food is relatively—not absolutely—safe. What appears to threaten food, threatens all in a very direct and visceral way. An understanding of basic definitions concerning safety and toxicity is crucial. First all compounds, no matter how salutary, can be ingested in some manner or quantity that can be harmful—in other words, toxic. Toxicity is the capacity of a substance to produce some adverse effect or harm. Even vital dietary components such as water and vitamins can be consumed at toxic levels. Too much pure water can cause renal shutdown; excessive vitamins can cause minor problems, such as flushing or nausea, or extremely adverse events such as liver damage, teratogenicity, 375
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and death. The 1538 Paracelsus motto, “only the dose makes the poison” operates for food components (Jones 1992). Second, what is safe for one is not safe for everyone. Individuals with allergies, inborn or acquired errors of metabolism, or certain diseases can ingest a food in a usual and customary manner and suffer adverse, and even, in rare instances, fatal outcomes. Thus foods that are safe for most are not safe for all. Third, how the food is used or produced may alter its safety. Combinations of a certain food and a drug or herb or in a bizarre or poor diet can render an otherwise safe food harmful. In certain growing or production conditions, a food may acquire an unexpected contaminant such as a mycotoxin or a prion. Thus, what is usually safe harbors a masquerading toxin. Because absolute safety is unattainable, relative safety is what is sought. Relative safety is the probability that no harm will come when the food is consumed in a usual and customary manner. Even relative safety, when it comes to food, is a big order because it requires constant diligence by all parties who come in contact with the food. Any glitch in the system, from the field to the table, can introduce a potential hazard. The environment and the starting raw material must be optimal. There should not be high levels of naturally occurring toxicants in the crop or animal itself. Safety must be maintained by controlling conditions such that the possibility of contamination is minimized. Plant materials must be free of infestations, harmful residues, mold, and mycotoxins. Animal materials should not contain any veterinary drug residues or any abnormal constituents transferred from feedstuffs. Metal particles, weed contaminants, or other incidental components from harvesting or processing must be vigilantly prevented and tested. During transportation from the field to plant or market, carriers must handle the food to maintain its quality. Care must be exercised so that proper temperatures and moisture levels are maintained, and that no contamination or pathogenic growth occurs during any point of the storage, shipping, and processing. Prevention of natural deterioration and further contamination is often done through packaging. Packaging materials and other applied techniques and handling procedures must not introduce risks of their own. Once in the consumer’s hands, food must be cooked and handled properly in the home or food service operation. This means food must not only be safe but that it must provide the expected nutrients. Unfortunately, both the home and restaurant are sites for food mishandling. Thus while maintenance of food safety from farm to fork is expected and taken for granted, it actually requires both care and vigilance, and even with the utmost care there is still an element of risk. While most scientists know that safe does not mean not risk free, this concept is extremely difficult to convey to consumers.
14.2 CONSUMER ATTITUDES TOWARD THE FOOD SAFETY PROBLEM The difficulty in convincing consumers that no food is risk free stems from both food practices and attitudes. First, most consumers in industrialized nations are far removed from the production and processing of food, and even the preparation of
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food. The modern strategy of procuring ready-prepared foods as take out or frozen preprepared products means that many children rarely observe meals being prepared. Thus cooking in many homes has become a process of heating items to serving temperature, usually in a microwave. In this scenario, safe preparation and food handling techniques may no longer be passed from generation to generation. Second, consumer concerns about food may, in part, result from a concern about today’s scientific and technological complexity, a lack of trust in big business and its advertising, and in government’s ability to protect food. For some, science has become the problem, not the answer. Even for those with faith in the promise science holds, scientific complexity is confusing. Two experts espouse different assumptions; extrapolations and interpretations reach diametrically opposed positions often using the same data. Disagreement of scientists is not the only source of confusion. Media savvy, self-appointed experts or consumer groups’ spokespeople with easily understood messages, albeit specific agendas, add to the multiplicity of positions consumers hear. Messages from these sources are seemingly straightforward and demonstrative. These slanted messages do not necessarily present all sides of the data, unlike those from ethical scientists, and thus fail to communicate either conflicting evidence or flaws in the research. It becomes a nearly impossible task for the consumer to sort through the cacophony of voices to find the truth. Groups that position themselves as anti-big business and technology and proconsumer and environment, whether justified or not, garner consumer trust. This is particularly true in areas where the fear factor is high or the technology is unfamiliar, such as irradiation and biotechnology. Oddly, consumer trust in such groups may be misplaced. Consumers often feel that the activist group is on their side and that those in government or industry must be espousing a position that furthers an agenda that is probusiness and not in the interest of the consumer. Yet activist groups often pick the most egregious examples or cite studies that mainstream science discredits for use in a press release. This strategy not only wins them a primetime news story, but also creates consumer angst resulting in further contributions to their cause. Recent scares, such as Sudan Red, dioxin, and mad cow disease, have fueled the lack of trust in government food agencies whose charge it is to protect the public. The case of Sudan Red showed that illegal additives were regularly entering the food supply in countries where its use is prohibited. The dioxin example gave the public justification in their lack of trust because food authorities failed to inform the public when they were first aware of potential dioxin contamination of feed and food. Mad cow disease forced the government to reverse statements that a food or food component is safe, eroding public trust. Communication about food may create misunderstanding. Direct conversations with scientists often fail to enlighten consumers because scientists often pepper the information with jargon and complexities that only further confuse and frustrate. Media stories about nutrition and food safety are often simplistic, boring, incomplete, or biased. Media sound bites distill years of study and lengthy discussion into a 60-second spot, and in the process omit important aspects that are key to a full understanding of the study. A news item may be taken out of context, overgeneralized or it may reflect the findings of a single study. In some cases the reported study is not in agreement with the whole body of scientific literature, but this is not mentioned
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in the story. Even if a study is accurately reported, frequently the news commentator has neither the required depth of understanding nor the time to interpret what a particular finding means to someone who eats the food once a week. Activist groups and advertisers may be selective about which studies they use. Studies that support their point of view, but do not fairly represent the full body of knowledge, may be used by some groups and bloggers. Fear is heightened when statements about potential toxicity feature vulnerable groups such as children and pregnant women. Inadequate scientific literacy also increases fear of chemicals and technology. Many consumers are unaware that human life has always entailed exposure to chemicals, and that everything ingested and inhaled is composed of chemicals. Elusive to most consumers is the fact that naturally occurring chemicals are abundant and many can be more toxic than synthetic ones. Most consumers believe the converse—that natural chemicals are innocuous and synthetic ones are nefarious. Ironically, for food chemicals, much more is known about chemicals added to food than the chemicals inherently present in natural foodstuffs. Adding to the fear of technology is a longing for a less complicated time better known as “the good old days.” This nostalgia fails to note the realities of the past, with shorter life spans, longer hours of preparation in the kitchen, limited availability of produce in the late winter months in the northern climes, and other detractors such as lack of food testing and regulation.
14.3 TESTS TO DETERMINE FOOD SAFETY Human beings have always been intuitive toxicologists, relying on their senses of sight, taste, and smell to detect harmful or unsafe food, water, and air. When asked, some consumers still feel that they are the prime determiners of food safety and try to rely on themselves. Scientists and savvy consumers have come to recognize that our senses are not adequate to assess the dangers inherent in exposure to a chemical substance, especially one in which the ill effect is either cumulative or delayed. Thus the sciences of toxicology and risk assessment have developed a protocol for testing and assessing the safety of foods and their constituents. Toxicity tests are performed on all compounds used as intentional additives and pesticides (Dybing et al. 2002). Substances must first undergo acute toxicity tests, which use at least two species of experimental animals to determine the lethal dose 50 (LD50)—the dose that kills half of the animals. If the substance is regarded as being of low toxicity, then metabolic tests are undertaken in several animal species. These tests track the fate of the compound in the body. If metabolites are formed, their fates and toxicity must also be determined. A variety of species are used to test for differences in metabolism and to determine which species will be most similar to humans. Subacute tests are then performed. These require the feeding of a range of doses below the LD50 to at least two species for two to three months. A threshold or noobserved-adverse-effect level (NOAEL) is determined from the highest dose that produces no harm in the most sensitive species. Chronic tests follow. These involve feeding a compound for a lifetime at doses 1000 to 100 times more than a human
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would likely ingest. These tests also involve two to three species. These tests not only assess the health of the animal over its lifespan, but also determine if there are any reproductive or offspring abnormalities. Safe levels for use are determined upon completion of testing and determination of the NOAEL. The acceptable daily intake (ADI) is then determined by dividing the NOAEL by a safety factor (Figure 11.1). The ADI is expressed as milligrams of the test substance per kilogram of body weight per day. The safety factor, often 100, is arbitrary and may vary according to the test material and circumstances. The rationale for 100 is that if the average sensitivity of humans to a particular compound is 10 times greater than for that of the most sensitive test animal, and if the most sensitive humans are 10 times more sensitive than the test animals; the use of the factor 10 × 10 = 100 would mean that the most sensitive individual could safely ingest the amount equivalent to the ADI. The threshold of toxicological concern (TTC) may be determined when it is not possible to set an ADI for constituents used in very small amounts, such as flavor compounds. TTC refers to the establishment of a level of exposure for all chemicals, even if there are no chemical-specific toxicity data, below which there would be no appreciable risk to human health. The main advantage of the use of TTCs is that the risk of low exposures can be evaluated without the need for chemical-specific animal toxicity data (Renwick 2005; Kroes et al. 2005). European Union (EU) countries use a decision tree approach (Szponer et al. 2003). Carcinogens and prions need special treatment. Some scientists adhere to the idea that there is, for some carcinogens, no threshold or tolerance level, but currently more scientists believe that there is a threshold level and that with proper understanding of the compound and proper animal experiments, this level can be determined (Waddell 2005). For carcinogens, the 100-fold safety factor may be inadequate, and factors as high as 5000 have been proposed. Tolerable daily intakes have been set for carcinogens in food (such as Aflatoxin B1) that are DNA adducts. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically (Jeffrey and Williams 2005). Prions—misfolded proteins that lead to neurological disorders—also require special precautions, partly because the mode of transmission is still under investigation. Research is needed to determine why some individuals are susceptible and others may not be, and delineation of roles played by other cellular components such as chaperone proteins. The current strategy is to keep prions out of the food supply rather than setting an ADI or TTC. Novel foods also require special safety assessment protocols (Edwards 2005). A case-by-case approach is needed, which must be adapted to take into account the characteristics of the individual novel food. A thorough appraisal is required of the origin, production, compositional analysis, nutritional characteristics, any previous human exposure, and the anticipated use of the food. The information should be compared with a traditional counterpart of the food, if this is available. In some cases, a conclusion about the safety of the food may be reached on the basis of this information alone, whereas in other cases it will help to identify any nutritional or
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toxicological testing that may be required to further investigate the safety of the food. The importance of nutritional evaluation cannot be overemphasized. This is essential in order to avoid dietary imbalances that might lead to interpretation difficulties, but also in the context of its use as food, and to assess the potential impact of the novel food on the human diet. The traditional approach used for chemicals with a large safety margin relative to the expected exposure cannot be applied to novel foods. The assessment of safety in use should be based upon a thorough knowledge of the composition of the food, evidence from nutritional, toxicological, and human studies, expected use of the food, and its expected consumption. Safety equates to a reasonable certainty that no harm will result from intended uses under the anticipated conditions of consumption. For all foods and components, the final step is to consider potential consumption estimates of the foods or commodities that might contain the chemical or novel food. For microconstituents, this allows calculation of the maximum residue level (MRL). Constant monitoring and reevaluation of these estimated intakes make certain that estimates reflect real exposure (Caldas and Souza 2004). For some items accurate exposure estimates may require development of new sampling procedures to accurately assess food product consumption data for high-risk groups and greater consideration of food purchased away from home. Even with the best scientific methods, extrapolations and judgments are required to infer human health risks from animal data. Basic toxicological concepts, assumptions, and interpretations were found, according to a recent survey, to differ greatly between toxicologists and laypeople (Low et al. 2004), as well as differences among toxicologists working in industry, academia, and government. Toxicologists were found to be sharply divided in their opinions about the ability to predict a chemical’s effect on human health on the basis of animal studies and other laboratory methods being employed in lieu of animal tests (Ames and Gold 2000). This difference of opinion creates public policy tensions. Renowned toxicologist Bruce Ames stated that minimizing minuscule hypothetical risks may damage public health by diverting resources and distracting the public from major risks. In addition, some epidemiologists state that the avoidance of possible risks from processes like irradiation may continue to allow hazards from foodborne disease that could be significantly reduced by use of irradiation.
14.4 FOOD SAFETY CONCERNS Foodborne disease has always topped the list of food safety concerns for most government bodies around the world. Outbreaks due to Salmonella and other organisms such as Listeria and E. coli have placed foodborne disease at the top of the list of consumer food safety concerns (Kirk et al. 2002). This has not always been the case. Chemicals and pesticides still manage to cause great concern. Scientific ability to detect substances to the attogram level fuels consumer unease. In cases where the consumer used to believe that the chemical was not in the food and is now there at very small levels is disquieting for consumers, and consumers and scientists alike are unable to interpret the effect, if any, of the small level of such a chemical.
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Bovine spongiform encephalopathy (BSE), foods produced by biotechnology— known to the consumer as genetically modified organisms (GMOs)—and food as a vehicle for bioterrorism add to the growing list of consumer fears. How a food safety concern is viewed varies by the perspective of the viewer. Scientific groups use documented hazards, known deaths, or cases of illness to extrapolat
