Food Flavors: Formation, Analysis and Packaging Influences (Developments in Food Science - 40)

DEVELOPMENTS IN FOOD SCIENCE 40

FOOD FLAVORS: F O R M A T I O N , ANALYSIS A N D PACKAGING INFLUENCES

This Page Intentionally Left Blank

DEVELOPMENTS IN FOOD SCIENCE 40

F O O D FLAVORS: FORMATION. ANALYSIS A N D PACKAGING INFLUENCES PROCEEDINGS OF THE 9TH INTERNATIONAL FLAVOR CONFERENCE* THE GEORGE CHARALAMBOUS MEMORIAL SYMPOSIUM * LIMNOS, GREECE, 1-4 JULY 1997 Edited by E.T, CONTIS College of Arts and Sciences 411 Pray-Harrold, Eastern Michigan University, Ypsilanti Ml 48197, USA C.-T. HO Department of Food Science, Cook College, Rutgers University 65 Dudley Road, New Brunswick, NJ 08901-8520, USA C.J. MUSSINAN International Flavors and Fragrances, Inc., Research & Development, 1515 Highway 36, Union Beach, NJ 07735, USA T.H. PARLIMENT Kraft Technology Center, 555 So. Broadway, Tarrytown, NY 10965, USA F. SHAHIDI Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, A1B 3X9, Canada A.M. SPANIER U.S.DA. Agricultural Research Service SRRC, 1100 Robert E. Lee Blvd. New Orleans, Louisiana 70124, USA

1998 ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon - Singapore - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211,1000 AE Amsterdam, The Netherlands

Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for.

ISBN: 0-444-82590-8 © 1998 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. © The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper) Printed in The Netherlands

DEVELOPMENTS IN FOOD SCIENCE Volume 1 Volume 2 Volume 3

Volume 4 Volume 5

Volume 6 Volume 7

Volume 8 Volume 9

Volume 10

Volume 11

Volume 12

Volume 13

Volume 14 Volume 15

Volume 16 Volume 17

Volume 18

Volume 19 Volume 20

J.G. Heathcote and J.R. Hibbert Aflatoxins: Chemical and Biological Aspects H. Chiba, M. Fujimaki, K. Iwai, H. Mitsuda and Y. Morita (Editors) Proceedings of the Fifth International Congress of Food Science and Technology I.D. Morton and A.J. MacLeod (Editors) Food Flavours Part A. Introduction Part B. The Flavour of Beverages Part C. The Flavour of Fruits Y. Ueno (Editor) Trichothecenes: Chemical, Biological and Toxicological Aspects J. Holas and J. Kratochvil (Editors) Progress in Cereal Chemistry and Technology. Proceedings of the Vllth World Cereal and Bread Congress, Prague, 28 june-2 July 1982 I. Kiss Testing Methods in Food Microbiology H. Kurataand Y. Ueno (Editors) Toxigenic Fungi: Their Toxins and Health Hazard. Proceedings of the Mycotoxin Symposium, Tokyo, 30 August-3 September 1983 V. Betina (Editor) Mycotoxins: Production, Isolation, Separation and Purification J. Hollo (Editor) Food Industries and the Environment. Proceedings of the International Symposium, Budapest, Hungary, 9-11 September 1982 J. Adda (Editor) Progress in Flavour Research 1984. Proceedings of the 4th Weurman Flavour Research Symposium, Dourdan, France, 9-11 May 1984 J. Hollo (Editor) Fat Science 1983. Proceedings of the 16th International Society for Fat Research Congress, Budapest, Hungary, 4-7 October 1983 G. Charalambous (Editor) The Shelf Life of Foods and Beverages. Proceedings of the 4th International Flavor Conference, Rhodes, Greece, 23-26 July 1985 M. Fujimaki, M. Namiki and H. Kato (Editors) Amino-Carbonyl Reactions in Food and Biological Systems. Proceedings of the 3rd International Symposium on the Maillard Reaction, Susuno, Shizuoka, Japan,1-5 July 1985 J. Skoda and H. Skodova Molecular Genetics. An Outline for Food Chemists and Biotechnologists. D.E. Kramer and J. Listen (Editors) Seafood Quality Determination. Proceedings of the International Symposium, Anchorage, Alaska, U.S.A., 10-14 November 1986 R.C. Baker. P. Wong Hahn and K.R. Robbins Fundamentals of New Food Product Development G. Charalambous (Editor) Frontiers of Flavor. Proceedings of the 5th International Flavor Conference, Porto Karras, Chalkidiki, Greece, 1-3 July 1987 B.M. Lawrence, B.D. Mookherjee and B.J. Willis (Editors) Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Congress of Essential Oils, Fragrances and Flavors, Washington, DC, U.S.A., 16-20 November 1986 G. Charalambous and G. Doxastakis (Editors) Food Emulsifiers: Chemistry, Technology, Functional Properties and Applictations B.W. Berry and K.F. Leddy Meat Freezing. A Source Book

Volume 21 Volume 22 Volume 23 Volume 24

Volume 25 Volume 26 Volume 27 Volume 28 Volume 29 Volume 30

Volume 31 Volume? 32

Volume 33

Volume 34 Volume 35

Volume 36

Volume 37A+B

Volume 38

Volume 39 Volume 40

J. Davidek, J. Velisek and J. Pokorny (Editors) Chemical Changes during Food Processing V. Kyzlink Principles of Food Preservation H. Niewiadomski Rapeseed. Chemistry and Technology G. Charalambous (Editor) Flavors and „Off-flavors '89. Proceedings of the 6th International Flavor Conference, Rethymnon, Crete, Greece, 5-7 July 1989 R. Rouseff (Editor) Bitterness in Foods and Beverages J. Chelkowski (Editor) Cereal Grain. Mycotoxins, Fungi and Quality in Drying and Storage M. Verzele and D. De Keukeleire Chemistry and Analysis of Hop and Beer Bitter Acids G. Charalambous (Editor) Off-Flavors in Foods and Beverages G. Charalambous (Editor) Food Science and Human Nutrition H.H. Huss, M. Jakobsen and J. Listen (Editors) Quality Assurance in the Fish Industry. Proceedings of an International Conference, Copenhagen, Denmark, 26-30 August 1991 R.A. Samson, A.D. Hocking, iJ.1.Pitt and A.D. King (Editors) Modern Methods in Food Mycology G. Charalambous (Editor) Food Flavors, Ingredients and Composition. Proceedings of the 7th International Flavor Conference, Pythagorion, Samos, Greece, 24-26 June 1992 G. Charalambous (Editor) Shelf Life Studies of Foods and Beverages. Chemical, Biological, Physical and Nutritional Aspects G. Charalambous (Editor) Spices, Herbs and Edible Fungi H. Maarse and D.G. van der Helj (Editors) Trends in Flavour Research. Proceedings of the 7th Weurman Flavour Research Symposium, Noordwijkerhout, The Netherlands, 15-18 June 1993 J.J. Bimbenet, E. Dumoulin and G. Trystram (Editors) Automatic Control of Food and Biological Processes. Proceedings of the ACoFoP III Symposium, Paris, France, 25-26 October 1994 G. Charalambous (Editor) Food Flavors: Generation, Analysis and Process Influence Proceedings of the 8th International Flavor Conference, Cos, Greece, 6-8 July 1994 J.B. Luten, T. Borresen and J. Oehlenschlager (Editors) Seafood from Producer to Consumer, Integrated Approach to Quality Proceedings of the International Seafood Conference on the occasion of the 25th anniversary of the WEFTA, held in Noordwijkerhout, The Netherlands, 13-16 November 1995 D. Wetzel and G. Charalambous t (Editors) Instrumental Methods in Food and Beverage Analysis E.T. Contis, C.-T. Ho, C.J. Mussinan, T.H. Parliment, F.Shahidi and A.M. Spanier (Editors) Food Flavors: Formation, Analysis and Packaging Influences Proceedings of the 9th International Flavor Conference The George Charalambous Memorial Symposium

FOREWORD The 9th International Flavor Conference: George Charalambous Memorial Symposiiim was held July 1-4, 1997 at the Porto Myrina Palace on the Island of Limnos, Greece. This conference was organized as a tribute to Dr. George Charalambous who organized the previous eight conferences. Unfortunately, George passed away in November of 1994, only a few months after the last conference which was held on the island of Kos, Greece. The 9th Conference venue was the island of Limnos, site of the oldest city in Europe, Poliochni (opposite the city of Troy), with the conference cer'.er ard h^h' ix^ I' ruins of the Temple of Artemis. The 9th Conference follo^ved tl e ixmiiaX an 11 ^di io* •^^ the previous meetings. More than 90 papers/posters were presented by scies tis.s ?;:*;. nineteen countries. Dr. Apostolos Grimanis, a radioanalyilcal clsen -st anc. u in -i - W . of the Radioanalytical Laboratory at the National (enter fc lei "at/K K:;Sr. "Demokritos" in Athens, and cousin of Dr. Charalambc^'US, oner d he n -Q Ap w tribute to George. The paragraphs below are excerpts fron h\^ ?.»nr: trl ;.

"George was bom in Alexandria, Egypt. However, both liis parent;- were Grce ,s, co: - ., from Mytilene, capital of the Aegean island of Lesvos, Greece, k I.Tit v Je ' Charalambous since my childhood. He served in the Greek Nav^ dusin. * e ^e-.-vn World War. His battleship was sunk after an air raid, and George was one of th^ ^ jr, \ . members of the crew who survived. He was on a wooden plank in the Medirei an ,ai v , for three days, watching the sharks pass by." George studied Chemistry (B.Sc.) and Industrial Chemistry (Ph.D.) at the UiUve/si ' Edinburgh in Great Britain. In 1956, George started to work at Anheuser BMSC*. i: v Louis, Mo. He eventually became one of the directors of the company. I^.^ ^ .diivC '-.' organize the international flavor conferences in 1978. "I will remember George for his fine character, his devotion to science and to his family, and his love for Greece and the USA. He was a nice man and an excellent scientist. George will remain in our thoughts and in our hearts forever. I am sure that George's colleagues will continue to organize successftil international flavor conferences in Greece." The Conference Committee is pleased to announce that the Division of Agricultural and Food Chemistry (American Chemical Society) has agreed to sponsor a Fellowship in George's honor. The Charalambous Fellowship is established in recognition of his tremendous contributions to the Division over many years. The Conference Committee would also Uke to make preliminary announcement of the 10*^ International Conference to be held tentatively in the year 2000 on the island of Santorini, Greece. The Editors

This Page Intentionally Left Blank

ACKNOWLEDGEMENTS The Conference Committee gratefully acknowledges the generousfinancialcontributions of the following;

Hershey, USA Kato Worldwide The Procter & Gamble Company The Society of Flavor Chemists

The Conference Committee further acknowledges support by the sponsor:

Agricultural and Food Chemistry Division of the American Chemical Society

This Page Intentionally Left Blank

CONTENTS Foreword

vii

Acknowledgements

ix

Overview Thirty Years of the AH-B Theory T.E. Acree, R.S Shallenberger, and S. Ebeling The Gatt-Trips Agreement-What it is and How has it Changed the Playing Field for alle Applicants for United States Patents S.P. Ludwig and A.C. Gogoris

1

15

Flavornet: A Database of Aroma Compounds Based on Odor Potency in Natural Products H. Arn and T.E. Acree

27

Beverage Flavor Emulsion - A Form of Emulsion Liquid Membrane Microencapsulation •C.-TTan

29

New Beverages: The Flavored Coffee . M. Bononi, E. Lubian, S. Martello and F.Tateo Indicators for Evaluation of Lipid Oxidation and Off-Flavor Development in Food F Shahidi

V-^

55

Analysis of Flavors Aroma Analysis of Coffee Brew by Gas Chromatography-Olfactometry ... K.D. Deibler, T.E. Acree and E.H. Lavin

69

Electronic Nose Versus Multicapillary Gas Chromatography: Application of Rapid Differentiation of Essential Oils T.Talou, S. Maurel and A. Gaset

79

Quantitation of Potent Food Aroma Compounds by Using Stable Isotope Labeled and Unlabeled Internal Standard Methods M. Preininger

87

Simplification of Complex Flavor Mixtures Via Micro Extraction Class Separation TH. Parliment

99

Xll

A Simulated Mouth to Study Flavor Release form Alcoholic Beverages S.J. Withers, J.M. Conner, J.R. Piggott and A. Paterson Comparisons of Volatile Compounds Released During Consumption of Cheddar Cheeses by Different Consumers C.M. Delahunty, RJ. O'Riordan, E.M. Sheehan and RA. Morrissey Effect of Adsorbent Particle Size on the Water-Ethanol Separation by Cellulosic Substrates G. Vareli, RG. Demertzis and K. Akrida-Demertzi Influence of Extraction Procedure on the Aroma Composition of Thymus Zygis L\ and Mentha Pulegium L M. Moldao-Martins, R. Trigo, M.A. Nolasco, M.G. Bernardo Gil and M.L Beirao Da Costa Hypericin and Hypericin-Like Substances: Analytical Problems RTateo, S. Martello, E. Lubian and M. Bononi

111

117

125

133

143

Sensory Evaluation Determination of the Cause of Off-Flavors in Milk by Dynamic Headspace GC/MS and Multivariate Data Analysis R.T Marsili and N. Miller

159

Sensory Properties of Musty Compounds in Food E. Chambers IV, E.C. Smith, LM. Seitz and D.B. Sauer

173

Evaluation in Score of the Intensity of Salty and L/Anan?/Tastes R. Kuramitsu

181

Sensory Characteristics of Chemical Compounds Potentially Associated with Smoky Aroma in Foods D.H. Chambers, E. Chambers IV, LM. Seitz, D.B. Sauer, K. Robinson and A.A. Allison Identification of Tasty Compounds of Cooked Cured Ham: Physico-chemical and Sensory Approaches J. Valentin, A.S. Guillard, C. Septier, C. Salles, and J.L. Le Quere Isolation of a Peptidic Fraction from the Goat Cheese Water-Soluble Extract by Nanofiltration for Sensory Evaluation Studies N. Sommerer, A. Garem, D. Molle, C. Septier, J.L Le Quere and C. Salles

187

195

207

Xlll

Effect of Distillation Process Factors on Ouzo Flavor Examined by Sensory Evaluation A. Geronti, C. Spiliotis, G.N. Liadakis and C.Tzia

219

Formation of Inosinic Acid as the Taste Compound in the Fermentation of Japanese Sake K. Fujisawa and M. Yoshino

227

Aroma, Meat Volatile Composition of Southern European Dry-Cured Hams RJ. Dirinck and F van Opstaele

233

Role of Sodium Nitrate on Phospholipid Composition of Cooked Cured ... Ham. Relation to its Flavor A.S. Guillard, I. Goubet, C. Salles, J.L. Le Quere and J.L. Vendeuvre

245

Influence of Fat on the Flavor of an Emulsified Meat Product F.RV. Chevance and L.J. Farmer

255

Aroma-Impact Compounds in Cooked Tail Meat of Freshwater Crayfish (Procambarus clarkii) K.R. Cadwallader and H.H. Baek

271

Comparison of Flavor Characteristics of Domestic Chicken and Broiler as Affected by Different Processing Methods A. Apriyantono and Indrawaty

279

Aroma, Fruits and Vegetables Comparison of Flavor Components in Fresh and Cooked Tomatillo with Red Plum Tomato R.J. McGorrin and L. Gimelfarb

295

Effect of Thermal Treatment in the Headspace Volatile Compounds of Tomato Juice M. Servili, R. Selvaggini, A.L. Begliomini and G.F. Montedoro

315

Fresh-Cut Pineapple {Ananas sp.) Flavor. Effect of Storage A.M. Spanier, M. Flores, C. James, J. Lasater, S.W. Lloyd and J.A. Miller

331

GC-MS Analysis of Volatile Compounds in Durian {Durio zibethinus Murr.) 345 J. Jiang, S.Y. Choo, N. Omar and N. Ahamad

XIV

The Effect of Drying Treatment on the Flavor and Quality of Longan Fruit C.Y. Chang, C.H. Chang, TH. Yu, L Y Lin and YH. Yen

353

Effect of Processing Conditions on Volatile Composition of Apple Jellies and Jams M. Moldao-Martins, N. Moreira, I. Sousa and M.L Beirao Da Costa

369

The Relationship between Ethylene and Aroma Volatiles Production in Ripening Climacteric Fruit S.Grant Wyllie, J.B. Golding, W.B. McGlasson and M. Williams

375

Aroma, Miscellaneous Sensory Characterization of Halloumi Cheese and Relationship with Headspace Composition J.R. Piggott, A. Margomenou, S.J. Withers and J.M. Conner

385

Comparison Study of UHT Milk Aroma L. Hashim and H. Chaveron

393

Some Toxic Culinary Herbs in North America A.O. Tucker and M.J. Maciarello

401

Influence Of Preparation on the Aroma Compounds in an Oatmeal Porridge M.J. Morello Characterization of Flavor of Tea Produced Different Tea Area M. Kato and M. Omori Studies on the Formation of Special Aroma Compounds of Pouchung Tea made from Different Varieties YS.Chen, H.J.Tasy andTH.Yu Egyptian Onion Oil N.A. Shaath and FB. Flores

415 423

431

443

Maillard Chemistry Melanoidins in the Maillard Reaction T. Obretenov and G. Vernin Formation of Volatile Sulfur Compounds in Reaction Mixtures Containing Cysteine and Three Different Ribose Compounds D.S. Mottram and I.C. Nobrega

455

483

XV

Flavor Formation from the Interactions of Sugars and Amino Acids under Microwave Heating TH. Yu, B.R. Chen, L Y Lin and C.-T Ho

493

Characterization of Intermediate 3-Oxazolines and 3-Thiazolines from the Reaction of 3-Hydroxy-2-Butanone and Ammonium Sulfide C.-T. Ho, J. Xi, H.-Y Fu and T.C. Huang

509

Mechanistic Studies of the Formation of Thiazolidine and Structuraly Related Volatiles in Cysteamine/Carbonyls Model System T.C. Huang, YM. Su, L.-Z. Huang and C.-T Ho

519

Effect of Antioxidants on the Formation of Volatiles from the Maillard Reaction A. Arnoldi, M. Negroni and A. D'Agostina

529

Formation of Flavors in Foods and IVIodel Systems The Use of Roasting Kinetics Data to Characterize Natural and Artificial Chocolate Aroma Precursors G.R Rizzi and RR. Bunke

535

Contribution of Muscle and Microbial Aminopeptidases to Flavor Development in Dry-Cuyred Meat Products M. Flores, Y Sanz., A.M. Spanier, M-C. Aristoy and F. Toldra

547

Effect of Adding Free Amino Acids to Cheddar Cheese Curd on Flavor Development H.M. Wallace and PR Fox

559

The Influence of Fat on Deterioration of Food Aroma in Model System During Storage M. Chen and G.A. Reineccius

573

The Effect of the Addition of Supplementary Seeds and Skins During Fermentation on the Chemical and Sensory Characteristics of Red Wines E. Revilla, J.M. Ryan, V. Kovac and J. Nemanic

583

Factors Influencing Food Flavors Role of Phenolics in Flavor of Rapeseed Protein Products M. Naczk, R. Amarowicz and F Shahidi

597

XVI

Effect of Ethanol Strength on the Release of Higher Alcohols and Aldehydes in Model Solutions H. Escalona-Buendia, J.R. Piggott, J.M. Connor and A. Paterson Ultrasonic Inactivation of the Soybean Trypsin Inhibitors H.H. Liang, R.D. Yang and K.C. Kwok

615

621

Evaluation of Shelf Life of Flavored Dehydrated Products using Accelerated Shelf Life Testing and the Weibull Hazard Sensory Analysis 627 M. Bill and RS.Taoukis Behavior of Histamine During Fermentation of Fish Sauce Determined by an Oxygen Sensor Using a Purified Amine Oxidase N.G. Sanceda, E. Suzuki and T. Kurata

639

Effect of Crystallization Time on Composition of Butter Oil in Acetone FM. Fouad, O.A. Mamer, F Sauriol and F Shahidi

647

Antimicrobial Effect of Volatile Oils of Garlic and Horse-Radish G. Patkai, J. Monspart Senyi and J. Barta

659

Auto-Oxidation Changes of the Flavor of Monoterpenes During their Auto-Oxidation under Storage Conditions J. Pokorny, F. Pudil, J. Volfova and H. Valentova

667

Effect of Rosemary and 1,4-Dihydro Pyridines on Oxidative and Flavor Changes of Bergamot Oil F Pudil, J. Volfova, V. Janda, H. Valentova and J. Pokorny

679

Effect of a-Tocopherol (Vitamin E) at the Retention of Essential Oil, Color and Texture of Chios Mastic Resin During Storage D. Papancolaou, M. Melanitou and K. Katsaboxakis

689

Dietary Oil and Endogenous Antioxidants in Hyperlipemia: Uric Acid TR. Watkins, D.K. Kooyenga and M. L. Bierenbaum

695

Changes in Citrus Hystrix Oil During Auto-Oxidation

707

F. Pudil, H. Wijaya, V. Janda, J. Volfova, H. Valentova and J. Pokorny Packaging Studies on the Development of a Quick Test for Predicting the Sorption Properties of Refillable Polycarbonate Bottles RG. Demertzis and R. Franz

719

XVll

Recycling Old Polymers in Bi-layer Bottles. Effect of the Volume of the Solid Food on the Contaminant Transfer I.D. Rosea and J.M. Vergnaud

735

Polypropylene as Active Packaging Material for Aroma Sorption from Model Orange Juice A. Feigenbaum, R. Lebosse and V. Ducruet

743

Identification of the Source of an Off-Odor in Premiums Intended for Use with Dry Mix Beverages D. Apostolopoulos

753

Effect of Microwave Heating on the Migration of Dioctyladipate and Acetyltributylcitrate Plasticizers from Food-Grade PVC and PVDC/PVC Films into Ground Meat 759 A.B. Badeka and M.G. Kontominas Effect of Ionizing Radiation on Properties of Monolayer and Multilayer Flexible Food Packaging Materials K.A. Riganakos, W.D. Koller, D.A.E. Ehlermann, B. Bauer and M. Kontominas

Author Index Subject Index

767

783 787

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

Thirty years of the AH-B theory T. E. Acree^, R. S. Shallenberger^, and S. Ebeling^, ^Department of Food Science & Technology, Cornell University, Geneva, NY, 14456 "University College Cork, Cork, Ireland. Abstract Thirty years ago two of us (Shallenberger & Acree) published a paper entitled the "Molecular Theory of Sweet Taste" in Nature[l]. The model developed in that paper for sweetness was based on a structure-activity relationship between the simplest sweet tasting compounds and their structural features of the stimulants and has become known as the AH-B theory. The theory described with considerable success the structural features necessary for sweetness but it was not sufficient to predict sweetness. That is, not all compounds that satisfied the theory tasted sweet nor was the theory able to predict potency level especially for the very high potency sweeteners subsequently synthesized. However, all sweet compounds seemed to have an identifiable AH-B feature. This paper will review the last thirty years in sweetness research and discuss the role of the AH-B theory in its development. 1. 1963 - 1971 There are two motives that have driven structure-activity research (SAR) into sweetness. One is the desire to predict the taste of molecules from their structure (this is primarily of commercial value), and the other is to understand taste in terms of its chemical ecology and how it evolved (this is primarily of academic interest). In a paper publish in 1963 Robert Shallenberger summarized the relationship between sugar structure and sweet taste response in terms of the idea of a functional group for sweetness called the "saporfic group": a pair of vicinal hydroxyl groups [2].

OH OH A fact that did not seem to support this hypothesis was that most molecules containing a saporific group were not sweet. Clearly, the taste active structure that caused the sweet response was more complicated than just a pair of vicinal hydroxyIs. This was demonstrated clearly by mannose which could be prepared in two crystalline diastereoisomeric forms a- and P-. Both a- and P-D-mannose have three glycol groups but a- is sweet and P- is bitter. In fact all mono- and disaccharides, sugar alcohols and similar polyols are characterized by the presence of multiple glycol groups. Therefore, some relationship between the glycol hydroxyl groups determines sweetness and in the case of mannose bitterness. Although one glycol group in a - D-mannose has a dihedral angle greater that 60^ assuming the preferred chair conformation, exactly how this subtile structural difference could cause such a profound difference in taste was difficult to envision?

H

OH

a - D - mannopyranose (sweet)

H

H

P - D - mannopyranose (bitter)

The prevailing hypothesis, then as it is now, was that the taste active compounds acted as a ligand that bound to a receptor protein on the surface of a taste receptor cell causing an allosteric effect on the inside of the cell inducing a change in ionic conductance resulting in depolarization. For the idea to work the receptor protein - ligand complex must undergo a change in at least its tertiary structure. However, these changes could include changes in the ligand as well as the receptor protein. Therefore, the conformation of the ligand as it approaches the receptor may not be as important as the conformation it can assume in the receptor - ligand complex. Predicting activity from the most stable structure in solution may be the wrong approach. This led to the idea that the

3

glycol group induces sweetness when it can assume a dihedral nearer to the gauch (60°) configuration than either the eclipsed (0°) or anticlinal (180°). In this view a dihedral angle somewhat less than gauch must be obtainable and the energy necessary to do this would be the determinant for sweet taste.

^^OH gauch (60°)

^^OH eclipsed (0°)

^ OH anticlinal (180°)

Between 1963 and 1968, while a graduate student in Shallenberger's laboratory, Terry Acree studied the secondary and tertiary structure of the monosaccharides in solution in an attempt to find a more successful structural description to predict sweetness. Although this work provided some greater detail about sugars in solution [3-6], it did not yield any better predictors of sugar sweetness. Meanwhile, in 1967 Shallenberger and Acree published a theory of sweet taste chemistry that was based on simpler and more rigid structures than the sugars. At that time, it was commonly accepted that the sweet taste mechanism evolved to detect biologically important primary metabolites such as sugars and hydrolyzed polysaccharides at molar concentrations and that the sweetness of non-sugar molecules was perhaps an artifact [7, 8]. However, it was also assumed that these non-sugar sweeteners acted on the same sweet receptor and t h a t their structure could be used to predict t h e structure of a "saporific" molecules including sugars. The simplest series of molecules t h a t have a clear sweet taste are t h e chloromethanes. They are rigid, and the analogues chloroform, methylene chloride and chloromethane shown below are slightly sweet whereas methane and carbon tetrachloride are not sweet. CI

CI^H>AHJ'^

CI

H^H>AHJ'^

H

H^H>AH>

chloroform methylene chloride chloromethane The structure common to these sweet analogues is the presence of a hydrogen bis to a chlorine. Comparing this to the structure of sweet glycols with a gauch dihedral angle we concluded that a hydrogen bondable proton located about 3 A (0.3 nm) from an electron rich orbital also capable of forming a hydrogen bond

was required for sweetness. The proton donor was called the AH group and the proton acceptor was called the B group: thus defining the "saporific group" as an AH-B (3 A). Examining the structure of a diverse group of sweet tasting molecules, Shallenberger and Acree concluded that AH-B could be a necessary condition for sweetness but clearly not a sufficient condition to guarantee sweetness. There were other structural features that rendered most molecules with an AH-B at 3A non-sweet or at least dominated by some other taste property usually bitterness. In 1969 with the help of C. Y. Lee, Shallenberger and Acree published a paper based on the taste of amino acids that identified the minimum requirements for sweetness among the chiral amino acids [9]. Starting with glycine and alanine they pointed out that since all of the D- amino acids with side groups larger than a methyl group or at least as large as an isobutyl group (leucine) were non-sweet while their enantiomers ( t h e L-amino acids) tasted sweet. The small achirial glycine with only a proton side group and the slightly larger Dand L-alanine with only a methyl group for a side chain were all distinctly sweet. Furthermore, glycine is functionally sweet in foods like the cooked crustatae, shrimp and lobster.

D - Alanine (sweet)

L - Alanine (sweet)

The simplest conclusion was that there was a steric barrier that inhibited the binding of the D- isomer but allowed the binding of the L- isomer. Alternatively, it could be argued that the side group of the L- isomers bound to a lipophylic part of the receptor resulting in what was reported by Kier in 1972 as a threepoint attachment theory for the "glycophore" in which X in the figure below is a lipophilic group [10] .

AH

.26 nm

It seems reasonable to assume that some multiple attachment process will contribute to the chirality of sweetness, but the fact that glycine is the sweetest amino acid and it has no side chain is still puzzling. Furthermore, the observation that the enantiomers of the momosacchrides are equally sweet while the diastereoisomers tasted different does not speak for a simple chiral component. For example, the two enantiomers of glucose shown below are both equally sweet while the two anomers (diastereoisomers) of mannose taste different. OH

HO' H H

a-D-glucopyranose

OH

H HO

a-L-glucopyranose

We can summarize the taste of polyols as follows: 1. Sweet ligands are bipolar hydrogen bonding units: AH-B 2. Enantiomers of sugars are equal tasting. 3. Diastereoisomers (anomers) of sugars can have different tastes. However, the taste of the amino acids were summarized differently: 1. Sweet ligands are bipolar hydrogen bonding units: AH - B 2. Enantiomers of the amino acids are different tasting if they have a side group larger than alanine. The contradictions created by these two summaries were the subject of numerous studies and structural activity relation investigations but none could resolve them with a single ligand receptor model.

2. 1972-1991 Over the next 20 years, the apparent contradictions posed by the tastes of amino acids and sugars eventually resulted in several descriptions of a receptor site structure that would accommodate a variety of "saporific groups". By 1991, these ideas reached their greatest degree of complexity in the structure simulation studies in Belitz'[ll] laboratory and the multi-attachment theory of Tinti and Nofri[12]. These workers approached the problem by creating graphic representations of the active site on the sweet receptor in terms of the types of functional groups that might interact and their spatial arrangements. Shown below is the model developed by Tinti & Nofre [12] in which the various spheres represent different functional groups that may be involved in the ligand binding.

Common to all of these receptor site models and multi-attachment theories are two assumptions: 1) the presence of an AH-B or equivalent and 2) the assumption that not all attachments are required for binding to take place. That none of these complex models describe necessary and sufficient conditions for taste is their weakest feature. Information about the nature of the receptor protein, the number of transduction mechanisms involved and the relationship between sweet and bitter taste biochemistry would certainly help. The AH-B model for the ligand binding to the receptor provided a reasonable idea for a transduction mechanism: the disruption of a hydrogen bond on the receptor protein on the outside of the cell followed by an allostericUy induced change inside the cell[13]. However, the details of this part of the transduction process shown below are too vague to guide SAR modeling and too speculative add anything to the study of transduction biochemistry.

Receptor site

H

Saporific ligand

There were two other facts about taste that were puzzHng. The first was the discovery in the early 1980's of a sweetness inhibitor by Michael Lindley [14, 15]. The phenoxyalkonic acids inhibited both the amino acid based sweeteners (aspartame) and the polyol sweeteners (sucrose) in exactly the same way with exactly the same competitive inhibition. This would strongly suggest that the same limiting steps were being inhibited and therefore the transduction mechanisms for both types of sweeteners were perhaps the same or at least shared some transduction components. Furthermore, that sweetness inhibition occurred put into question any modeling based on the taste intensity of a series of molecules. If sweetness intensity is a balance between inhibitory elements and stimulant structures, perhaps on the same molecule, then interpreting sweetness intensities in terms of ligand binding affinities would be erroneous. The complex chemistry of most natural products predicts that inhibition is most surely is an important component of many real food systems. The second set of puzzeling facts about taste was the inability of many sweeteners, the amino acid based sweetners for example, to produce as intense a sweetness as sucrose. Polyols are not very potent sweeteners nor would they need to be if their taste was simply an indication of metabolically important concentrations. However, as shown below sucrose, fructose, etc., are one of the most intense sweeteners where as the the extremely potent sweeteners like aspartame are never as sweet as the polyols [16].

Intensity 100 •

High Intensity (sucrose)

80

High Potency (aspartame)

-4

T -2 0 Log (Concentration)

This distinction between high intensity sweeteners as opposed to high potency sweeteners would tend to indicate different transduction mechanisms. However, as Lindely pointed out in a paper presented at the first American Chemoreception Science symposium meeting in 1975 [17] "Assuming that there is in fact a direct relationship between structure and taste, I think the only conclusion to be drawn from this [contradictory facts] is that there is something missing." Exactly what was missing became clearer when evidence for a transduction mechanism based on G-proteins found in the taste membranes of many non-human models began to accumulate. 3. 1991- 1997 The present theory of t a s t e transduction, recently summarized by Lindmann[8, 18], indicates that high intensity sweeteners (polyols in particular) react with a seven-transmembrane receptor protein (SR) which is associated with a G- protein inside the cell. The diagram below shows a schematic of the olfactory receptor protein found in the rat (adapted from Krieger [19]).

membrane

^COOH It is typical of the 7-transmembrane receptor proteins usually associated with

G-proteins. In the case of olfaction the present speculation is that the cytoplasmic loops are associated with the G-protein inside the cell and the extra cellular loops are involved in forming the ligand binding site. Modifications in the tertiary structure of the external loops presumably provides the energy to create an allosteric change in the cytoplasmic loops activating the G-protein. Although the details of this part of the process are still unclear, the general idea seems convincing since it has been repeated in so many different chemo-sensory systems [20]. In a more recent review, Naim[21] summarized some truly exciting ideas about sweet transduction based on studies from of non-human systems. In simple terms measurements of intracellular transduction second messengers, calcium ion, inosotol triphosphate (IPS) and cyclic adenosine monophosphate (cAMP) indicates the presence of multiple mechanisms on multiple receptors. For example, after reaction with a receptor cell saccharin caused the accumulation of IPS and sucrose the accumulation of cAMP inside the cell indicating that non-polyol sweeteners are involved in a different transduction process. The figure below shows a modification of the scheme for sweet taste transduction in the rat circumvallate taste papillia proposed by Naim. AA

Si^ar

AA

Sensory Nerve Hber-

The scheme shows a taste cell with two taste receptor proteins: SR, a sugar receptor protein that uses cAMP as a second messenger and NSR, a non-sugar re-

10

ceptor protein that uses IPS as a second messenger. NSR responds to saccharin, small peptides and similar compounds. Both of these mechanisms appear to be on the same sensory cell. Also shown in the diagram are the a, (3 and y subunits of the G-protein that are putatively activated by the receptor protein. The interesting twist to this picture is the possibility that some sweet-tasting compounds labeled here as AA (called amphipathic compounds by Naim, i.e. having both polar and non-polar properties) induce transduction by moving across the receptor cell membrane and reacting directly with the P-y subunit. These compounds are then acting like many drugs that enter cells, modify their behavior and stimulate responses that were evolved to detect the presence of different ligands. In the case of sweetness: caloric polyols. The implication for SAR of sweetness created by the possibility multiple receptors with multiple mechanisms is profound. We would have to conclude that the multiple attachment theories must represent a melange of receptor structures and this would explain the "necessary but not sufficient..." nature of their predictive powers. We can then speculate that the following scheme for sweet taste in which high potency sweeteners induce sweetness by disrupting the transduction process at the G-protein while high intensity sweeteners react with the sweet receptor protein would explain their different dose-response behavior. Higli Fotemy " l ^ ^ Inteitaitj-

Finally, the creation of knock-out mice by Wong et al that lack a-gusducin (presumably the a subunit of the sweet taste G-protein) inhibited both second messenger formation at the cellular level and the taste response to bitter (denatonium benzoate and quinine sulfate), high-intensity sweeteners (sucrose) and high potency sweeteners (a guanidine sweetener: SC45647). This strongly indicates that both sweet reception and bitter reception share same transduction components and that the non-sugar sweet receptor system is related to the bitter receptor if not in fact the same as shown in the diagram below [22].

11

Non-sugar

Non-sugar I

Exactly how the second order neurons interpret this multiple receptor - multiple mechanism process is a little difficult to imagine. However, we should be able to predict that if sweetness inhibitors act by inhibiting the G-protein complex they would have the same effect on both high potency and high - intensity sweeteners. Furthermore, they should also inhibit bitter compounds in a similar fashion. This, however, has yet to be determined. After thirty years, the AH-B theory remains a possible explanation for the ligand binding chemistry that induces some sweet taste responses but it seems to have become a minor part of what has evolved into a complicated yet elegant story chemo-sensory response.

4. References

1. 2. 3.

4. 5. 6.

Shallenberger, R.S. and T.E. Acree, Molecular Theory of Sweet Taste. Nature, 1967. 216(5114): p. 480-2. Shallenberger, R.S., Hydrogen Bonding and the Varying Sweetness of the Sugars. Journal of Food Science, 1963. 28(5): p. 584-589. Acree, T.E., R.S. Shallenberger, and L.R. Mattick, Mutarotation ofD-galactose. Tautomeric composition of an equilibrium solution in pyridine. Carbohyd. Res., 1968. 6(4): p. 498-502. Acree, T.E., Tautomerism of D-glucose, D-mannose, and D-galactose. 1969. 29(11). Acree, T.E., et al., Thermodynamics and kinetics of D-galactose tautomerism during mutarotation. Carbohyd. Res., 1969. 10(3): p. 355-60. Acree, T.E., Chemistry of sugars in boric acid solutions. Advan. Chem. Ser.,

12

7. 8. 9.

10. 11.

12.

13.

14. 15.

16.

17. 18.

N o , 1973. . Moncriff, R.W, The Chemical Senses. 3 ed. 1967, London: Leonard Hill. Lindemann, B., Taste Reception. Physiol. Rev., 1996. 76(3): p. 719-766. Shallenberger, R.S., T.E. Acree, and C.Y. Lee, Sweet Taste of D and LSugars and Amino-acids and the Steric Nature of their Chemo-receptor Site. Nature, 1969. 221(5180): p. 555-556. Kier, L.B., A molecular theory of sweet taste. J. Pharm. Sci., 1972. 61: p. 1394-7. Rohse, H. and H.-D. Belitz, Shape of Sweet Receptors Studied by Computer Modeling, in Sweetners: Discovery, Molecular Design, and Chemoreception. ACS Symposium Series, D.E. Walters, F.T. Orthoefer, andG.E. DuBois, Editor. 1991, Am. Chem. Soc: Boston, p. 176-192. Tinti, J.-M. and C. Nofre, Why Does a Sweetener Taste Sweet? A New Model, in Sweeteners: Discovery, Molecular Design, and Chemoreception. ACS Symposium Series, D.E. Walters, F.T. Orthoefer, andG.E. DuBois, Editor. 1991, Am. Chem. Soc: Boston, p. 176-192. Acree, T.E. A Molecular Theory of Sweet Taste - Amino Acids and Peptides. in Joint Symposium on Carbohydrate /Protein Interactions: American Association of Cereal Chemists. 1971. Excelsior Springs MO: Lindley, M.G.,. 1986, Europe. Lindley, M.G., Phenoxyalkanoic Acid Sweeteness Inhibitors, in Sweetners: Discovery, Molecular Design, and Chemoreception. ACS Symposium Series, D.E. Walters, F.T. Orthoefer, andG.E. DuBois, Editor. 1991, Am. Chem. Soc: Boston, p. 251-260. DuBois, G.E., et al., Concentration-Response Relationships of Sweeteners, in Sweeteners: Discovery, Molecular Design, and Chemoreception. ACS Symposium Series. 1991, Am. Chem. Soc: Boston, p. 251-260. Lindley, M.G. and G.G. Birch, Structural functions of taste in the sugar series. J. Sci. Food Agric, 1975. 26(1): p. 117-24. Lindemann, B., Chemoreception: Tasting the sweet and the bitter. Curr. Biol., 1996. 6(10): p. 1234-1237.

19. Krieger, J., et al., Cloning and Expression of Olfactory Receptors, in Adv. in Biosciences, R. Apfelbach, et al., Editor. 1994, Elsevier Science Inc.: Oxford. 20. Brand, J.G. and A.M. Feigin, Biochemistry of sweet taste transduction. Food Chem., 1996. 56(3): p. 199-207. 21. Naim, M., et al. Molecular aspects of Sweet Taste Transduction, in Contrib.

13 Low- Non-Volatile Mater. Flavor Foods. 1996. Allured, Carol Stream, 111. 22. Wong, G.T., K.S. Gannon, and R.F. Margolskee, Transduction of hitter and sweet taste by gustducin. Nature, 1996. 381(6585): p. 796-800.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

15

THE GATT-TRIPS AGREEMENT - WHAT IT IS AND HOW IT HAS CHANGED THE PLAYING FIELD FOR ALL APPLICANTS FOR UNITED STATES PATENTS S. Peter Ludwig and Adda C. Gogoris Darby & Darby PC, 805 Third Avenue, New York, New York 10022-7513, Telephone: 212-527-7700, Internet Web Site: http://www.darbylaw.com

Abstract Entry into force of the GATT-TRIPs agreement on intellectual property has changed the rules for obtaining patents in the United States. These changes include a revision in the manner in which the term of U.S. patents is calculated as well as the introduction of a simplified and low-cost provisional patent application. The provisional application can be used to secure an early invention date, without triggering the beginning of the patent term (which now commences on filing of a nonprovisional U.S. patent application). The amended U.S. law also makes it possible for inventors based outside the U.S. to prove the date of invention by relying on acts of invention outside the territory of the United States. Since U.S. patents are awarded to the first party to invent the subject matter, this change is of great importance to both U.S. and non-U.S. inventors and businesses. Non-U.S. based inventors in particular must now maintain adequate invention records in the same way that U.S. inventors have been required to for more than 200 years.

INTRODUCTION Patent practice in the United States was radically altered after the U.S. adopted the changes mandated by the GATT-TRIPs agreement [1]. The TRIPS agreement was an effort by members of GATT to establish minimum standards for the protection of intellectual property. The TRIPS-prompted changes to the U.S. Patent Law were signed into law on December 8, 1994, and are contained in the Uruguay Round Agreements Act (URAA) [2]. Most significantly the URAA revised the term of U.S. patents and afforded inventors working outside the U.S. rights similar to those given to inventors working within the U.S. for establishing priority of invention.

16 Title V of the URAA makes five significant changes to U.S. patent law.

They

are: •

The term of a U.S. patent, previously 17 years from the issue date, has been changed to 20 years from the earliest U.S. filing date;

•

U.S. and foreign inventors can file a simplified, lower-cost provisional application;

•

Patent term extensions are now available for up to five years if issuance of a patent is delayed by an interference proceeding, a government secrecy order, a successful appeal to the Board of Patent Appeals and Interferences, or the Federal Courts;

•

Inventors working outside the U.S. can refer to work carried out in any World Trade Organization (WTO) country in order to establish a date of invention; and,

•

The statutory definition of infringement has been broadened by giving U.S. patent holders the right to exclude others from importing infringing products into the U.S. and offering such products for sale in the U.S.

Each of these changes is discussed below and accompanied by a brief outline on how each can affect scientific and business operations.

CHANGE TO THE PATENT TERM Since the inception of the U.S. Patent Law, the term of a U.S. patent has been 17 years from the date the patent is granted. Thus, in the past, time spent prosecuting a U.S. patent application in the Patent and Trademark Office (USPTO) did not affect the term of any patent that was ultimately granted. Under prior U.S. law an inventor could (and often did) maintain an application in the USPTO for 10 or more years before the patent was granted, without compromising the full 17 year patent term starting with the grant date. The era of the guaranteed 17 year U.S. patent term ended with the enactment in the U.S. of the URAA. The patent provisions of the URAA became effective on June 8, 1995 [3]. Article 33 of the TRIPs agreement provides for a patent term of 20 years beginning on the filing date of the earliest patent application for the invention [4].

17 The URAA changes the term of U.S. patents by amending the U.S. Patent Law (35 U.S.C. § 154) to provide for a patent term of 20 years that is calculated from the date on which the application was filed in the USPTO. Determination of the effective filing date of a U.S. patent application is critical for measuring the date on which the 20 year period commences. For original U.S. patent applications filed under 35 U.S.C. §111(a), the patent term ends 20 years from the date on which the application is filed in the USPTO. For continuing or divisional applications (i.e. those claiming the priority of an earlier co-pending U.S. patent application), the patent term ends 20 years from the filing date of the first U.S. or international application from which priority is claimed under 35 U.S.C. §120, 121, or 365(c) [5]. The term of a patent issuing on a continuing application is now measured from the filing date of the earliest application in the chain leading up to the patent grant, regardless of what type of continuing application is filed (continuation, continuation-in-part, or divisional) [6]. The URAA also includes transitional provisions. Thus, for U.S. patents issued before June 8, 1995, or patents granted on U.S. patent applications on file prior to June 8, 1995, the patent term is the longer of 17 years from the grant date, or 20 years from the earliest claimed U.S. filing date. In order to determine the effective life of a U.S. patent, it is now necessary to ascertain the earliest filing date (application date) which the patent claims. The claimed initial filing date must be checked to determine if it is before or after June 8, 1995. With this information in hand, the expiration date of the patent can be ascertained from the face of the patent document, but only in the absence of certain proceedings such as an interference or a successful appeal during the pendency of the application. If there was such a proceeding, it or a portion of it may have tolled the running of the 20 year term for up to five years. Thus, determining the expiration date of a patent can be quite complex. The following examples illustrate how the new provisions operate. patents or patent applications on file prior to June 8, 1995 [7]. •

First, for

Patent application A is filed on June 10, 1976 and issues on June 10, 1978. Since the earliest application on which the patent was granted was filed prior to June 8, 1995, the patent term is computed using the transitional provisions of the URAA. Patent A expires on June 10, 1996, 20 years from the filing date. The guaranteed patent term of 17 years from the date of issue (June 10, 1995) is shorter than the term computed by reference to the filing date.

18

•

Patent application B is filed on June 1, 1995, and issues on June 10, 2000. Patent B expires on June 10, 2017, 17 years after the date of issue. The alternative, 20 years from the date of filing, is earlier (June 1, 2015).

For patent applications filed on or after June 8, 1995, the following examples illustrate how the new law will operate. •

Patent application C is filed on June 8, 1995, and issues on June 8, 1996. Patent C expires on June 8, 2015, 19 years after issuance. The patent term is 20 years from the filing date of the earliest U.S. application from which the patent claims priority.

•

Patent application D is filed on June 8, 1995, but issues on June 8, 1999. Patent D expires on June 8, 2015, 16 years after issuance. The patent term is computed by reference to the filing date.

•

Parent patent application E is filed on May 1, 1994, and issues on May 1, 1998. A first divisional application. E l , is filed on June 7, 1995, and issues as patent El on December 1, 1996. A second divisional application, E2, is filed on August 1, 1995, and issues on February 1, 1999. (a)

The parent patent E expires on May 1, 2015, 17 years from the date of issue. This patent is entitled to the transitional provisions.

(b)

The first divisional patent. E l , expires on May 1, 2014, 20 years from the filing date of the application on which the parent patent was granted. The filing date of the parent application was the earliest effective filing date to which the El patent is entitled.

(c)

The second divisional application, E2, also expires on May 1, 2014, 20 years from the filing date of the parent application but only 15 years ( + 3 months) from its issuance date. The term of the E2 patent begins on the filing date of the earliest application (i.e. the grandparent E application) from which the E2 patent claims priority.

Patent term extensions for up to 5 years can be granted if issuance of the patent is delayed as a result of: 1. a patent interference; 2. a government secrecy order; or, 3. a successful appeal (e.g. from a rejection of the patent application by the USPTO) to the Board of Patent Appeals and Interferences or to the Federal Courts [8]. In the

19 case of appellate review, the extension period begins on the date a notice of appeal is filed and extends to the date (no more than five years later) on which a final decision is rendered in favor of the patent applicant. This extension is reduced by any time attributable to appellate review that falls within the three years immediately following the U.S. filing date, or any time during which the patent applicant did not act with due diligence as determined by the Commissioner of Patents. An additional 5-year maximum extension (not related to the URAA) is also available for delays resulting from premarket regulatory review of a product, such as a drug product [9]. These provisions cover only some common types of delay, and they do not address all of the possible situations that can delay grant of a patent. The absence of a more general remedy for delays, coupled with the 20-year patent term from filing creates a new sense of urgency among U.S. patent applicants and patent practitioners to advance prosecution of pending U.S. patent applications as rapidly as possible. Consider these guidelines for obtaining U.S. Patents with maximum effective term: Complete all non-provisional U.S. patent applications (i.e. file all formal papers such as declarations, power of attorney, and the like) along with, or within sixty days after the filing of a patent application, in order to avoid delaying the commencement of substantive examination. Respond promptly to Patent Office Actions and avoid requesting extensions of the response time; Where appropriate, use telephone calls and/or personal interviews with U.S. Patent Examiners to speed prosecution; File separate applications covering closely related subject matter together in order to obtain the same filing dates; Pay the Government Issue Fee as soon as possible after receiving a Notice of Allowance for a pending patent application that has been found to be allowable. In certain cases, where this makes business sense, enter the National Stage of an International (PCT) application early: the entire international stage is subtracted from the 20-year term.

20 TAKE FULL ADVANTAGE OF THE 20-YEAR TERM The URAA created a new form of U.S. patent application, the so-called "Provisional Patent Application" [10]. The Provisional Application is informal, simple, and inexpensive to prepare and file, but creates significant rights in favor of the applicant. Most important, the Provisional Application can establish an invention date in the U.S., but its filing does not start, and its one-year provisional application term does not count as part of the twenty year patent term. The Provisional Application includes only a specification and drawings; no patent claims are required. In addition, formal papers such as an oath, declaration, or Information Disclosure Statement are not needed. The government filing fee for a Provisional Application is U.S.$150 ($75 for a small entity). Provisional Patent Applications are not examined, but are simply retained by the USPTO. One year after filing, the Provisional Application is deemed to have been abandoned without the possibility of revival, unless a complete (non-provisional) application has been filed in the USPTO prior to the expiration of this time period. The Provisional Application is a form of "national" priority document intended to place U.S. inventors in the same position as foreign inventors (who can rely on their national patent applications to establish priority of invention in the U.S. without starting the twenty year from filing U.S. patent term. The U.S. Provisional Application can serve as the basis for claiming priority (under the Paris Convention) for purposes of filing foreign patent applications (outside the U.S.). The Provisional Application can also serve as the basis for establishing a date of invention in the U.S. Because the 20-year patent term starts from the filing date of the complete (nonprovisional) U.S. application, mat the filing date of the Provisional Application, the Provisional Patent Application effectively postpones the start of the patent term [11]. Why file a Provisional Application? A Provisional Application: •

Provides a mechanism whereby patent applications can enjoy the benefit of a priority year without starting the clock on the 20-year from filing U.S. patent term;

•

Can assist the applicant to prove an early date of invention that may in turn be useful in establishing senior party status in an interference proceeding in the U.S., or in establishing a filing date in other countries which follow the firstto-file principle;

21 •

Permits examination of a patent application to be deferred for up to one year, allowing for time to raise capital, or to continue research and acquire additional supporting data.

Since the Provisional Application will not be examined, the grant of a patent will be postponed for one year. This may in certain cases be an advantage, in other cases a disadvantage. In addition, filing of the Provisional U.S. patent application commences the convention priority year. That is, the Provisional Application can serve as the basis for claiming convention priority and for filing foreign patent applications. This can be of strategic importance because almost all jurisdictions other than the U.S. award the patent to the first to file, not the first to invent.

DATE OF INVENTION - 35 U.S.C. § 104 The URAA makes it possible for the first time for non-U. S. inventors to establish a date of invention using the same procedures as inventors working in the U.S. The date of invention is important not only in the effort to obtain a patent (an early date of invention defeats a competitor's later activities) but also in establishing that a competing inventor is not entitled to a patent for the invention. Under U.S. Law, the patent for an invention is awarded to the first patent applicant to make the invention, rather than the first person to file a patent application for the invention [12]. This has been the case since the inception of the U.S. Patent Law. U.S. practice contrasts sharply with almost every other jurisdiction in which the patent is awarded to the first party to file a patent application for an invention, i.e., in the U.S. it is a race to the invention, whereas elsewhere it is a race to the Patent Office. Under U.S. Law, the act of "invention" has two elements, conception and reduction to practice and the patent for an invention is granted to the first party to conceive the invention and diligently reduce it to practice. Conception is the mental part of inventive activity and involves the formulation and disclosure by the inventor of a complete idea for a product or process [13]. The idea must be sufficiently complete to permit a person of ordinary skill in the art to reduce the concept to practice [14].

22

Reduction to practice can be either "constructive" (achieved by filing a patent application) or "actual" ("the inventor constructs a product or performs a process that is within the scope of the patent claims and demonstrates the capacity of the invention to achieve its intended purpose") [15]. Under prior U.S. law, an inventor could not rely on activity done outside the U.S. to establish a date of conception or reduction to practice. Prior to the URAA the only route available to foreign inventors for establishing a date of invention in the U.S. was either to (a) introduce the invention into the U.S., (b) rely on the filing date of their home country patent application, or (c) rely on the filing date of their U.S. patent application. This situation was considered unfair to non-U. S. inventors because in most cases it precluded use of work carried out in their own laboratories to establish a date of invention, while inventors working in the U.S. could and did refer to such work for this purpose. NAFTA changed this policy for inventors working in Mexico and Canada by enabling an applicant or patentee to rely on activities in a NAFTA country to prove a date of invention in proceedings before the United States Patent and Trademark Office (USPTO), the Courts, or before any other competent authority [16]. Article 27.1 of the GATT-TRIPs agreement extended this protection to inventors working in any member country of the World Trade Organization (WTO) and provided that "patents shall be available ... without discrimination as to the place of invention" [17]. As a result of the URAA, section 104 of the U.S. Patent Law was amended to permit inventors in WPO member countries to establish a date of invention by reference to acts of inventions carried out in such countries. Because these changes with respect to establishing acts of invention have come into force only recently (from January 1, 1996 under the URAA), it is only recently that applicants have attempted to establish a date of invention for a U.S. patent application based on work carried out in either a NAFTA or WTO member country. These policy changes have already had a dramatic impact in the patent arena outside the U.S. in two ways: First, inventors and scientists working outside the U.S. will have to maintain invention records in the same way as U.S. inventors have been required to for more than 200 years. Second, a large increase is likely in the number of patent interferences, i.e. proceedings employed by the USPTO to determine which one of two competing applications is entitled to the patent for an invention. Patent interferences are conducted at the USPTO, and have as their sole objective to determine priority of invention, a process often lasting for years. In an interference proceeding how does an inventor prove that he or she was the first to invent? In most instances by producing a written record of works on the

23

invention from a notebook or similar journal. The change brought about by the URAA now highlights for non-U.S. inventors the importance of maintaining adequate notebook records. The best time-tested approach is for an inventor to keep careful notes of work carried out on the invention in a bound notebook, in which each page is dated and signed. Significant developments should be witnessed by a third person signing and dating in writing in the notebook that he or she read and understood the development. A full treatment of the means that could and should be adopted to prove work on an invention is beyond the scope of this paper. However, some highlights of the procedures to employ in maintaining a laboratory notebook that will be useful to establish priority of invention in the event of a dispute, are outlined below [18]: •

Use as the work record a permanently bound notebook with consecutively numbered pages;

•

Enter ideas, calculations and experimental results into the notebook as soon as possible, preferably on the same date they occur, so that the laboratory notebook becomes a daily record of the inventor's activities;

•

Make all entries in permanent black ink and as legible and complete as possible. Abbreviations, code names or product codes should not be employed unless clearly defined;

•

Draw a line through all errors, do not erase;

•

Entries should be made without skipping pages or leaving empty spaces at the bottom of a page;

•

Pages should never be torn or removed from the book;

•

Have each page signed by the inventor and dated at the time an entry is made. No entry should be changed or added to after signature. If there is new or additional information or corrections, a new entry should be made;

•

Have each page periodically witnessed, signed and dated by a third party who understands the inventor's work but who is not a contributor to the project. This should preferably occur weekly and certainly no less frequently than bimonthly;

24

•

Completed notebooks should be indexed and stored in a safe location and, thereafter, handled in accordance with the company's established record retention and destruction policy for such documents. Never:

•

Make illegible entries (they are worthless);

•

Have unsigned or undated pages (they are almost worthless);

•

Have notebook pages which have not been witnessed (they are almost as bad as unsigned and undated pages). Avoid:

•

Waiting a long time between entry of the information and signing of the pages on which the entry is made;

•

Consecutive notebook pages which are not dated in chronological order;

•

Missing notebook pages, erasures and deletions.

By following this careful record keeping practice an inventor has a better chance of having the ammunition needed to win a patent interference proceeding [19]. In light of the URAA, inventors working in Europe, Asia and elsewhere can now refer to work carried on outside the U.S. to establish an invention date. The number of inventors who may seek to provoke interference proceedings in the USPTO and have a good chance of prevailing has thus vastly increased [20].

SCOPE OF INFRINGING ACTIVITY The definition of U.S. patent infringement has been expanded by the URAA. Prior to enactment, if a party imported into the U.S. a product covered by a U.S. patent, neither the importation, nor the offer of the product for sale constituted patent infringement. The URAA adds to 35 U.S.C. § 154 the right to exclude others from "offering for sale" or "importing into the United States" an invention that is covered by a U.S. patent. In the case of process patents, the patent holder is given the right to exclude others from "offering for sale" in the U.S. products made anywhere in accordance with the U.S. process patent. The U.S. Patent Law (35 U.S.C. § 271)

25 now permits patent holders to sue for infringement in the event an infringing invention is offered for sale in, or imported into the U.S. [21]. Because importation or offering for sale now constitute acts of infringement, it is easier for a patent holder to seek relief against infringing products that are imported into the U.S.

CONCLUSION The GATT-TRIPs agreement has already had a major impact on U.S. inventors, scientists and businesses. Patent attorneys, businessmen, engineers and scientists must continue to remain aware of how these changes will affect them. To review: •

The term of a U.S. patent is now 20 years, measured from the U.S. filing date of the earliest U.S. patent application for an invention;

•

U.S. patent applicants can file a simplified, low cost Provisional Application;

•

The date of invention can now be established by reference to activity outside the U.S.; and

•

The Statutory definition of Infringement has been broadened to include the right to exclude others from importing infringing products into the U.S. and offering products for sale in the U.S.

References 1. 2. 3.

4.

General Agreement on Tariffs and Trade (GATT) - Trade Related Aspects of Intellectual Property Rights (TRIPs). Uruguay Round Agreements Act, Pub. L. No. 103-465, 108 Stat. 4809, enacted on December 8, 1994. See, U.S.C. §154(A)(2) Term.-"Subject to the payment of fees under this title, such grant shall be for a term beginning on the date on which the patent issues and ending 20 years from the date on which the application was filed in the United States..." See, Agreement on Trade-Related Aspects of Intellectual Property Rights, Including Trade in Counterfeit Goods. WTA/GATT (1994) (http://ra.irv.no/trade_law/documents/freetrade/gatt/art/iialc. htmP: Article 33: Term of Protection... "The term of protection available shall not end before the expiration of a period of twenty years counted from the filing date."

26 5.

6. 7. 8. 9. 10. 11.

12.

13. 14. 15. 16. 17.

18. 19.

20. 21.

See, 35 U.S.C. § 154(1)(2)-... "or, if the application contains a specific reference to an earlier filed application or applications under section 120, 121, or 365(c) of this title, from the date on which the earliest such application was filed." See, Charles E. Van Horn, Effects of GATT and NAFTA on PTO Practice, 11 J. Patent and Trademark Office Society 231, 239-40 (1995). See, Martin Voet, Rod Berman, and Michael Gerardi, Patent practitioners - don't let GATT get you, 47 Managing Intellectual Property 20, 20-21 (1995). 35 U.S.C. § 154(b). 35 U.S.C. § 156. 35 U.S.C. §lll(b). See also, 35 U.S.C. § 111(b)(6): This section provides for an alternative procedure for filing a provisional application by allowing a complete application to be converted to a provisional application within 12 months after filing. The conversion is effected by petition to the Commissioner. See generally, 35 U.S.C. § 102(a), (e), and (g): The prior art provisions all provide that a person is entitled to a patent unless there is some evidence of prior invention by another before the date of invention by the applicant. Chisum on Patents (1997), 3:10.04. Chisum on Patents (1977), 3:10.04. Chisum on Patents (1997), 3:10.06. See, Charles E. Van Horn, Effects of GATT and NAFTA on PTO Practice, 11 J. Patent and Trademark Office Society 231, 233. (1995) See, Agreement on Trade-Related Aspects of Intellectual Property Rights, Including Trade in Counterfeit Goods, WTA/GATT (1994) rhttp://ra.irv.no/tradeJaw/documents/freetrade/gatt/art/iialc.htmP: see also, 35 U.S.C. § 104(a)(1), which has now been amended to read: "In proceedings in the Patent and Trademark Office in the courts, and before any other competent authority, an applicant for a patent, or a patentee, may not establish a date of invention by reference to knowledge or use thereof, or other activity with respect thereto, in a foreign country other than a NAFTA country or a WTO member country, except as provided in sections 119 and 365 of this title." See, http://www.darbylaw.com/note.html. See, Jerry Voight, Succeeding in US patent interference, 57 Managing Intellectual Property 33 (1996): for a complete description of that uniquely American proceeding call the patent interference. In addition, the cost and complexity of interferences is likely to increase because of the need to translate laboratory notebooks and other documents into English. 345 U.S.C. §271.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

27

Flavomet: a database of aroma compounds based on odor potency in natural products H. Am^ and T.E. Acree"^ *Swiss Federal Horticultural Research Station ^Department of Food Science & Technology, Cornell University, Geneva NY 14456

Abstract For thirty years gas chromatography-olfactometry (GCO) has been an essential tool in the determination of the most potent odorants in natural products. It is presimaed that these odorants create the olfactory precepts t h a t affect memory, attention and behavior. In order to facilitate the identification of odorants by GCO, a database was created firom the published Uterature on odorants detected by quantitative GCO. Software was developed to generate hypertext markup language (HTML) files that organized and displayed the data extracted firom files exported firom a standard database program. The resulting World Wide Web (WWW) site displays odorant retention indices in both Kovats and ethyl ester units, associated aroma descriptor, a protein data bank (pdb) structural file, molecular weight, CAS registry number and published source. The specific descriptor used by the authors to describe the quality of the detected odorant was assigned to a genus based on the ASTM D-66 categories of food odors extended to include non-food smells.

Web Design Flavomet data is stored and maintained in a database file where it can easily be accessed, modified and sorted. To update, the Flavomet data is selected, sorted and exported into a flat text file. The flavorEngine is a program that generates n + 5 HTML files from the export files where n is the number of compounds to be listed. The present form of Flavomet has 346 compounds listed. Its permanent WWW address is:

http://www.nysaes.comell.edu/flavornet

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

29

Beverage Flavor Emulsion - A Form of Emulsion Liquid Membrane Microencapsulation Chee-Teck Tan International Flavors & Fragrances, Inc. New Jersey 07735 USA

Abstract Beverage flavor emulsions are a unique class of emulsion. They are required to be stable in concentrate form and also in the diluted finished beverage. Because the addition of weighting agents is regulated by governments, the density of the flavor oil plus weighting agent cannot reach that of the sugar solution in the beverage. Based on the Stokes' law, these emulsions will not be stable in the sugar solution due to the difference of the density of the oil phase and the water phase containing sugar. This study shows a film of gum arabic or modified starch is formed on the surface of oil droplets and encapsulates the oil droplets. At the proper size of oil droplet, the film contributes enough weight to make the emulsion oil droplets stable. The formation of interfacial film on an oil droplet was studied under a microscope. In this t3rpe of emulsion, oil droplets are encapsulated by emulsion liquid membrane. The encapsulated flavor oil in the droplets is released when the beverage is consumed.

1. INTRODUCTION Among all the soft drinks consumed worldwide, citrus flavors are the most popular of all flavors, and orange flavor is the favorite. These citrus flavors are composed of citrus oils extracted from the rinds of the fruits. Like all essential oils, citrus oils are not water-soluble. For this reason, they can not be used directly in the soft drink as the oil is not miscible with sugar solution. Two common methods are used to utilize citrus oil to flavor the beverages. The first method involves separating out the water-soluble fraction of the essential oils by extraction and distillation, and the second method is to convert the oil into a water dispersible emulsion such as a beverage flavor emulsion. In this paper we will discuss the basics of oil droplet stability in the emulsion including a microscopic study of gum arabic and starch film formation on the droplets in the emulsion.

30

1.1 COMPOSITION OF BEVERAGE EMULSIONS Beverage emulsions are a unique class of emulsions. They are different from other food emulsions in that they are consumed in a highly diluted form rather than in their original concentrate form. For beverage emulsions, the flavor oils, such as orange oils, are first prepared into an emulsion concentrate, which is later diluted in sugar solution to produce the finished beverage. In soft drinks, the beverage emulsion is diluted several hundred to several thousand times to provide flavor, color, and a cloudy appearance for the beverage. A beverage emulsion must be stable in both the concentrate and diluted forms. The requirements of the beverage industry are that they should be stable for at least six months or longer. Beverage flavor emulsions are oil-in-water (OAV) emulsions. The oil phase consists of flavor oils and weighting agents. Flavor oils are usually composed of citrus oils for citrus flavor. Orange oils are most commonly used. Weighting agents are used to increase the density of the total oil phase. Weighting agents are materials soluble in oil and have no flavor of their own but should have density higher than the flavor oils. However, there are government regulations limiting the amount of weighting agents that can be used in the beverages. The water phase usually consists of various types of hydrocolloids, citric acid, preservatives, and colorings. The most commonly used hydrocolloids are gum arabic and specially modified starch. Artificial emulsifiers or surfactants are not used in beverage emulsions. Since there are few natural emulsifiers available for use in soft drinks, the emulsifying function in the emulsion depends on gum arabic [1-3] or modified starches [4].

1.2. THE STABILITY PROBLEM OF BEVERAGE EMULSIONS The instability commonly observed in both the emulsion concentrate and the finished soft drinks can be described as: a) creaming, b) flocculation, and c) coalescence. These phenomena leading to instability are described as follows: a) Creaming - Creaming is the separation of one emulsion into two emulsions. The upper portion of the emulsion is richer in oil phase than the original emulsion, and the lower portion of the emulsion is richer in the water phase than the original emulsion. Because the separation is gradual, there is no well-defined separation line between the portions in the emulsion. b) Flocculation ~ Flocculation occurs when oil droplets form aggregates or clusters without coalescence but still retain their original identities. Although the flocculation generally changes the physical properties of the emulsion, the particle size distribution remains unchanged. In the soft drink system, the oil droplet aggregates gradually rising to the neck of the bottle and form a ring. The rate of the oil droplet aggregates rising to the top of the bottle is accelerated in systems in which the density difference of the aggregated oil droplets from the water phase is sufficiently large. However,

31 because the interaction forces between the droplets are weak, the aggregates can be readily redispersed. c) Coalescence - In this stage, there is localized disruption of the sheaths around neighboring droplets of the aggregates, and the oil droplets merge together to form a large droplet. This leads to a decrease of the number of oil droplets and eventually causes the breakdown of the emulsion in the soft drink system. The most critical criterion of the quality of a beverage emulsion is its stability in the diluted state as in the soft drinks. In soft drinks, the emulsion concentrate is dispersed in sugar solution at a ratio varies from 1:300 to 1:2000, depending on the flavor oil concentration in the emulsion. At this stage, the emulsion concentrate is actually dispersed in a second water phase, which has a different composition from that of the original water phase of the concentrate. In this new water phase, it usually contains 10 to 12% of sugar with the exception of diet beverages where artificial sweeteners are used in place of sugar. Because of the finished beverages contain 10 - 12% sugar, the major cause of instability is the density difference between the oil phase of the emulsion and the sugar solution of the beverage which is the new water phase. This is clearly demonstrated in the Stokes' law:

2gr'(Prp2) V =

(1)

In Eq. (1), V is the rate of oil droplets separation (creaming), g is the acceleration of gravity, r is the droplet radius, p^is the density of the oil phase, p2 is the density of the water phase, and rig is the viscosity of the water phase. Stokes' law shows that the velocity of droplet, v, is directly proportional to the density difference between the oil phase and the water phase, and the square of the radius of the droplet. It is also inversely proportional to the viscosity of the water phase r\^. The equation clearly shows that the approaches to make a stable emulsion in beverage are to reduce the density difference between the oil phase and the water phase to as close to zero as possible, and to make the particle size as small as possible. The viscosity of the water phase is related to the sugar concentration in water and is considered as a constant. In a typical orange flavor beverage, orange oils of the emulsion typically have a density of 0.845 g/ml. The sugar solution of beverage has a density range from 1.038 to 1.047 g/ml for 10% and 12% sugar solutions, respectively. In this case, the oil phase density, p^is smaller than that of the water phase, pg According to Stokes' law, if the resulting sign of v is negative, creaming or ringing will occur. Because of the density difference, weighting agents are needed to adjust the density of the oil phase to be as close as possible to that of the sugar solution in order to achieve good stability.

32

Four weighting agents are commonly used by the soft drink industry. They are ester gum (density = 1.08 g/ml), SAIB (sucrose acetate isobutyrate, density = 1.15 g/ml), dammar gum (density = 1.05 g/ml), and BVO (brominated vegetable oil, density = 1.33 g/ml). The usage of these weighting agents in the beverages are regulated differently in different countries. Brominated vegetable oil is the highest density weighting agent and was the first one used to increase the density of orange oils since 1940. The permission of using BVO was withdrawn in the UK in 1970, and a limitation of 15 ppm of its use in the finished beverage was set for the USA and many other countries. The maximum permitted limits of the other newer weighting agents in beverages are also regulated. For example, the maximum usage of ester gum is set at 100 ppm and no SAIB permitted in the USA. Considering the regulations on the uses of weighting agents and using ester gum in orange oil in a typical orange flavored beverage as an example. The orange oil could be weighted to have density to about 0.95 g/ml in order to have no more than 100 ppm of it in the beverage. According to Stokes' law, an oil phase with density of 0.95 g/ml will separate quickly in a water phase with density of 1.05 g/ml. In the preparation of orange flavored beverage, we were able to prepare emulsion stable in both the concentrate and diluted forms when the oil droplets were made to an optimal size. That is, the droplet size is so small that the gum arabic or starch film formed at the interface will add weight to the droplet. With this additional weight, it will make the density of the total droplet so close or equal to that of the water phase and make the emulsion stable. We assumed the film thickness remains constant over the range of oil droplets particle size. Therefore, the smaller the droplet, the more weight gain will be contributed from the film as illustrated in Figure 1. In actual preparation

0.05 I ' " ! " Gum Arabic layor

0.05 I'Ti - Gum Arabic layor

oil droplet density = 0.95 g/ml gum arabic layer density = 1.10 g/ml droplet A density = 0.987 g/ml droplet B density = 1.037 g/ml

Figure 1. Weight contribution to orange oil droplets of different sizes from the gum arabic interfacial membrane.

33

of orange oil flavored emulsions this assumption has been proved to be true for achieving the emulsion stability.

2. INTERFACIAL FILM FORMATION STUDY In the literatures, it was reported that gum arable formed film on paraffin oil [5], and on tetradecane droplets [6]. Since these studies used pure liquid hydrocarbon as the oil phase, we would like to see if gum arable will form film on orange oils as it is one of the most important flavor oils in beverage. The following experiments were made to study some physical chemical properties of the films formed at the interface of orange oil droplets in gum arable and modified starch solutions. 2.1. Materials Because gum arable and modified starch are most commonly used in the preparation of beverage emulsions, water phases were prepared from these two hydrocolloids, separately. The gum arable used was spray dried, low bacteria, beverage grade supplied by Meer, Inc., New Jersey. The modified starch. Purity Gum 1773, was supplied by National Starch and Chemical Co., New Jersey. It is an octenyl succinated modified starch. For preparing the water phases, 20 percent solution of gum arable and 14 percent solution of modified starch were prepared separately with deionized water. For the oil phase, cold pressed Florida orange oil was used. Ester gum 8BG (Glyceryl abietate) obtained from Hercules, Inc., Wilmington, Delaware was used as the weighting agent. Ester gum was added into the oil in order to simulate the real beverage emulsion condition. It was added to the oil at the weight ratio of 50:50 and mixed at room temperature until completely dissolved. The density of the weighted orange oil was 0.95 g/ml. 2.2. Methods In this study, a glass ring of 1.5 cm in diameter x 1 cm height with a hole of about 0.5 mm diameter drilled at the middle of the side of the ring was prepared. The side hole was made to permit the insertion of a micro S3n:*inge. This ring was then set and glued on a microscopic slide. The study was carried out by placing the glass ring directly under the objective lens of the microscope equipped with a camera. The camera was made interchangeable with a video camera for making sequential study. After the well was filled with the gum solution, a drop or orange oil was introduced in the oil from the micro syringe. The film formation on the interface of the oil droplet was observed under the microscope and recorded by the camera or the video camera.

34

2.3. Results In gum arabic solution, after the oil droplet had been aged for about 30 minutes in the gum solution, a skin-like film was observed on the droplet. When the oil droplet was gradually pulled back to the syringe, the film exhibited as a small collapsed balloon (Figure 2A and 2B). From an accidental ejection of an aged oil droplet into the gum solution, a sausage like droplet was seen as in Figure 3. The oil was very well encapsulated by the gum arabic film, which performed like a sheath and kept the oil from going back to the lowest surface energy level of a spherical droplet. These micrographs indicate the gum arabic film is rigid and strong. In the octenyl succinated modified starch solution, the film formation at the interface of the oil droplet was not as easily observed visually under the microscope as in the gum arabic solution by the same technique of withdrawing the oil back to the S5n:*inge (Figure 4A and 4B). However, the film formation was observed as a hazy film on the oil droplet when tilting the light at an angle after the oil droplet had been aged for about 30 minutes. The starch film seemed to be much more elastic than the gum arabic film. During withdrawal of the oil droplet back to the syringe, the film on the oil droplet shrank, as the oil droplet became smaller. A wrinkled film was seen only at the very end of withdrawal of the oil droplet back to the syringe. By gradually ejection, several oil droplets were dispersed into the starch solution. After they were aged together for several hours no coalescence occurred among these particles. This indicated that there was film formed on each droplet and thus preventing coalescence and the formation of a large oil droplet. This sequence was recorded in Figure 5. These droplets were flat topped because they were touching the glass cover slide on top of the glass ring cup.

3. DISCUSSION The difference observed in the characteristic of films formed by gum arabic and modified starch are apparently due to the physicochemical nature of the two hydrocoUoids. The precise mode of action of gum arabic and modified starch in stabilizing flavor oil emulsions is still far from being fully understood. It has been demonstrated that in gum arabic it is the protein-containing high molecular weight fraction, which adsorbs most strongly at the oil-water interface, and is probably mainly responsible for the emulsifying and stabilizing properties of the gum. The modified starch used in this study is a starch derivative with balanced lipophilic and hydrophilic groups on the starch molecules [7]. It is a low viscosity octenyl succinated starch. It seemed to behave very much like an emulsifier besides as a stabilizer. Orange flavor emulsions made with gum arabic and modified starch, if they were properly formulated and processed, are very stable. There are almost no particle size change in storage during a six-month period as analyzed by Coulter counter. Model LS-130. A typical orange oil/gum arabic emulsion had a mean particle size of 0.364 |Lim when fresh and 0.410 |Lim after aged for six months at

35

A

B

Figure 2. Orange oil droplet in gum arabic solution: A) Full oil droplet; B) Oil droplet shown with wrinkled membrane after oil had been partially withdrawn.

Figure 3. A sausage like oil capsule formed by injecting an aged oil droplet in gum arabic solution.

36

Figure 4. Orange oil droplet in sodium octenyl succinated starch solution: A) Full oil droplet; B) Oil droplet shown with wrinkled membrane after oil had been partially withdrawn.

Figure 5. Orange oil droplets with membrane aged in sodium octenyl succinated starch solution.

37 Differential Volume %

A

OrangeOil/Gum Arabic Emulsion, 2 days old.

Particle size - Mean: 0.264 urn; Median: 0.208 urn

O.c

0.6

1

10

20

40

oO

100

200

^100 600

lOOO

Particle Diameter (urn)

Differential Volume %

B

Orange Oil / Gum Arabic Emulsion, 6 months old.

Particle size - Mean: 0.320 pm; Median: 0.216 |.im

IrmiTTn 0.4

0.6

1

4 6 10 20 Particle Diameter (|.im)

40

60

100

200

QOO 600

lOOO

Figure 6. Particle size distribution histograms of an orange oil/gum arabic emulsion stored at room temperature: A) 2 days old; and B) six months old.

38 Differential Volume %

Orange Oil / Modified Starch Emulsion, 2 days old.

Particle size - Mean: 0.364 ^im; Median: 0.358 urn 0.4 0.6

1

40

60

100

200

400 600

1000

Particle Diameter ().im)

Diflerential Volume %

Q

Orange oil / Modified Starch Emulsion, 6 months old.

Particle size - Mean: 0.410 (.im; Median: 0.401pm

0.2

0.4

0.6

1

40

60

100

20':'

400 600

1000

Particle Diameter (|.im)

Figure 7. Particle size distribution histograms of an orange Oil/modified starch emulsion stored at room temperature: A) 2 days old, and B) six months old.

39 ambient condition. A typical orange oil/modified starch emulsion had a mean particle size of 0.264 jim when fresh and 0.320 jim after six months aging. In both the gum arabic and the modified starch emulsions there is only very slight change in the particle size. The changes are so small and the effect to the emulsion stability in beverage is negligible. The particle size distribution histograms of these two emulsions when freshly prepared and after six months in storage are shown in Figure 6 and 7. In the preparation of beverage using the emulsion concentrate, the emulsion concentrate is diluted in sugar solution 300 to 2000 times depends on the flavor strength in the emulsion. It is equivalent to dispersing the emulsion oil droplets into a new water phase. This new water phase is a 10 to 12 percent sugar solution with no gum arabic or modified starch in it. In a recent study of a tetradecane/gum arabic emulsion, it was reported that the gum arabic film adsorbed on the tetradecane oil droplets at the oil-water interface is thick and strong. The film is very resilient with respect to desorption by dilution of the aqueous phase [6]. Since orange oil is an essential oil and tetradecane is a saturated hydrocarbon the following study was conducted to find out whether the gum arabic and modified starch films adsorbed on the orange oil droplets have the same property as gum arabic film on saturated hydrocarbon. Because the main concern of the flavor and beverage industry is the stability of the emulsion in the finished beverage, this study was carried out by diluting the emulsions in 12 percent sugar solution at the dilution ratio of 1 to 600. The oil droplet particle size change was determined on the emulsion concentrate, freshly diluted within 2 hours, and after aged from 1 day to 90 days. The results are shown in table 1 and Figure 8. The particle size measurements show that the droplet size became slightly larger than the original emulsion concentrate particle when the emulsion concentrate was first diluted in sugar solution. Both gum arabic and modified starch emulsion particles behaved the same. The particle size became larger when the oil droplets were first introduced into the sugar solution indicates that the film on the oil droplets swelled in the sugar solution. Apparently, in the emulsion concentrate there were high concentrations of gum arabic or starch and it makes the film at the interface more compact in structure. However, after aged for one day the particle size gradually became smaller. It may be explained that a small amount of the loosely attached outer layer of the film materials on the droplet sloughed off into the sugar solution. The rate of slough off or desorption was faster in the first ten days and became stabilized about after 40 days for gum arabic, and 50 days for starch. From then on, until the end of this study, the 90 days aged beverages, showed no particle size change. From observing the aged beverages on shelf there was no creaming or oil separation in the bottles. It may be concluded that there are stable films formed on the oil droplets, and the films provide two functions to the oil droplets: 1) providing additional weight to the oil droplets, and 2) preventing oil droplets from coalesce to form larger particles.

40

Table 1 Oil droplets particles size change during aging in sugar solution

Days in Solution

Gum Arabic Emulsion fim

0 (Emulsion) 0.1 1 5 10 15 20 25 30 40 50 60 90

V.f

Starch Emulsion |im

0.563 0.577 0.593 0.589 0.548 0.558 0.548 0.551 0.549 0.529 0.533 0.532 0.539

0.325 0.346 0.361 0.343 0.345 0.353 0.340 0.339 0.333 0.325 0.229 0.300 0.310

•

0.6 •

^"^

•

M

c o E QT

W

-*

0.3 •

Ci

u t Q.

—•—Modified Starch Emulsion • • 0

Gum Arabic Emulsion

•

40

50

60

70

Age in Sugar Solution, days

Figure 8. Oil droplet particle size change during aging in sugar solution

41

4. EMULSION LIQUID MEMBRANE ENCAPSULATION On the interface of orange droplets, the film can be called as a membrane. A membrane can be viewed as a semipermeable barrier between two phases. This barrier can restrict the movement of molecules across it in a very specific manner. The membrane must act as a barrier between phases to prevent intimate contact [8]. A typical emulsion is produced from mixing two immiscible phases with a surfactant. This emulsion is then dispersed in a continuous phase it produces also emulsion liquid membrane [9]. Emulsion liquid membrane (ELM) are double emulsions formed by mixing two immiscible phases and then dispersing the resulting emulsion in another continuous phase under agitation. The applications of emulsion liquid membranes have included selective recovery of metal ions, separation of hydrocarbons, removal of trace organic contaminants, and encapsulation of enzymes or whole cells. The beverage flavor emulsion is definitely fit to be classified as an emulsion liquid membrane. In actual practice of the flavor and beverage industry, the flavor oil is microencapsulated by emulsion liquid membrane. Once the flavor emulsion concentrate is made, it is then dispersed in another continuous phase, the sugar solution, to become the beverage. When the beverage is consumed, the flavor in the liquid membrane encapsulation is released by the contact with the enzymes in the mouth. Therefore, we are calling the beverage flavor emulsion is a type of liquid membrane microencapsulation. 5. ACKNOWLEDGMENT The author deeply appreciates the constructive comments on the preparation of this paper by Dr. Lewis G. Scharpf, and the help of Dr. Siew L. Chung for her experimental emulsion work 6.

REFERENCES

1. R.C. Randall, G.O Phillips,, and P.A WiUiams,. Food Hydrocoll., 2 (1988), 131-140. 2. E. Dickinson, V.B. Galazka, and D.M.W. Anderson, Carbohyd. Polym., 14 (1991), 385-392. 3. A.K.Ray, P.B. Bird, G.A. lacobucci, B.C. Clark, B.C., Jr. Food Hydrocolloid 9 (1995), 123-131. 4. D.M.W Anderson, and W. Weiping, Int. Tree Crops J., 7 (1991), 29-40. 5. E. Shotton,. and R.F White,. Stabilization of emulsion with gum acacia in Rheology of Emulsion (P. Sherman, ed). Pergamon Press, Oxford, England. 1963. 6. E. Dickinson, D.J. Liverson, and B.S. Murray, Food HydrocoUoids, 3 (1989), 101-114, 7. C. G. Caldwell and O.B. Wurzburg (to National Starch and Chemical Corp.), U.S. Patent 2,661,349, (1953).

42

8. R.D Noble, and D. Way, ACS Symposium Series 347, American Chemical Society, Washington, DC (1987). 9. D.L. Reed, A.L. Bunge, and R.D.Noble, In Liquid Membranes (R.D. Novle and D. Ways, eds). ACS Symposium Series 347. American Chemical Society, Washington, DC (1987).

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

43

New beverages: flavored coffee, M. Bononi, E. Lubian, S. Martello and F. Tateo D.I.F.C.A.-Sezione di Chimica Analitica Agroalimentare ed Ambientale, University of Milan, Via Celoria n.2, 20133 Milan, Italy

Abstract The attempt, some years ago, to boost the consumption of soft drinks by introducing new products on the market led to the production of tea-based flavored beverages such as: "peach-flavored tea", "lemon-flavored tea", etc. Now a plan is afoot to market coffee flavored with mint, hazelnut, toasted almond, coconut, chocolate, Irish cream, cinnamon, etc. This paper deals with enhancing technology for coffee flavoring, quality control and standardization of sensory parameters. The authors present the formulation criteria to be adopted for the best utilization of flavoring substances.

1. INTRODUCTION The decision to market tea-based drinks, or with a reference to tea in their names, represented a significant attempt to exploit a "nervine" image to increase the consumption of non-alcoholic beverages. Such a market, traditionally based on the image of "fruit juices" (orange, Ume, etc.) or on "fancy" flavors (ginger, cola, etc.) called for decisive innovation, and the tea-based beverages proved to be commercially viable in terms of consumption. A market therefore developed for beverages such as "peach-flavored tea", "lemon-flavored tea" and so on, peak consumption being reached in 1992. The research carried out in D.LF.C.A.'s Analytic Agroalimentary and Environmental Chemistry Section with regard to "analysis problems, in the characterization of aromas for peach-flavored tea drinks" was also checked didactically (1), and in 1995 F. Tateo, L. M. Di Cesare, G. Cantele and M. Bononi pubUshed a work "On the Methods of Extraction and Evaluation of the Volatile Compounds Constituting the Aroma of Tea Beverages" (2). Flavored coffees are of interest in the marketplace, particularly in Brazil where such interest in their consumption has traditionally been based on the image of products claimed to be "tonics" (drinks containing Guarana, etc.). "Cafe do Ponto S/A- Sao Paulo, Brazil" has recently launched a line of coffee (toasted and ground) with various aromas in packs for domestic use. The incentive for consumption was achieved through the parallel distribution, with "espresso

44

coffee", to the chain of bars called "Cafe do Ponto", of "mint-flavored", "toasted almond-flavored", "cinnamon-flavored", "chocolate-flavored", "coconut-flavored", "hazelnut-flavored", "Irish cream-flavored", and "walnut-flavored" coffee. The analytical experiments referred to in this paper were carried out on the "mint-flavored coffee" produced by "Cafe do Ponto". A number of qualitative parameters were evaluated in accordance with E.E.C. directives on aromas (3), and the advisability was examined of producing mint-flavored coffee by applying criteria other than those employed by the present producer. Production alternatives have been considered as regards the composition of "mint" flavoring suitable for the purpose, after evaluation of organoleptic acceptability. The series of experiments carried out have highUghted the possibility of producing a mintflavored coffee with analytical specifications conforming to E.E.C. directives (3). At the same time, the appUcation of two different extraction techniques has produced conditions useful for quality control of the aromatized product. Based on the same "aromatization" concept, Kjraft Jacobs Suchard AG (Switzerland, 8032 Zurich) has launched on the market a series of soluble powder products for making coffee with the following aromas: vanilla, amaretto, and chocolate. Preparing these drinks involves simply dissolving the powder in hot water. Of the two production trends considered, the better one seems to be that of aromatizing a ground coffee base rather than using a soluble product. This paper describes part of our research on the production of toasted, ground and aromatized coffee. The work included analytical research on products of Brazilian origin as well as the development of a proposal for new products, in keeping with the legislation of a large number of countries.

2. EXPERIMENTAL 2.1. Methods and Instruments a) Extraction of the volatiles of aromatized coffee was carried out using a Likens-Nickerson concentrator/extractor: 30 g of aromatized, toasted and ground coffee (dispersed in 500 mL of distilled water) being extracted with 30 mL of 97% n-hexane. The extraction was conducted for about six hours. After cooling to room temperature, the hexane was dried with anhydrous Na2S04. HRGC/MS analysis was carried out as described in c). b)The ethanol extract was prepared by extracting 10 g of aromatized coffee with 15 mL of 96% ethanol (extraction with a dynamic system) for about 24 hours. The extract was then filtered and subjected to HRGC/MS analysis as in c) c)HRGC and HRGC/MS analyses: instruments and operating conditions. -HRGC: a gas chromatograph, HRGC MEGA 2 SERIES (Fisons-Instruments) equipped with a SUPELCO SPBTM-5 column (30 m x 0.32 mm i.d., 0.25 ^im film thickness), and a FID detector were used. Oven temperature was programmed as foUows: 50°C (8 min), 50°-120*^C at 2.5^C/min, 120°-140°C at l°C/min, 140°230°C at 2.5°C/min, 230°C (28 min). Injector and detector temperatures were

45

200°C and 220°C. Inlet pressure of the hydrogen carrier gas was 24 KPa. 1 |iL of sample was injected splitless (30")-HRGC/MS analyses were carried out on a SHIMADZU QP-5000 mass selective detector directly coupled to a SHIMADZU GC-17A gas chromatograph. An HP 101 column (25 m x 0.20 mm i.d., 0.20 ^im film thickness) and a SUPELCO SPB™-5 column (30 m x 0.32 mm i.d., 0.25 |im film thickness) were used. Oven temperature was programmed as follows: SO^'C (8 min), 50°-120°C at 2.5°C/min, 120°-140°C at rC/min, 140°-230°C at 2.5°C/min, 230°C (28 min). Injector and detector temperatures were set respectively at 220°C and 250°C. Inlet pressure of the helium carrier gas was 50 KPa. 1 \xL of sample was injected spUtless (30").

3 RESULTS AND DISCUSSION. Figures 1 and 2 show two "Cafe do Ponto" extracts obtained as described in a) and b). Identification of the peaks was hmited to the components derived from the mint aroma utilized for aromatization. Figure 3 shows an expansion of the area in Figure 1 containing the more characteristic compounds of the essential oil of mint eluted (0-50 min). Figure 2 clearly shows the presence (r.t. 70-120 min) of compounds that are less volatile than those typical of mint: these include caffeine. Propylene glycol is also in evidence. From the HRGC/MS analysis, it appears that the "Cafe do Ponto" product contains a solution in propylene glycol of the essential oil of mint. Propylene glycol and pulegone were identified quantitatively in the EtOH extract (ethyl laurate as the internal standard). The following results refer to 1 kg of mint flavored coff'ee: 7759 mg/kg for propylene glycol, 9 mg/kg for pulegone. The propylene glycol content is unacceptable according to Italian law (4), which lays down a maximum of 1000 mg/kg in foodstuffs. The pulegone content is acceptable . The marked presence of carvone in the GC chromatogram in respect of "Cafe do Ponto" indicates, moreover, that the essential oil may be derived from some chemotype of Mentha longifolia (for example var. crispa) or from essential oils of mint enriched in carvone. The use of solvents other than propylene glycol for aromatization was investigated. Two types of toasted, ground coffee were utilized: "Lavazza-Qualita Oro" and "Illy espresso". The solvents investigated as alternatives to propylene glycol were chosen from those tolerated in large quantities by Italian law. Solubility tests in glycerol and triacetate of glycerol were carried out on the trirectified essential oil of mint . The high solubiUty (1:1) of the essential oil of mint in triacetin has been previously demonstrated (5), but there does not appear to be any published data regarding its solubility in glycerine. Experiments have shown the low solubility of mint oil in glycerine (1:15, mint oil-glycerine). The experiments were aimed at identifying the maximum quantity of essential oil dispersible in toasted and ground coffee, utilizing solutions of trirectified mint oil

Menthone Isomenthone+Menthofuran Neo-menthol 4. Menthol 5. Isomenthol 6. Pulegone 7. Carvone 8. Piperitone 9. Neo-menthyl acetate 10.Menthyl acetate 11. Isomenthyl acetate 12. p-Bourbonene 13. Caryophyllene 1. 2. 3.

4

24 0

4R 11

72 0

96.0

Tbr (mw)

Figure 1. GC profile of "Caf6 do Ponto" extract obtained by Llkens-Nickerson concentratorlextractor. Operating conditions are described in section 2.1.

o

a

X!

C

^j

O 0

00

0^

^

rH

«^ «^

OS TH

I

g ^i >i a fi

r>.

4

+

tf>

2 ^ o 'S

»0

I

fi S c o o 6 o o <^ 3 3 . & ^

rH ffsl CO "^

O

o

Menthone Isomenthone+Menthofuran Neo-menthol Menthol 5. Isomenthol 6. Pulegone 7. Carvone 8. Piperitone 9. Menthyl acetate 10. Caryophyllene 11. Caffeine 1. 2. 3. 4.

1

I

24.0

0.0

48.0

71.0

96.0

20.0

'I-itnc (min)

o

.S

a;

en

O

C3

o

O

o

OS

o

o

o

P

47

PM

O

O

US bJD •iH

Figure 2. GC profile of "Cafe do Ponto" ethanol extract. Operating conhtions are described in section 2.1.

4

48

P

00

Menthol

\

I Menthone

Neo-menthyl acetate

Isomenthone Menthofuran

% ' ,

Menthyl acetate /

Neo-menthol

\ k p m e n t h y l acetate

.w

L in.w

40.w

3o.no

1O.W

Tinu (min)

S fl

O

I

0

o

o

-rH

0

bJD

•PH

Figure 3. Expansion of the GC profile in Figure 1between 0-50 minutes.

49

in triacetin and glycerol. The solutions were prepared at the highest possible concentrations. Account has to be taken of the maximum quantity of glycerine and triacetin permitted in foodstuffs: 2000 mg/kg is the limit for glycerine, while for triacetin Italian law requires SMP (sound manufacturing practice) (4). For the glycerine solution, the quantity of mint oil dispersed in the toasted and ground coffee was 133.3 mg/kg. The product was designated GL. Since the aromatic impact of this was insignificant, another experiment was carried out using a triacetin solution. In this second experiment, the quantity of triacetin used was equal to 1.8 g/kg of coffee. This dosage results in an additional equal quantity of essential oil of mint. The mint aroma, at this dosage, turns out to be sufficiently perceptible (comparison with "Cafe do Ponto"), but an underljdng side taste renders the beverage prepared quite unacceptable (product denoted TR). The underlying side taste in the TR does not stem from the essential oil, but is most likely due to the diluent, triacetin. It is thus possible to conclude that: - Glycerine cannot be used as a solvent due to the low solubility of the essential oil in it. - Triacetin cannot be used as a solvent due to the side taste it creates. Finally, the aromatization of coffee was attempted by using various blends with ethanol as the solvent. The best results were obtained with a blend (denoted O.A.), characterized by the total-ion chromatogram in Figure 4. The operating conditions are described in c). O.A. is a "nature-identical" aroma, i.e. a blend consisting of: - "oil of mint" as base - enrichment with carvone - enrichment with anethole - enrichment with menthyl acetate - EtOH as diluent Figure 5 shows the GC profile of the volatile fraction obtained by LikensNickerson extraction of the aromatized lUy coffee. The numbered peaks were identified by GC/MS analysis. The results of the organoleptic analysis of five preparations of aromatized coffee are given in Table 1. The results were obtained by a panel test involving 40 tasters. In the final analysis, "aromatized coffee" is not a substitute for traditional Italian "espresso" coffee. It is, however, an alternative drink, aimed at those consumers who do not use coffee as a stimulant, but just as a pleasant drink.

50

nc

1

1

3

10 1

k

y

13

9 \

11 /

^21 / .A ,)l /

.

_^

^^

Figure 4. Total ion chromatogram of O.A. utilized to aromatize "Lavazza-Qualita Oro" and "Illy espresso" coffee. Operating conditions are described in section 2.1. 1) eucalyptol, 2) limonene, 3) menthone, 4) isomenthone, 5) menthofuran, 6) isomenthol, 7) menthol, 8) neo-menthol, 9) pulegone, 10) carvone, 11) anethole, 13) menthyl acetate, 14) isomenthyl acetate.

o O

bJD O

2 fl S 0 fi o fl ( fl S o fl »o ;o t^

a

00

.

Qs S Q O in ^ 0. U PH

N eo ^

O 0 TH

o o o

iH

fi

0 fi V

crt 0

a 0) 0

s

2 fi P 0 TH

. o TH C5

CD

.^

s o CD

o •iH

CD

XI

o q

51

. 0

(D

0

s « OS

o

0 -S >

-iH

o cc O

(J bJD • i-i

fl

O g 0

0)

in

fo 0

52

Table 1 Comparison of the fundamental organoleptic characters (CP: Cafe do Ponto; G.L: coffee aromatized with mint essential oil in glycerine; TR: coffee aromatized with mint essential oil in triacetin; A e B, Lavazza and Uly coffee respectively aromatized with O.A.). CP GL TR A B •Intensity of mint aroma +++

+/-

+++

++

++

•Underlying side taste

-f/-

+

-

-

•Sensation of freshness ++ imparted by the mint aroma

-

-

+

+

•Overall judgement

Not good

Not good

Good

Good

-

Fairly good

4. CONCLUSIONS 1) The use of propylene glycol, declared as "propylene glycol (umectant)" on the label of "Cafe do Ponto", is an impediment to the introduction and marketing of the product in many countries. 2) The alternative use of glycerine or triacetin as solvent/diluent of the mint aroma (even with the use of a good mint essential oil) is not an acceptable solution for the various reasons described here. 3) The use of mint aroma with a high carvone content, in "Cafe do Ponto", conveys the aroma of "crispa" mint (a sensation of freshness). 4) The use of the solvent/support "EtOH" is preferable from all points of view. 5) The use of essential oil of mint "enriched" with limited quantities of carvone and other compounds results in an acceptable product. 5. R E F E R E N C E S 1

2

3

Graduation Thesis in Food Technology of G. Cantele, (1994-1995), Problematiche analitiche sulla caratterizzazione degli aromi mile bevande "te allapesca"y Tutor and Science Dir. F. Tateo (University of Milan) F. Tateo, L. M. Di Cesare, G. Cantele and M. Bononi, Sui metodi di estrazione e valutazione dei composti volatili costituenti I'aroma di bevande al te, Atti del 2° Congresso Nazionale di Chimica degli Alimenti, Giardini Naxos, 24-27 Maggio, 1995. EEC Directives n. 88/388 and 91/71-Gazzetta Ufficiale delle Comunita

53 europee. Supplemento Ordinario alia Gazzetta Ufficiale della Repubblica Italiana n. 39, 17/2 1992, Decreto Legislative n. 107 del 25/01/1992. F. Tateo, E. Ciserchia, L. Triangeli and E. Verderio, Criteri di base per Vimpiego di diluenti nella produzione di aromatizzanti, Bollettino Chimico Farmaceutico 126, 217-227, 1987.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

55

Indicators for evaluation of lipid oxidation and off-flavor development in food F. Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John's, NF, AlB 3X9 Canada

Abstract Lipid oxidation is a major cause of food quality deterioration. Its effects are manifested in off-flavor development with production of potentially toxic and carcinogenic products. Methods to monitor lipid oxidation include evaluation of changes in the starting lipids, production of lipid hydroperoxides and their breakdown products. Different classical methods as well as use of individual aldehydes such as propanal and hexanal for assessing oxidation of edible oils and muscle foods will be presented. While propanal served as a good indicator forflavordeterioration of oils andfishmeat which are rich in omega-3 fatty acids, hexanal proved to be a reliable indicator for assessing the degree of oxidation of meat lipids and those rich in omega-6 fatty acids.

1.

INTRODUCTION

Lipid oxidation is a major cause of food quality andflavordeterioration. Oxidation of lipids is primarily dependent on the degree of unsaturation of their fatty acid constituents, but is also affected by other components present in the food matrix as well as conditions under which the product is stored. The relative rate of autoxidation on the basis of oxygen uptake for oleate, linoleate and linolenate is in the order of 1:40-50:100 [1]. Polyunsaturated fatty acids such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), containing 4, 5 and 6 double bonds, are much less stable than linoleic and linolenic acids. Hydroperoxides are the primary products of lipid oxidation, but hydroperoxides, despite their deleterious effects on health [2, 3] have no effect on flavor quality of foods. However, these unstable molecules decompose readily to form a myriad of products such as aldehydes, ketones, alcohols and hydrocarbons, amongst others. Some of these secondary oxidation products have threshold values in the parts per billion range and thus have a major impact onflavordeterioration of foods in which they are present. Significance of lipid oxidation is not limited to the development of off-flavors in food, but it also affects the nutritional value of food as it brings about loss of essential polyunsaturated fatty acids, fat-soluble vitamins, protein value and destruction of pigments, when present [4, 5]. Products of lipid oxidation have also been implicated as having toxic, carcinogenic, mutagenic and teratogenic properties. In living organisms, reactive radical species are inactivated by the action of antioxidant enzymes and this might be assisted by the presence of natural antioxidants. However, in the elderly, where the ability of enzymes in combating reactive radical species is diminished, a number

56 of diseases such as inflammation, arthritis, diabetes, schemia and cancer may become prevalent, all of which hasten the process of aging in humans [6]. Therefore, it is essential to examine indicators that might be used to assess the oxidative state of food and biological systems.

2.

OXroATIVE STATE AND OFF-FLAVOR GENERATION

Methodologies that maybe employed for evaluation of oxidative state of food lipids and offflavor development may consider changes in chemical, physical, or organoleptic properties offood lipids. Therefore, changes in the starting material as evidenced by the weight gain and/or oxygen uptake, changes in the fatty acid composition and iodine value as well as generation of free radicals (which is evident from electron paramagnetic resonance studies), may be pursued. Furthermore, primary products of oxidation may be assessed by measuring peroxide value (PV), individual hydroperoxides, conjugated dienes and trienes. The secondary products of oxidation, some of which are mainly responsible for off-flavor development, could be quantitated by determination of generated hydrocarbons such as ethane or pentane, individual volatile carbonyl compounds such as malonaldehyde, propanal and hexanal, as well as total carbonyl compounds. The latter are represented by/7-anisidine value (p-AnV) and 2-thiobarbituric acid (TBA) value, 2,4-dinitrophenylhydrazones formed from the reaction of carbonyl compounds with 2,4dinitrophenylhydrazine, as well asfluorescentcompounds (1 -amino-3 -iminopropene derivatives). Finally, overall changes occurring in food lipids may be estimated from consideration of TOTOX (total oxidation; 2 P V +/?-AnV) value, nuclear magenetic and infrared spectral changes, Rancimat and oxidative stability instrument (OSI) data. 2.1. Primary changes Methods that measure primary changes of lipids may be classified as those that quantify loss of reactants (unsaturated fatty acids), addition of oxygen or changes in iodine value or formation of primary lipid oxidation products which are hydroperoxides [7-10]. With respect to fatty acid composition, only lipids with a high proportion ofunsaturated fatty acids or a reasonable content of highly unsaturated fatty acids might lend themselves to this method of analysis. Thus, marine lipids and highly unsaturated vegetable oils might serve as best candidates for which changes in fatty acid composition could be used as a valid indicator. Table 1 shows changes in fatty acid composition of seal blubber oil during intense heating of rendering, in the presence of catalytic hemoproteins. Similarly, changes in iodine value (IV) due to loss of unsaturation may be employed to monitor oxidation of food lipids [9]. During oxidation, the oxygen above the lipid surface slowly reacts with lipids and changes in the oxygen pressure may be measured quantitatively. The drop in pressure of the system may be quantified as oxidation proceeds. It is generally accepted that addition of oxygen to lipids and formation of hydroperoxides is reasonably quantitative during initial stages of autoxidation. Therefore, measurement of induction period from relevant data is possible. Weight gain data and oxygen depletion/uptake, or change in IV, are other means by which oxidation status of bulk oils could be estimated. In this respect, 0.5% weight gain data might be used as a measure of induction period of an oil [8,10,11]. Obviously, highly unsaturated oils are most susceptible to weight gain and oxygen uptake. Figure 1 shows the weight gain data of representative marine oils in comparison with those from selected vegetable oils. Marine oils tested exhibited a sharp increase in weight by the end of the induction period. This method may

57 also be used to compare the relative efficiency of different antioxidants in an oil sample. Reduction in the headspace oxygen for edible oils has also been demonstrated in the literature [12]. Table 1 Fatty acid composition of seal blubber oil as affected by steam injection heating for 2 h in the presence of hemoprotein Fatty Acids 14:0 16:0 16:1 18:0 18:1 18:2 18:3 18:4 20:4 20:5 22:5 22:6

Fully rendered

Raw

9.09 8.44 2.28 1.27 15.9 1.36 0.95 1.57 0.34 6.25 2.46 4.86

6.32 8.10 11.50 1.02 26.4 1.25 0.54 1.63 0.53 9.11 4.78 9.55

RBD-Menhaden oil RBD-Cod liver oil RBD-Seal blubber oil RBD-Canola oil RBD-Soybean oil 72

90

108

Storage Time (h) Figure 1. Weight gain data of selected edible oils during storage under Schall oven conditions.

58 With respect to quantitation of primary products of lipid oxidation, namely the hydroperoxides, it is instructive to note that their production is generally concurrent with the appearance of conjugated dienes and trienes. Figure 2 shows production of 9- and 13hydroperoxides from linoleic acid, both of which contain conjugated double bonds in their chemical structure [13]. Thus, content of conjugated dienes/trienes might correspond with that of peroxide value [14].

13

12

GOGH

10 9

Linoleic Acid

Initiator

K-

Delocalized Pentadienyi Radical

GO'

R R"H

OOH

13-Hydroperoxide

9-Hydroperoxide

Figure 2. Concurrent formation of hydroperoxides and conjugated dienes from linoleic acid. Determination of peroxide value (PV) of lipids is carried out by an iodometric titration. The iodine produced is then titrated with a standardized solution of sodium thiosulfate as given below [15, 16]. ROOH + 2 H ' + 21 I2 ^ 2S2O;

I2 + ROH + S4O; ^ 2 1 -

nfi

59 Figure 3 displays the formation of peroxides during storage of edible oils under Schaal oven conditions. Correlation of peroxide values with the content of conjugated dienesfromthis work was linear for each oil examined (results not shown). It is worth noting that the correlation parameters depend on the type of oil examined and these might also be influenced by the presence of other constituents in the assay medium.

50

75

100

Storage Time (h) Figure 3. Formation of hydroperoxides of selected edible oils during storage under Schall oven conditions.

2.2. Secondary changes A widely used method of determination of secondary oxidation products is the 2thiobarbituric acid (TBA) test. In this test, malonaldehyde, a product of oxidation of Upids, as well as alkenals and alkadienals interact with the TBA reagent to form a pink chromogen which absorbs at 530-532 nm [17]. The intensity of this pigment in solution is used as an index of oxidative status of foods. Figure 4 shows the reaction of malonaldehyde with the TBA reagent. Depending on the material under investigation, the procedure used for determination of TBA reactive substances (TBARS) varies. Thus procedures used for TBA determination of bulk oils [16] are different from those employed for evaluation of oxidative state of muscle foods [18]. Furthermore, several different procedures may be used for determination of TBA values in meat

60

IIJ H-C-CH2-C;;:Ho^

. Hj,0 '2'-' T

Malonaldehyde

O H o II
9LO HO^N

SH

HO'CN-^S-H

2-Thiobarbituric Acid

^ H o ^ . ^^^H H-C-CH = C

O

V

I

^

Jl HS^N"

OH

^ OH

OH

S^N^OH

O^N-^S

^

S^N-^O

HO^N"^S

H

Figure 4. Reaction of malonaldehyde with 2-thiobarbituric acid reagent.

and seafoods. Each method has its own advantages and disadvantages, details of which are beyond the scope of this article. Figure 5 displays the variation of TEARS of several oils with storage time under accelerated conditions. Similar to the results obtained for peroxide values, there was a steady increase in the TEARS values of oils, but vegetable oils represented by canola and soybean oils, were much less susceptible to oxidation than marine oils tested. However, there are certain limitations when using the TEA test for evaluation of the oxidative state of foods because of the chemical complexity of systems involved. Nonetheless, relationships have been found between TEA values and the development of undesirable flavors or flavor thresholds in fats and oils [15, 19]. In cured meats, TEA values may be affected by the presence of residual nitrite, amongst other ingredients. Interaction of nitrite (reactive species N2O3) with malonaldehyde results in the formation of an oxime (Figure 6) which leads to the underestimation of TEARS values [20,21]. In such cases, sulfanilamide has been added in order to scavenge free nitrite present in the samples (see Figure 7). However, in many cases, residual nitrite in cured meats might have been depleted or present in only minute qualities. In such cases, malonaldehyde may interact directly with sulfanilamide and thus result in the underestimation of TEARS [20-22]. Evidence for the formation of adducts of malonaldehyde with primary amines has come from fluorescent studies of conjugated Schiff bases formed as well as other spectrometric studies [23, 24]. It should also

61

• • V • o

RBD-Menhaden oil RBD-Cod liver oil RBD-Seal blubber oil RBD-Canola oil RBD-Soybean oil

O

E (D

27

:*E 25

50

75

100

125

Storage Time (h) Figure 5. Formation of 2-thiobarbituric acid reactive substances (TEARS) during storage of selected edible oils.

" > - ; - < ' ' Malonaidehyde

p-Hydroxyacrolein

°-.'<;-.-'"

ti ^r

C-nJtroso form

Figure 6. Interaction of malonaidehyde with nitrite.

62

'j'

H

NV— <j/ \ ^

C\

H j N O g S - ^

'

^

H

^ H

Malonaldehyde

ti

Sulfanilamide H

1

f

HgNOgS ' " ' ^

f^ Ji

V

HgNOgS " ' ' ^ ^

!

tl " X NH

(^ HgNOgS ^ ^ ^ /

^ C H

OH 1

H 1 N

JJ

n LA

I

^-^

^

HgNOgS '^

SO2NH2

l^

^ SOgNHg

Figure 7. Interaction of malonaldehyde with sulfanilamide.

be noted that Schiff base formation, evidenced by fluorescent data, might be used as a measure of lipid oxidation, when lipids are present together with proteins. Another method by which oxidation of food lipids might be assessed is the/7-anisidine test. This method is very similar to the TBA procedure in that a conjugated l-amino-3-iminopropene derivative is formed between one mole of malonaldehyde and two moles ofthep-anisidine reagent [25]. Thus, /7-anisidine values (p-AnV) of edible oils show steady increase during their storage. List et al [26] have reported a highly significant correlation between/?-ansidine values of salad

63 List et ah [26] have reported a highly significant correlation between p-ansidine values of salad oils processed fi-om undamaged soybeans and their flavor acceptability scores. In order to determine individual carbonyls, as secondary oxidation products, in lipidcontaining foods, one might pass the volatiles of the test material through an acidic solution o f 2,4-dinitrophenylhydrazine [ 2 7 , 2 8 ] . The resultant 2,4-dinitrophenylhydrazone mixture may then be fihered off and the total solids determined or used to release the carbonyl compounds for further studies. In addition, 2,4-dinitrophenylhydra2ine may be employed for stripping o f test materials fi-om carbonyl compounds in order to study the chemical nature o f the remaining volatiles. On the other hand, static headspace gas chromatographic analysis may be used to directly quantify selected aldehydes [29, 31]. W e have used hexanal and propanal as indicators for monitoring oxidative status of foods rich in omega-6 and omega-3 fatty acids respectivelv (see Figures 8 and 9). ' ^ It should be noted that a linear relationship generally holds between the TEARS and each o f the hexanal and propanal contents o f foods. However, it is recommended that appropriate aldehydes be used for monitoring oxidation of food lipids [ 2 9 , 3 1 ] . Thus, the use of propanal for studying fish oils, marine foods and research related to feeding trials involving omega-3 fatty acids is appropriate. In the case of meat, vegetable oils and related commodities, where omega-6 fatty acids are dominant, hexanal would serve as a more appropriate indicator.

O

I E c (D C

o O

CO

c CO X

a> X

50

75

100

Storage Time (h) Figure 8. Formation of hexanal in selected edible oils during accelerated storage at 65°C.

64

50 75 100 Storage Time (h) Figure 9. Formation of propanal in selected edible oils during accelerated storage at 65°C. During prolonged storage of food lipids, caution should be exercised when interpreting numerical values of TBA or individual volatile contents. In the first few days of storage the content of TEARS as well as hexanal and pentanal (not shown) increased in a linear fashion, after 5-7 days, their concentration may begin to decline. An example of this latter situation has been shown for cooked ground pork (Figure 10). Thus, the absolute values obtained for the content of each aldehyde cannot be correlated with the length of storage period since it is not known which side of the hill one might be. 2.3. Overall changes In order to monitor overall changes in the oxidative state of foods, the industry has traditionally used other indicators. These include TOTOX value, defined as 2PV +^AnV, and resultsfi-omRancimat or Oxidative Stability Instrument (OSI) studies. The TOTOX value is often considered to have the advantage of combining evidence about the past history of an oil (as reflected in/?-anisidine value) with its present state (as evidenced by the pV) [7, 32, 33]. Thus, TOTOX value may provide a measure of the total oxidation of an edible oil, however, despite its practical advantages, its chemical significance is questionable as variables with different dimensions are combined. For the Rancimat or OSI, the oil is oxidized in the presence of oxygen to produce acids, mainly formic acid. From changes in the conductivity oif sample, induction period of different oils may be extrapolated for comparative purposes [33]. Spectrometric methods such as electron paramagnetic resonance (EPR) [34, 35], Fourier transform infrared (FTIR) spectroscopy [36] and proton nuclear magnetic resonance (^HNMR)

65

E 3 c o 2 c

15

0)

o c o O 75 c CO X

a>

I '•S3 iS o >

0

7

14

21

Storage Period (Days) Figure 10. Variation in the content of hexanal during storage for cooked ground pork.

spectroscopy [37-40] may be employed to determine the oxidative state of food lipids. Proton NMR provides data on changes of the relative number of aliphatic, olefinic and diallylmethylenic protons during storage and processing of lipids. Table 2 provides the chemical shifts of different groups of protons in a triacylglycerol molecule. During oxidation of lipids with unsaturated fatty acids, there is a decrease in the relative number of olefinic and diallymethylene protons and a corresponding increase in the proportion of aliphatic protons in lipids under investigation. Wanasundara and Shahidi [3 8] have shown changes in the relative proportion ofprotons belonging to each group in a triacylglycerols of food lipids (Table 3). Furthermore, the ratio of aliphatic to olefinic (R^) and aliphatic to diallymethylene (R^d) increase steadily during the oxidation of selected vegetable and marine oils. In addition, a linear relationship existed when plotting R^^ and R^ values against corresponding TOTOX values. Therefore, NMR methodology may be used

66 as a rapid method for determining oxidative state of lipids and to estimate the overall changes in the primary and secondary oxidation products. Table 2 Proton nuclear magentic resonance (^HNMR) chemical shifts of various groups of triacylglycerols Group

Chemical shift, ppm

CH3 (CH^),CH2 - C = a - CH2 = C - CH2 - C H2C-

0.7-1.0 1.1-1.8 1.8-2.2 2.2-2.4 2.6-2.9

I

4.0-4.4

-CH2CH C = CH C

5.1-5.4 5.1-5.4

H-CI

C

Table 3 Changes in the proportion of different groups of triacylglycerols in selected fi-esh and oxidized edible oils as determined by ^H NMR Olefinic (o)

Oil

Diallylmethylene (d) Fresh

Aliphatic (a)

Fresh

Oxidized

Canola

7.12

6.00

2.18

1.54

79.28

84.19

Soybean

8.12

7.24

4.00

3.40

75.64

80.64

Fish Oil

11.44

9.47

7.46

6.70

78.86

83.78

Seal Oil

11.00

9.28

5.54

5.37

73.29

81.57

Oxidized

Fresh

Oxidized

In conclusion, there are a number of indicators that might be used for evaluation of oxidative state and off-flavor development in foods. Choice of an appropriate indicator is important. Furthermore, it is recommended that at least two indicators be used. Of course, the ultimate test is correlation of any of these indicators with sensory characteristics of test material.

67 3.

REFERENCES

1 2 3

RJ. Hsieh and J.E. Kinsella, Adv. FoodNutr. Res., 33 (1989) 233. A. Nishikawa, R. Sodum and F.-L. Chung, Lipids, 27 (1992) 54. F.-L. Chung, H.-J. Chen, J.B. Guttenplan, A. Nishikawa and G.C Hard, Carcinogenesis, 14 (1993)2073. E.R. Sherwin, J. Am. Oil Chem. Soc, 55 (1978) 809. R.J. Hamilton, In Rancidity in Foods, ed. by J.C. Allen and R.J. Hamilton, Applied Science Publishers, New York, 1983, pp. 1-20. F. Shahidi, In Natural Antioxidants: Chemistry, Health Effects and Applications, ed. by F. Shahidi, American Oil Chemists' Society Press, Champaign, 1997, pp. 1-11. U.N. Wanasundara and F. Shahidi, J. Food Lipids, 2 (1995) 7. U.N. Wanasundara and F. Shahidi, J. Am. Oil Chem. Soc, 71 (1994) 817. B.J.F. Hudson, In Rancidity in Foods, ed. J.C. Allen and J. Hamilton, Applied Science Publishers, London, 1983, pp. 47-58. U.N. Wanasundara and F. Shahidi, J. Am. Oil Chem. Soc, 73 (1996) 1183. P.J.Ke and R.G. Ackman, J. Am. Oil Chem. Soc, 53 (1976) 636. A.R. Wewela, In Natural Antioxidants: Chemistry, Health Effects and Applications, ed. by F. Shahidi, American Oil Chemists' Society Press, Champaign, 1997, pp. 331-345. M.K. Logani and RE. Davis, Lipids, 15 (1980) 485. F. Shahidi, U.N. Wanasundara and N. Brunet, Food Res. Inter., 27 (1994) 555. J.I. Gray, J. Am. Oil Chem. Soc, 55 (1978) 539. AOCS, Official Methods and Recommended Practices ofthe American Oil Chemists' Society, 4* ed., ed. by D. Firestone, American Oil Chemists' Society, Champaign, 1990. B.G Tarladgis, AM. Pearson and L.R. Dugan, J. Sci. FoodAgric, 15 (1964) 602. F. Shahidi and C. Hong, J. FoodBiochem., 15 (1991) 97. Z.J. Hawrysh, In Canola andRapeseed: Production, Chemistry, Nutrition and Processing Technology, ed. by F. Shahidi, VanNostrand Reinhold, New York, 1990, pp. 99-122. F. Shahidi, L.J. Rubin, L.L. Diosady and D.F. Wood. J. Food Sci., 50 (1985) 274. F. Shahidi and R.B. Pegg and R. Harris, J. Muscle Foods, 2 (1992) 1. R.B. Pegg, F. Shahidi and C.R. Jablonski, J. Agric Food Chem., 40 (1992) 1826. W.R. Bidlack and AL. Tappel, Lipids, 8 (1973) 203. C.J. Lillard and A.L. Tappel, Lipids, 6(1971)715. lUPAC, Standard Methods for the Analysis of Oils and Fats and Derivatives, 7*^ edition, Blackwell Scientific Publication, Oxford, 1987. GR. List, CD. Evans, W.K. Kwolek, K. Warner and B.K. Boundy, J. Am. Oil Chem. Soc, 51 (1974) 17. S.R. Meyer and L. Rebrovic, J. Am. Oil Chem. Soc, 72 (1995) 385. N. Yukawa, H. Takamura and T. Matoba, J. Am. Oil Chem. Soc, 70 (1993) 881. F. Shahidi and R.B. Pegg, J. Food Lipids, 1 (1994) 177. F. Shahidi, U.N. Wanasundara, Y. He and V.K.S. Shukla, In Flavor and Lipid Chemistry of Seafoods, ed. by F. Shahidi and K.R. Cadwallader, ACS Symposium Series 674, American Chemical Society, Washington, D.C., 1997, pp. 186-197. A.J. St. Angelo, J.R. Vercellotti, M.G Legengoe, C.H. Vinnelt, J.W. Kuan, C. Janies, and H.P. Dupuy, J. Food Sci., 52 (1987) 1163. J.B. Russell, In Rancidity in Foods, ed. by J.C. Allen and J. Hamilton, Applied Science Publishers, London, 1983, pp. 21-45.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

68 33 F. Shahidi and U.N. Wanasundara, FoodSci. Technol Int., 2 (1996) 73. 34 K.M. Schnaich andD.C. Borgi, InAutoxidation in Food andBiological Systems, ed. by M.G. Simic and M. Karel, Plenum Press, New York, 1980, pp. 45-70. 35 M.J. Davies, Chem. Phys. Lipids, 44 (1987) 149. 36 F.R. van de Voort, A.A. Ismail, J. Sedman and G. Emo, J. Am. Oil Chem. Soc, 71 (1994) 243. 37 F. Shahidi, Inform, 3 (1992) 543. 38 U.N. Wanasundara and F. Shahidi, J. Food Lipids, 1 (1993) 15. 39 U.N. Wanasundara, F. Shahidi and C.R. Jablonski, Food Chem., 52 (1995) 249. 40 H. Saito and M. Udagawa, J. Sci. FoodAgric, 58 (1992) 135.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

69

Aroma analysis of coffee brew by gas chromatography-olfactometry K. D. Deibler, T. E. Acree, E. H. Lavin Depart, of Food Science & Technology, Cornell University, Geneva, NY 14456 Abstract During the study of coffee flavor, the processes of brewing, extraction and sampling cause losses of the aroma compounds present in coffee grounds. In this study, coffees from two brewing methods were extracted, serially diluted and each dilution sniffed twice using the gas chromatography-olfactometry (GCO) technique called CharmAnalysis . Among the hundreds of volatile chemicals present, 18 of the thirty most potent odorants were identified by comparing the mass spectra, odor activity and Kovat's retention indices with those of authentic standards. Our studies have verified the presence of previously identified aroma compounds among the most potent odorants in coffee and show the differences between the two brewing methods tested.

1. INTRODUCTION According to legend, coffee was discovered by an Arab goat herder named Kaidi. He noticed that his goats became frisky and danced around the fields after chewing on the berries from coffee bushes. After watching this, an abbot gave some of the berries to neighboring monks, who prayed all night without falling asleep. The first coffee drink, a steeped water broth, was consumed around the year 1000 AD. Arabs from the port of Al Mukkah (Mocha) on the Red Sea became the sole source for the world's coffee controlling the lucrative coffee market by only permitting the export of boiled or roasted beans. In the 1600's, smugglers broke the Arabian monopoly in coffee growing. They took seven seeds of unroasted coffee beans from the port of Mocha to the western Ghats of southern India. In the early 1700's, the Dutch began cultivating descendants of the original plants in Java [1, 2]. Today coffee is the second most important trade commodity, second to oil [3]. Coffee shops grew 20% annually from 1991 to 1995 with an expected four fold increase by 1999 making coffee shops the fastest-growing type of food and drink outlet in the United States [4]. However, coffee houses or bars are not a new phenomenon. New York colonists first brought coffee to their breakfast table in about 1668 to replace beer. Coffeehouses became the centers of cities' business.

70

political and social life during colonial times. Court trials and city council meetings were held in early coffee houses. Paul Revere plotted the American Revolution at the Green Dragon Coffee House in Boston [2]. Breakfast remains the most popular time of day for coffee consumption in the US [5]. Coffee sales in the United States reached $7.4 billion in 1995 with a 2 cup per person daily consumption [4]. Consumer tests show that the taste of coffee is the most important factor in purchasing coffee, thus understanding the aroma profile of coffee is imperative [6]. The two commercially consumed varieties of coffee come from Coffea arabica and Coffea canephora var. robust a. Most supermarket coffees are a blend of the two and most instant coffees are made from Robusta beans. Robusta beans are generally considered inferior to the more expensive Arabica beans. Coffee grows in the regions between the Tropic of Cancer and the Tropic of Capricorn. Many countries' economy depends on its sales of coffee beans. Beans grown at lower altitudes are believed to be of lower quality with less flavor. Where the coffee is grown is very important to the quality. Table 1 shows commonly accepted characteristics of beans grown in various regions [7].

Table 1 Characteristics of coffees from different regions of the world. GENERAL AREA African Arabian Peninsula Hawaii Caribbean Indonesian Central American South American

COUNTRY OR TYPE Tanzanian, Kenya, Ethiopian Yemen (Mocha)

CHARACTERISTICS Heavy body; bright and floral; excellent for blending Heavy body but more aroma than African coffees. Kona No body; some aroma Jamaica Blue Mountain Balance of body and aroma Java, Sumatra, Celebes Balance of body and aroma; spicy Nicaraguan, Mexican, Some body, lots of aroma; Costa Rican, Guatemalan hints of cocoa Colombian, Brazilian Some body; lots of aroma; nutty

The coffee bean is actually half of a bean found inside a fruit called the coffee cherry. The coffee cherry is ripe when the skin is red and has two green beans inside. The fruit is picked by hand since the fruits ripen at different times on the same bush. The fruit is fermented to loosen the beans, which are then removed, washed and dried. There are two methods of extracting the green seed from the fruit: the wet method and the dry method. The wet method produces a higher acidity and cleaner flavor than the dry method which produces an increased body and earthy flavors [3]. The green bean is the commodity primarily traded. It is roasted by a roastmaster at 180 °C which is primarily w h e n

71

the characteristic aromas are formed. Formation pathways of many coffee odorants at roasting conditions have been discussed by Holscher [8], Baltes [9], and Tressl [10]. During roasting the composition of the beans dramatically changes; sucrose content drops from 7.3% to 0.3%, chlorogenic acid drops from 7.6% to 3.5% and protein content goes from 11.6% to 3.1%. Free amino acid levels also change greatly [11]. The length of time for roasting affects the amount of caffeine in the beans; the darker the roast the less the caffeine. Roastmasters use both smell and sight to determine when the type of roast they desire has been achieved. The roast is differentiated based on color from a Light city roast, city roast, Brazilian to Viennese, French roast, Spanish -Cuban and espresso being the longest roast time and darkest bean [7]. Due to the high quantity of unsaturated oils (13%), coffee beans are highly vulnerable to autoxidation. Different brewing methods call for different sizes of grinds. Grinds for espresso are much finer than those used for the long slow method of percolation which use a course grind. Contact with light and moisture affect the composition of the coffee bean while stored. All of these factors make coffee flavor highly variable. 1.1. Coffee Aroma and Brewing Method The enticing aroma of coffee cannot be characterized by a single chemical component but is a combined response to many different chemical components. More than 800 volatile compounds have been found in roasted coffee [12, 13]. Only a small number of these volatile compounds contribute to the aroma. Aroma profiles of the green beans, roasted beans, brewed coffee, Coffea arabica, and Coffea canephora var. Robusta have been evaluated [8,14-21]. The Werner Grosch group has quantitated 22 important odorants in coffee brews by stable isotope dilution assay and identified 32 of the 38 odorants detected. Stable isotope dilution assay, aroma extraction dilution analysis (AEDA), odor active value (OAV) analysis, and gas liquid chromatographic analysis have been conducted on coffee [22, 23]. Extraction temperature, time and particle size are among the ways brewing methods profoundly affect coffee flavor. In the US market filtered coffee (extracted between 2 and 10 min) falls in between the extremes of espresso (extracted in seconds) to percolator coffee (extracted between 15 to 30 min) and it is used widely around the world. In this study a laboratory method for brewing filtered coffee was developed to allow both controlled brewing time followed by rapid cooling and solvent extraction. Because the technique involved cooling under reduced pressure, the potential for aroma loss was examined by comparing the gas chromatography-olfactometry (GCO) data from solvent extracts of rapidly cooled coffee with coffee cooled in an ice bath. This quick brew method produces an extractable brew ideal for GCO analysis. Using the experimental brew method, shorter brew times (down to seconds); immediate and rapid cooling; controlled contact time of water and grinds; and controlled brew time are easy to achieve. This quick brew method uses apparatus available in most modern chemistry laboratories. It allows for the extraction into water of the coffee aromas with a minimized loss of aroma with the water vapor.

72

The aroma profile of a cup of coffee is variable and can be influenced by bean origin, annual weather conditions, roasting method and time, grind size, freshness, and brewing procedures. By using this experimental brewing method, brewing time and temperature can be easily controlled and comparisons may easily be made between experiments while producing an extractable simulation of a typical cup of coffee.

2. MATERIALS AND METHODS 2.1. Brewing Methods 2.1.1. Quick Brew The coffee grind to water ratio most commonly reported in the literature (0.035, [24, 25]) was used. Approximately 50 g of a blend of Brazilian, Guatemalan, and Colombian roasted Arabica coffee beans were ground in a Krups Type 203 for 8 sec to achieve a particle size range of 300-500 jim. The apparatus used for the experimental brew is shown in Figure 1. Distilled deionized water (1250 mL, 95 °C) was filtered over the roasted and ground coffee (45.0 g) on a UF-50 filter (Mr. Coffee., Inc., Bedford Heights, OH) in a 10 cm diameter Buchner funnel attached to a 20 cm water cooled condenser collected in a 2000 mL ice bath cooled vacuum flask. The condenser and exposed glassware other than the funnel were insulated and chilled with frozen chill packs. A minimal vacuum was pulled (0.13 atm) to achieve an increased flow rate (3.5 mL/sec) and reduced brewing time (6 min). 2.1.2. Conventional Brew Water temperature, grind size, water volume, filter paper, and grind quantity were held constant for the conventional brew method. The conventional brew had a flow rate of 1.0 mL/sec. After brewing, the coffee was chilled in an ice bath to 35 °C. 2.2. Aroma Extraction and Dilution Analysis The aroma extraction procedures are summarized in Figure 2. The brew (1.0 L) was successively extracted with a nonpolar solvent, Freon 113^^ (666 mL) and a polar solvent, ethyl acetate (666 mL). This successive extraction with two solvents (non-polar and polar) produces a greater volatile recovery than would have been achieved using a single solvent. Each solvent extraction was first stirred gently with a magnetic stirrer for 30 minutes, then separated in a separatory funnel, and dried by filtering over MgS04. The extracts were concentrated 243 times at 0.5 atm for freon and at 0.8 atm for ethyl acetate in a rotary evaporator. The concentrates were then diluted in increments of 3-fold. Gas chromatography-olfactometry (GCO) using CharmAnalysis was conducted on the dilutions down to the concentration in which no aroma could be detected [26]. A GCO run consisted of a 1 vil injection into a 0.25 mm x 10 m column coated with 0.52 micron OVIOI methyl silicone in an HP5890 gas

73

Water at 95X Coffee Grinds 45.0 g

Coffee Filter

Cold Packs Water

Vacuum 4 in Hg Coffee Ice Bath

Figure 1. Diagram of quick brewing method.

chromatograph modified by DATU, Inc. The temperature was held at 35 °C for 3 min, programmed at 6 °C/min to 225 °C. The injector temperature was 200 °C and the detector was held at 225 °C. Retention times of all odor active compounds were recorded on a Macintosh^^^ computer and converted to retention indices by linear interpolation of the retention times of a series of 7-18 carbon paraffin standards run under identical conditions and detected with a flame ionization detector (FID) [27]. The retention times of the n-paraffins were measured before each series of analyses and periodically between GCO analyses to account for any changes in the column. The OVIOI column was used because it elutes most odorants at the lowest possible temperature and can be temperature-programmed at high rates to minimize sniffer fatigue [26]. The same human subject was used for all GCO analyses. Multiple measures of each GCO analysis were conducted on 2 replicates of the brewing methods and extraction procedure, comprising a total of sixteen sets of dilutions. The corresponding data from the two solvents were grouped together to total all the aromatic components of the coffee brew. The resulting dilution analyses were converted into Charm units (the areas of the peak in the Charm chromatogram) a unitless ratio proportional to

74

the amount of eluting stimulus divided by its odor-detection threshold [27]. Odor spectra were generated from the Charm data using an exponent of 0.5 and normalizing to the most potent odorant. Chemical identification of odor ants in the coffee samples was based on an exact match of odor character and retention index with that of an authentic standard [27] [28]. Gas chromatography-mass spectrometry (GC-MS) correlation of authentic standards verified the chemical identification. The GC-MS was conducted at 70 electron volts on a mass range of 33-300 M / Z in an HP5970. The HP5890 GC was programmed to heat isothermally at 35 °C for 3 min and then increase at 4 °C/min to 240 °C. The same column type used for GCO except twice the length (20m) was used for GC/MS. The injector temperature was 200 °C and the detector was held at 250 °C.

1 Liter Coffee Sample added 666 mL Freon 1 13TM stirred gently 30 min separated and dried over MgS04 Freon 113TM

- added 666 mL ethyl acetate - stirred gently 30 min - separated and dried over MgS04 Water

Ethyl Acetate

Discan

3

^Concentrate 243X ^ by rotovap

f Serial Dilutions by factor of 3X

Concentrate 243X^ by rotovap

Serial Dilutions^ by factor of 3X

f CharmAnalysisTM ^

r CharmAnalysisTM ]

(Ethyl Acetate CHARM]

(Freon 113 CHARM)

i

CHARM GROUP TOTAL

Figure 2. Flow summary of solvent extraction of the coffee brews.

75 3. RESULTS AND DISCUSSION The thirty most potent aroma chemicals detected in the coffee extracts (spectral values greater than 1.0%) are listed in Table 2. The 18 odorants identified were also among the most potent odorants detected in coffee by Aroma Extraction Dilution Analysis (AEDA) in three other studies [22-24, 29]. In all three studies 2-furfurylthiol and P-damascenone were among the top three most potent odorants found in coffee. As shown in Table 2, the odorants were the same in both brews although they were ranked somewhat differently. Table 3 shows that quantitative GCO data is very noisy since the ranking variation in spectral values contributed by multiple measures (Al, A2) is almost as great as the variation contributed by the replicate samples (Al, Bl). Therefore, the ranking data should be accepted as approximations and perhaps listed as "most potent groups," not individual compounds. These errors partially result from using a human subject as a GC detector. To compare the yield of the two methods, total Charm (sum of the peak areas in the Charm chromatogram) for each grouped chromatogram was logarithmicly transformed (for normalization) and compared using analysis of variance (ANOVA). A significant difference between the extracted aromas from the two methods was detected at p=0.03. The experimental brew produced 100% greater total Charm than the conventional brewing method. The challenge with comparison of individual chemical responses is that the system is over defined; there are more variables (intensity measurements) than there are cases (brewing methods and replications). It would not be reasonable to increase the number of cases due to cost and time of each experiment. Spectral data was used since cluster analysis strongly indicated an increase in charm values between the duplicate. Zero charm values were replaced with a calculated upper limit equal to 3 s where s was the standard deviation in the blank. For this data s was taken as the median standard deviation, 2.3. Any charm value below 6.9 was thus replaced with 6.9 [27]. The spectral data was arcsine square-root transformed. Six chemicals (methional, E-2-nonenal, sotolon, guaiacol, 5-methyl-6,7dihydrocyclopyrazine, and Furaneol) were selected because they all varied in the same direction. The selection was required to reduce the number of variables. A factor analysis using Statistica resulted in three factors with an eigenvalue greater than 1.0 and also exhibited an apparent cut off on a Scree plot. The resulting factors explaining 86% of the variation were varimax rotated (Table 4). Multivariate analysis of variance (MANOVA) was conducted considering brewing method and duplication with factor 2 and 3. There was an overall intensity increase in the data from the first run to the duplicate. Based on the MANOVA and the factor analysis, it can be concluded that there is a 280% (p=0.03) increase in concentration of methional comparing the conventional brewing method to the quick brewing method. Sotolon demonstrated a 167% increase and cis-2-nonenal demonstrated a 100% decrease at a significance level of 15%. Using discriminate analysis, methional and cis-2-nonenal showed a significant change (p=0.5).

76 Table 2 Aroma occurrences resulting from CharmAnalysis of two brewing methods of coffee. Retention times were converted to retention indices (RI) by linear interpolation of the retention times of the series of 7-18 carbon paraffin. CHEMICAL STIMULANT

sotolon P-damascenone 2-furfurylthiol 4-vinylguaiacol 2-methyl-3furanthiol vanillin guaiacol furaneol methional 3-methoxy-2isobutyl pyrazine unknown unknown 2,4,5trimethylthiazole Abhexon unknown unknown unknown 4-ethyl guaiacol 5-methyl-6,7dihydrocyclopentapyrazine unknown unknown 2-ethyl-3,5-din\ethylpyrazine cis-2-nonenal unknown unknown unknown unknown unknown 2-isopropyl-3methoxypyrazine 2,3,5trimethylpyrazine

RI

EXPERIMENTAL CHARM OSV

CONVENTIONAL CHARM OSV

81 98 100 62 89

DESCRIPT

toast fruit toast cloves nuts

1057 1349 881 1279 844

46200 41123 37226 22327 19701

100 94 90 70 65

13937 20266 21092 7998 16740

1335 1066 1033 863 1160

18899 16159 15152 14221 8378

64 59 57 55 43

10773 12641 7064 3950 2989

1502 1252 965

5331 5285 4973

34 34 33

2016 1117 3035

31 burnt 23 floral 38 plastic

1156 990 1403 1222 1250 1110

2977 2493 2059 2001 1692 1613

25 23 21 21 19 19

4086 1118 2006 856 2027 983

44 23 31 20 31 22

1285 850 1045

1507 1280 907

18 17 14

808 1107 1295

20 cloves 23 stinky 25 burnt

1132 1206 1142 908 984 803 1076

866 865 656 589 547 480 464

14 14 12 11 11 10 10

1585 576 495 392 357 351 403

27 17 15 14 13 13 14

10

449

971

461

CAS NUMBER

28664-35-9 23726-93-4 98-02-2 7786-61-0 28588-74-1

71 vanilla 121-33-5 90-05-1 plastic n 58 caramel. 3658-77-3 3268-49-3 43 potato 24683-00-9 38 plants

13623-11-5

honey 698-10-2 plastic spice honey 2785-89-9 spice cotton 23747-48-0 candy

18138-04-0

18829-56-6 toast licorice cereal nutty plastic skunk green 25773-40-4

15 toast

14667-55-1

77

Table 3 Comparison of spectral results from GCO multiple measures (1 and 2) and brewing replicates (A and B) for the experin\ental brewing method extracts for the ten most potent components. Data are combined results from ethyl acetate and Freon 113^^ fractions. AROMA CHEMICAL A l 2-furfurylthiol p-damascenone 2-methyl-3-furanthiol sotolon guaiacol vanillin 4-vinylguaiacol furaneol methional 3-methoxy-2-isobutyl pyrazine

A2

100 19 56 lb 6 32 62 5 37 19

71 31 20 67 29 75 100 28 35 19

Bl 66 34 50 100 23 33 57 27 23 15

B2 100 35 13 31 11 40 7 14 11 10

STDev multiple measures 34 12 7 30 18 13 36 21 18 6

STDev replicates

STDev All

32 9 39 30 21 33 45 21 6 2

18 7 21 29 11 20 38 11 12 4

Table 4 Factor loading (variance maximized rotated) from factor analysis of selected chemicals' arcsine square-root transformed spectral data. AROMA CHEMICAL

FACTOR 1

FACTOR 2

FACTOR 3

methional furaneol sotolon guaiacol 5-methyl-6,7dihydrocyclopyrazine E-2-nonenal

-0.8 0.9 -0.1 0.9 0.6

0.0 -0.2 0.7 0.2 0.6

0.96 0.1 0.5 -0.3 -0.2

0.0

0.8

0.0

4. CONCLUSIONS CharmAnalysis and AEDA detect the same important aroma chemicals in coffee but variability in the data makes it difficult to obtain exact orders of importance. The experimental brewing method described here should minimize errors by providing better control of time and temperature. Although quantitative GCO is more error prone than other chemical measurements, it is useful for understanding the affects of various treatments on coffee aroma and provides direction for more precise chemical analysis such as isotope dilution analysis. 5. ACKNOWLEDGMENTS We are grateful for the financial and sample support from Nihon Tetra Pak.

78

6. REFERENCES 1. S. Braun, Buzz, The Science Lore of Alcohol and Caffeine. 1996, New York: Oxford University Press. 2. Krups, The Encyclopedia of Coffee and Espesso From Bean to Brew. 1995, Chicago: Trendex International, Inc. 160. 3. R. J. Clarke, and R. Macrae, Chemistry. Coffee. Vol. 1. 1985, New York: Elsevier Applied Science. 306. 4. Research Alert, Oct. 18 (1996) 5. C. A. National, Automatic Merchandiser, (1995) 38. 6. E. Maras, Automatic Merchandiser, (1996) 28. 7. T. Neuhaus, The Informed Baker.1996, Ithaca, NY: Cornell University. 8. W. Holscher, and H. Steinhart, Thermally Generated Flavors, Maillard, Microwaves, and Extrusion Processes, T. Parliment, M. Morello, R. McGorrin, Editor, (1994), American Chemical Society, 207-217. 9. W. Baltes, and G. Bochmann, Z. Lebensm Unters Forsch, 185 (1987) 5-9. 10. R. Tressl, Thermal Generation of Aromas,, (1989), ACS, 293-301. 11. I. Flament, and C. Chevallier, Chemistry and Industry, (1988) 592-596. 12. N. Imura, and O. Matsuda, Nippon Shokuhin Kogyo Gakkaishi, 39 (1992) 531-535. 13. A. Stalcup, K. Ekborg, M. Gasper, D. Armstrong, J. Agric. Food Chem., 41 (1993) 1684-1689. 14. W. Holscher, O. G. Vitzthum, H. Steinhart, The Cafe Cacao, XXXIV (1990) 205-212. 15. I. Blank, Sen, A., W. Grosch, Z. Lebensm Unters Forsch, 195 (1992) 239-245. 16. C. A. B. De Maria, L. Trugo, R. Moreira, C. Werneck, Food Chemistry, 50 (1994) 141-145. 17. W. Grosch, Trends in Food Sci. & Tech., 4 (1993) 68-72. 18. N. K. O. Ojijo and P. B. Coffee Research Foundation, Ruiru, Kenya., Kenya Coffee, vol. 58 (685) (1993) p.1659-1663. 19. N. qijo, Kenya Coffee, 58 (1993) 1659-1663. 20. O. Vitzthum, C. Weisemann, R. Becker, H. Kohler, The Cafe Cacao, XXXIV (1990) 27-32. 21. A. Williams, and G. Arnold, J. Sci. Food Agric, 36 (1985) 204-214. 22. P. Semmelroch, G. Laskawy, I. Blank, adn W. Grosch, Flavour and Fragrance J., 10 (1995) 1-7. 23. P. Semmelroch, and W. Grosch, J. Agric. Food Chem., 44 (1996) 537-543. 24. I. Blank, A. Sen, and W. Grosch, ASIC. 14 Colloque, San Francisco, (1991) 117-129. 25. T. Lee, R. Kempthorne, J. Hardy, J. of Food Sci., 51 (1992) 1417-1419. 26. T. Acree, J. Barnard, D. Cunningham, Food Chemistry, 14 (1984) 273-286. 27. T. Acree, and J. Barnard, Trends in Flavour Research, H. a. D. G. v. d. H. Maarse, Editor, (1994), Elsevier, 211-220. 28. L. Ettre, Chromatographia, 7 (1974) 38-46. 29. P. Semmelroch, and W. Grosch, Leben. Wiss.u-Technol, 28 (1995) 310-313.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

79

Electronic nose versus multicapillary gas chromatography: application for rapid differentiation of essential oils T.Talou, S. Maurel and A. Gaset Agro-industrial Chemistry Laboratory (UA ESFRA 31A1010), National Polytechnic Institute of Toulouse, ENCST 118 route deNarbonne, 31077 Toulouse Cedex4, France

Abstract Whithin the past five years, there has been a rapid development of electronic nose technology, i.e. multi gas sensor devices coupled to statistical results data processing, which provides the advantage for faster differentiation of complex mixtures of volatile compounds as compared to gas chromatography. A comparative study on the differentiation of essential oils representative of the major aromatic notes of « The Field of Odors® » [1,2] by electronic nose, equipped with an array of conducting polymers gas sensors and by gas chromatography was carried out. The new concept of multicapillary column allowing reduction of time analysis to a few minutes was used in this study.

1. INTRODUCTION Considerable interest has been expressed over the last ten years in the use of gas sensors together with associated pattern recognition technique to differentiate and identify complex mixtures of volatile compounds [3]. The major detection principle of such apparatus known as "electronic noses" (EN) is based on the reversible electrical resistance changes of the sensing elements (metal oxides or conducting polymers) in the presence of volatiles and on-line computerized statistical processing of the data (FDA, PCA, ANN, fuzzy logic, etc..) [4-6]. A number of publications have reported the application of different prototypes or commercial devices for odors differentiation of industrial products (raw, extracts, processed, packaged, etc ,...) [7-18], includingflavoringsand plant extracts (essential oils, concretes, oleoresins, etc ...) [19,20], but only a few have compared the efficiency of such potential alternative methods to classical ones, especially gas chromatography (GC) [ 21,22]. The main advantage of an electronic nose is the speed of the analysis (5-15 min) in comparison with GC methods (4590 min). Recently, multicapillary columns, i.e. a combination of 900 liquid-phase coated 40 (im capillaries in a single glass tube, are reported as being able to reduce the analysis time without sacrificing sample loading, resolution and efficiency.

80

In continuation to our previous research on differentiation of essential oils , the present study reports use of electronic nose with conducting polymers gas sensors (ENCPGS) versus gas chromatography with dynamic headspace concentration and multicapillary column separation (MGCDHC) for the differentiation of 32 different descriptors of 7 odors notes, belonging to a traditionnal flavorist's osmotheque.

2. MATERIALS AND METHODS 2.1 Aromatic samples Eight odors notes particularly used in perfumes and flavorings formulations were selected in our own osmotheque. It is a collection of natural extracts and synthetic molecules formulated according to both the professional olfactory reference work « The Field of Odors® » [1,2] and to the recommendations of the famous perfumers Carles[23] and Roudnitska [24,25]. These notes (anise, balsamic, minty, resineous, rustic, spicy, woody) are described by 32 different chemotyped essential oils and represented by 96 samples provided by 3 different suppliers (BERDOUES, Cugnaux, France; CRMM Lab., Toulouse, France; NATURLAND, St. Laurent du Var, France) in glass screw top container and stored at room temperature (25°C). Constituents of odors notes are provided below. Anise notes; Basil (Ocimum basilicimi), caraway (Carum carvi), cumin (Cuminum cyminum), estragon (Artemisia dracunculus) Balsamic notes; Copaiba balsam (Copaifera officinalis), roman chamomile {Anthemis nobilis\ cistus (Cistus ladaniferus\ Sumatra benzoin (Styrax benzoin), Minty notes; Commint {Mentha arvensis), green mint {Mentha spicata), peppermint {Mentha piperita), Poulio mint {Menthapulegium), sweet mint {Mentha suavolens) Resineous notes; Myrrh {Commiphora molmol), elemi {Canarium luzonicum), olibanum {Boswellia carterii), g2\bdimm\{Ferulagummosa) Rustic notes: hdi>/Qn6QX {Lavandula angustifolia), lawsindin {Lavandula hybrida), laurel (Laurus nobilis), hyssop (Hyssopus officinalis) Spicy notes; Pimenta berry {Pimenta dioica), canella {Cinnamomum verum), nutmeg {Myristicafragrans), black pepper {Piper nigrum), clove bud {Eugenia caryophyllus) Woody notes; Cedarwood {Cedrus atlantica), patchouli {Pogostemon cablin), pine {Pinus pinaster), WQXXVQX {Vetiverazizanoides), guaisicwood{Bulseniasarmienti)

2.2 Multicapillary Gas Chromatography with Dynamic Headspace Concentration (MGCDHC) Analysis were performed using a dynamic headspace injector DRI apparatus (Perichrom, Saulx-les-Chartreux, France) coupled to a gas chromatograph DN 200 (Delsi Instruments, Paris, France). Sample preparation: The DRI device was directly connected to a specially designed glass cell (250 mL capacity) in which 5^1 of essential oil was deposited with a microsyringe on a testing-strip. After static equilibration (15min), volatile compounds were concentrated on a Tenax TA trap, cooled at -20°C by circulation liquid nitrogen with a scavenger gas (Helium) at

a flow rate of 30 mL/min at room temperature (25°C) for 2 min. The trap was then heated to 250°C allowing direct injection of the volatiles into the multicapillary GC column. Sample analysis; The GC separation was performed on a multicapillary capillary wall coated column (AUtech CBWax 20M Multicapillary, Im length x 0.2^m film thickness , 900 capillaries X 43 ^im LD. each). The oven temperature was isothermal at 60°C. Column inlet pressure of carrier gas (helium) and splitter flow rate were respectively fixed at 17psi and 70mL/min. The FID temperature was 230°C. Each analysis was replicated three times. Data analysis: The recorded GC profiles were used as 'Finger Print' for direct comparison and differentiation of the essentials oils. Statistical data processing (PCA) was performed using STATBOX software (GRIMMER, Paris, France)

2.3 Electronic Nose vnth Conducting Polymer Gas Sensors (ENCPGS) The analysis were performed with an Aroma Scanner A20S/A8S (AromaScan pic, Crewe, U.K.), i.e. an analyzer system using an array of 20 conducting polymer gas sensors [26], and a sample station, the complete device being monitored by a dedicated software including data processing. The sample headspace was generated at a set temperature to reach equilibrium in a heated oven , a so-called sample station. After equilibration, the pouch was purged by a vacuum pump in order to deliver the headspace to the sensor array in a dynamic mode. The sensors were then cleaned and the sensors were made ready for the next sample. Sample preparation Essential oil (5|il) was deposit with a microsyringe on a testing-strip placed in a plastic pouch which was filled with 500 mL of purified air and then topped with a tight teflon cap. The pouch was first placed in the oven of the sample station at 25°C for 15 min for equilibration and then connected to the analyzer injection port. Sample analysis: The pump flow rate was fixed at 200 mL/min. The acquisition parameters, i.e. detection threshold and sampling interval were respectively fixed at 1.5 and Is. The sequence of analysis was: reference gas, 10s; sample, 120s; wash (2%butanol/ 98%water), 60s; reference gas, 120s. Carrier gas and reference gas were purified air. Each analysis was replicated three times. Data analysis.The slice section of the sampling time from which the database files were created was T= 40s to T=100s. These databases, based on the normahsed sensors response profiles, were averaged. Statistical data processing (PCA) was performed with STATBOX software (GRIMMER, Paris, France) The responses measured for the two techniques were: i) the capacity of differentiation of the 4-5 different samples of essential oils for each aromatic note, ii) the reproducibility of the technique itself (triplicated measures), iii)the variability of the essential oil content according to the suppliers origins.

82

3 . RESULTS AND DISCUSSION 3.1 Multicapillary Gas Chromatography with Dynamic Headspace Concentration (MGCDHC) Typical GC profiles obtained respectively with a multicapillary column and classic capillary column for IsLVQndin (Lavendula hybrida) essential oil are shown in Figure 1. By reducing the analysis time by a factor 10 without dramatically sacrificing resolution and efficiency, analysis carried out with multicapillary column allowed differentiation of the 4 descriptors of the rustic note on the basis of direct comparison of their Fingerprints. The distinct cluster populations resulting from the statistical data processing (retention times and peak area of the major compounds) as reported on Figure 2 clearly show: i) the good reproducibility of the method, despite the use of a manual dynamic headspace concentrator, ii) for this aromatic note, the absence of variability in the essential oils content according to their supplier origin, iii) a correct differentiation of the descriptors in spite of closed cluster populations due to a similar qualitative chemical composition of analyzed samples of lavender, lavandin, laurel and hyssop, i.e., linalool, linalyl acetate, camphor and eucalyptol.

1

/^

Sx

F2

+0,6 +0,4 Lavandin

Laurel

+0,2

.-0,2

V ^ ^ ^

Lavender

-0,4 -0,6

Hyssop

'

1 -0,8

\

H

h-

-0,6 -0,4 -0,2

;

+-

H — .— 1

1

!

+0,2 +0,4 +0,6 +0,8

Figure 2. Differentiation of rustic note descriptors by MGCDHC (3 different samples represented by symbols were analized three times for each chemotyped essential oil).

For the 6 other aromatic notes analyzed, the differentiation of their descriptors was successfully performed on the basis of their fingerprints, particularly for resineous, spicy and woody notes. Nevermind, for anise and minty notes, the variability intra descriptors, i.e. between the three samples of a same chemotyped essential oil, was quasi-equivalent to this one inter descriptors, i.e. between the different essential oils. Consequentively the dispersed and closed cluster populations did not allow to discriminate the different products with the high security level required for Quality Control purposes.

83 3.2 Electronic Nose with Conducting Polymer Gas Sensors (ENCPGS) Contrary to GC, electronic nose is not an intrinsically selective technique via column separation and/or specific detector, but a global method which needs a statistical data processing to allow classification or differentiation of samples. Consequently, if the direct comparison of the responses curves did not allow to clearly differentiate the descriptors of the same aromatic note, the normalized patterns may do so. But their differentiation and subsequent identification must be set up after statistical data processing, i.e. Principal Component Analysis , as recorded for rustic notes descriptors (Figure 3). In this case, the 4 distincts cluster populations obtained were closer than in the previous study, mainly due to the lower reproducibility of the method. Indeed, the control of the headspace generation in pouches was difficult in spite of the control of the thermodynamic equilibrium parameters (temperature and time) and sensors themselves appeared to vary over time (aging, pollution and/or hypersensibility to humidity). This was confirmed by analysis carried out at three months of intervals which were significantly different from those obtained the first time. The reduction of the number of sensors in the patterns used for the data processing i.e. selection of the more stable, sensitive and selective sensors, in order to limit this variation in time, did not allow increase in the reproduciblity rate.

-0,8 -0,6 -0,4

-0,2

+0,2 +0,4 +0,6 €,8

Figure 3. Differentiation of rustic note descriptors by ENCPGS (3 different samples represented by symbols were analized three times for each chemotyped essential oil).

Similarly, the problem of reproducibility was encountend and affected the differentiation of other aromatic notes, especially when heterogenity of the essential oils of the same genus was considered, i.e. anise and minty notes. On the other hand, no differentiation axis based on an increasing content of a key compound could be reported for any notes, as it was the case in previous preliminary studies [20].

84

4 . CONCLUSIONS In this study, the comparative use of MGCDHC and ENCPGS for the differentiation of 32 descriptors of 7 odors notes of the olfactory reference work « The Field of Odors® » allowed: i) confirmation of the strong relationship between the results obtained by head-space-GC and by electronic nose; ii) showing the value of multicapillary columns for rapid fingerprints comparison; iii) reporting that at equivalent analysis time multicapillary GC is in direct competition with the electronic nose; iv) demonstrating the necessity of increasing the reproducibility of electronic noses by abetter control of both headspace generated (with an headspace autosampler for example) and sensors themselves (robotized fabrication of the sensors, temperature control of the sensors array and hydrophobation of its surface, etc.).

Acknowledments This work is a part of the 'FLAVOR 2000' program carried out by the 'Electronic Nose Department' of the CATAR-CRITT Agroressources (Technological Research Centre of INPT-ENSCT) and sponsored by the Midi-Pyrenees County Council. The authors thanks Mrs. M. Doumenc, S. Breheret and B. Bourrounet for their participation to the present work

5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12

13 14

J.N. Jaubert, G. Gordon and J.C. Dore. Parfums, Cosmetiques, Aromes, 78, (1987)71. J.N. Jaubert, C. Tapiero and J.C. Dore, Perfiimer&Flavorist, 20 (1995) 1 T. Talou, Internet Web Site, http://www.inp-fc.fr/cirano (1997). J.W. Gardner and P.N. Bartlett,« Sensors and sensory systems for an electronic nose », NATO ASI Series, Kluwer Acad. Pub., London, (1992) 317. K.Persaud, Analytical Proceedings, 28 (1991) 339. K. Persaud and P.Pelosi, « Sensors and sensory systems for an electronic nose », NATO ASI Series, Kluwer Acad. Pub., London, (1992) 237. T. Aishima, J. Agric. Food Chem, 31 (1991) 752. J.L. Berdague and T. Talou, Sciences des Aliments, 13 (1993) 141. M. Egashira, Y. Shimizu and Y. Takao, Sensors and Actuators B, 1 (1990) 108. J.W. Gardner, H.V. Shurmer and T. T. Tan, Sensors and Actuators B, 6 fl992) 71. J.W. Gardner and P.N. Bartlett, « Olfaction and taste XI », Springer-Verlag, Tokyo, (1994) 690. R. Olafsson, E. Martinsdottir, G. Olafsdottir, P.I. Sigfusson and J.W. Gardner, « Sensors and sensory systems for an electronic nose », NATO ASI Series, Kluwer Acad. Pub., London, (1992) 257. F. Windquist, E.G. Homsten, H. Sungren and I. Lundstrom, Meas. Sci. Technol., 4 (1993) 1493. B. Bourrounet, T. Talou and A. Gaset, Sensors and Actuators B ,26-27 (1995) 250.

85 15 C. Di Natale, F. Davide, A. d'Amico, G. Sberveglieri, P. Nelli and G. Faglia, « Current status and future trends », Proceedings EURO FOOD CHEM VIII, (1995) GOCh, Vienna, 131. 16 B. Bourrounet, T. Talou and A. Gaset, Odors&VOC's J., 1 (1996) 334. 17 J.F. Clapperton, Odors and VOC's J., HS (1996) 22. 18 S. Breheret, T. Talou and A. Gaset, « Bioflavor'95 », Ed. INRA, Paris, (1995) 103. 19 B. Bourrounet, M. Cazagou and T. Talou, Rivista Italiana EPPOS, HS An.96 (1996) 566. 20 B. Bourrounet, T. Talou and A. Gaset, Odors and VOC's J., HS (1996) 34. 21 T. Talou, B. Bourrounet and A. Gaset, 2nd Int. Symp. Olfaction & Electronic Nose, Toulouse, France (1995) 22 T. Talou, J.M. Sanchez and B. Bourrounet, « Flavor Science:recent developments » A.J. Taylor and D.S. Mottram (eds), RSC, Cambridge, (1996) 277. 23 J. Carles, Recherches, 11 (1961) 8. 24 E. Roudnitska, Parfums, Cosmetiques, Aromes, 115 (1994) 47. 25 E. Roudnitska, Parfums, Cosmetiques, Aromes, 116 (1994) 45. 26 K. Persaud and P. Pelosi, WO Patent No.O 1599 (1986)

o a

^

I

O

CD

p

^

II

I-

I-

3

O <

O p^

B n

CD

C

0 3 ^

Ui

p

3 El

oil

sIs

1

c 3 -5

3

C1>

3: p

3

I'

CM

3

1=

O a B

E. 3 'hi-

^2 f^, o. . o •^ 2 . 3 Q r P 3 S^ p 3 to

o O

GO

W

3 o OS o r^ o ^ 3. 3

o

to K) O o

o

86

®

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

87

Quantitation of Potent Food Aroma Compounds by Using Stable Isotope Labeled and Unlabeled Internal Standard Methods M. Preininger Kraft Foods, Technology Center, 801 Waukegan Road, Glenview, IL 60025, USA

Abstract Potent food aroma compounds were quantified at ppb concentration level via Gas Chromatography / Ion Trap Mass Spectrometry in chemical ionization mode. Some analytes were selected based on the results of Aroma Extract Dilution Analysis of a food aroma source. These aroma compounds have been found in many different foods by other researchers. Quantitation results obtained from using synthesized stable isotope (deuterium) labeled standard compounds are compared with those obtained from using unlabeled internal standards in the same experiment. The isotope standards are analogous in chemical structure to the analytes. Advantages and disadvantages of the two different quantitation methods are discussed.

1. I N T R O D U C T I O N 1.1. Why quantify potent aroma compounds? In modern systematic food aroma analysis, potent aroma compounds are first detected by Gas Chromatography/Olfactometry methods [e.g. Aroma Extract Dilution Analysis, AEDA; Charm® Analysis; Osme; see (1, 2, 3)], and then identified. Quantitation of these potent odorants is necessary to determine whether they are contributing to a specific food flavor as important characteristic aroma compounds. Odorants exceeding their odor threshold concentrations manyfold are such aroma impact compounds and can be used as indicators to objectively describe food flavor quality. The study of their formation from precursors by using quantitative data may help to improve food flavor quality by optimizing food processing conditions to enhance formation of desired aroma compounds and to reduce formation of off-flavor compounds. Quantitative data of aroma impact compounds are also used by creative flavorists as a starting point for food flavor composition.

88

1.2. Quantitation methods As many aroma impact compounds occur at low ppb (jag/kg) concentration levels, reliable and sensitive quantitation methods are necessary. Using stable isotope labeled standards in Gas Chromatography/Mass Spectrom_etry (Stable Isotope Dilution Assay, SIDA) was shown to be a very accurate but expensive quantitation method because labeled standards, which are analogous in chemical structure to the analytes, must be synthesized (4, 5). However, analyte recovery factors depending on the particular aroma compound isolation method do not have to be determined in SIDA. On the other hand, usage of inexpensive unlabeled internal standards (UIS) in GC analysis requires determination of combined recovery and response factors (6) and are reported to yield concentration values considered of order of magnitude accuracy only (7). A method such as SIDA, independent from the sample preparation procedure, is desired for reliable key aroma compound quantitation.

2. OBJECTIVE How reliable is a simplified method? The purpose of this study is to gain an insight into the magnitude of error in quantitation of potent food aroma compounds when, instead of stable isotope labeled standards (SIDA), unlabeled internal standards (UIS) are used in GC/MS analysis without recovery factor determination. This information may be helpful in deciding when SIDA is recommended and when UIS provides results comparable to those obtained using SIDA. Costs and time required for purchase and synthesis of labeled standards, respectively, may be minimized.

3. METHOD Based on the results of AEDA of a food flavor source, potent aroma compounds occurring in many foods were selected as analytes. They were isolated from this complex food matrix and quantified at ppb concentration levels via SIDA and UIS in the same experiment using an ion trap GC/MS system in chemical ionization mode. Deuterium labeled standards were synthesized for SIDA. GC/MS response factors for UIS vs. analytes were determined for quantitative calculation.

89 4. EXPERIMENTAL 4.1. Synthesis of deuterium labeled standards [6,6,6-^H3\-hexanal (ds-hexanal) 2-(4-chlorohutyl)-l,3-dioxolane (I) was converted to 2-(4-iodobutyl)- 1,3-dioxolane (II) according to (8). II was deutero-methylated with ds-methylmagnesium iodide to III catalyzed by dilithium tetrachlorocuprate (8, 9). After hydrolysis the target compound, ds-hexanal, was isolated by distillation and purified by silica gel flashchromatography. Electron impact-mass spectra (EI-MS) data of ds-hexanal. m/z 85 (13 %, M-H2O+), 75 (20), 60 (17), 59 (61), 58 (19), 57 (33), 46 (21), 45 (45), 44 (100), 43 (49), 42 (31), 41 (35).

Nal; DMSO/toluene

cr \ ^ -s/ o

•

,/"\/^\x/^o

18-crown-6 (')

(11)

DsCMgl

O^A^

H2SO4 •

n ^

THF; L»2CuC|4

^

^

O

^ (III)

Figure 1.

Synthesis route oi [6,6,6-^Hs]-hexanal (ds-hexanal)

[^Hs}-3-(methylthio)vropanal (ds-methional) In a simplified method of (10), 3-mercapto-l-propanol (IV) was deuteromethylated by Grignard-Reaction with ds-methyliodide to ds-(methylthio)propanol (V) using phase-transfer catalysis. The alcohol V was then oxidized with pyridinium chlorochromate (PCC) to ds-methional and purified by silica gel flashchromatography. Electron impact-mass spectra (EI-MS) data of ds-methional: m/z 107 (43 %), 79 (28), 64 (28), 59 (11), 58 (10), 51 (100), 50 (20), 49 (21), 46 (23).

90

D3CI; KOH •

CHCI3,BU4NHS04 (IV)

(V)

PCC

Figure 2.

Synthesis route of [^H3\-3-(methylthio)propanal

(ds-methional)

[l,2-^H2and 3\'l-octen-3-one (d2/3-l-octen-3-one) Similar to (5) l-octyn-S-ol was catalytically deuterated to [1,2-^H2 and 3]'l-octen-3oly oxidized to d2/3-l-octen-3-one with pyridinium chlorochromate (10), and purified by siUca gel flash-chromatography. Byproducts, dn-3-octanones, could not be separated but do not interfere with d2/3-l-octen-3-one in GC/MS analysis. Electron impact-mass spectra (EI-MS) data of d2/3-l-octen-3-one: m/z 100 (14 %), 99 (17), 98 (11), 97 (13), 86 (13), 85 (10), 73 (97), 72 (72), 58 (100), 57 (82), 43 (43). \2,3-^H2y(E)-2-nonenaUd2-CE)-2-nonenal) This was synthesized according to (5). Electron impact-mass spectra (EI-MS) data of d2-(E)-2-nonenal: m/z 141 (0.3 %, M-1+), 124 (4), 113 (7), 99 (14), 97 (16), 85 (40), 84 (31), 72 (54), 71 (41), 59 (29), 57 (33), 55 (57), 43 (100), 41 (79). [^H4\-(E.E)-2A-decadienal and [^Hel-dimethvltrisulfide They were prepared by H. Guth and Ch. Milo, respectively. Unlabeled aroma standards and chemicals were purchased from Aldrich, Milwaukee, WI or Bedoukian, Danbury, CT. 4.2. Concentration of deuterium labeled standards, Response factors The concentration of the labeled standard solution was determined by GC/MS using the analogous, pure unlabeled compound as external standard at a peak area ratio of 1.0 for selected Cl-ion masses (see Table 2) of labeled/unlabeled compounds. A peak area ratio range of 0.15 to 14 (see Figure 3) shows a quantitation method error of maximum 10 % from the concentration value of e.g. d3-methional determined at a 1.0 ratio. The same GC/MS tuning conditions were applied as for the quantitation of the aroma compounds in the food sample. Therefore the response factor for labeled standard vs. analyte was assumed to be and set as 1.00 for the quantitative calculations of the sample.

91 4.3. Response factors for unlabeled standards vs. analytes A mixture of equal amounts of the unlabeled internal standards, 4-heptanone and 4-decanone (see Table 2), and the analytes (see Table 2 and 3), was analyzed by GC/MS under the same tuning conditions as applied for the quantitation of the aroma compounds in the food sample. Selected Cl-ion masses and calculated response factors are listed in Table 2 and 3.

MS-response 70 65 3 60 E o) 55 50 45 40 35 30 25 20

0.1

1 peak area ratio 10 (dS-methional / methional)

100

Figure 3. MS response expressed as concentrations of the ds-methional standard solution determined via GC/MS-CI using methional as external standard at different ratios vs. ds-methional (response factor set as 1.00).

4.4. Sample preparation The sample (ca. 250 g powdered flavor source; 56 % lipids, 30 % protein, 3 % water) was reconstituted to 35 % water and stirred (3 h at RT) under argon atmosphere with diethyl ether which was spiked with the internal deuterium labeled and unlabeled standard compounds (see Table 1). The ether extract was separated from solids by centrifugation, concentrated (40 °C), and high vacuum distilled at 2 to 4 x lO-^ Torr for 1.5 h (40 °C) and for 1.5 h at 60 °C. The neutral/basic volatile fraction was obtained by washing the distillate with sodium bicarbonate (0.5 mol/L), and concentrated to 3 mL by Vigreux-distillation for GC/MS quantitation of hexanal, 2- and 3-methylbutanal (see Table 2, # 1; Table 3, #12, 14). The sample was concentrated further to 200 |LIL by micro-distillation

92 for quantitation of 2-, 3-methylbutanal compounds.

(see Table 3; #13, 15) and for all other

Table 1 Stable isotope (deuterium) labeled and unlabeled internal standard compounds used for quantitation of potent food aroma compounds standard

total amount (jiig) added to 251.2 g sample

da-hexanal da-methional de-dimethyltrisulfide d2/3-l-octen-3-one d2-(E)-2-nonenal d4-(E,E)-2,4-decadienal 4-heptanone 4-decanone

800 18.65 3.97 1.71 23.36 8.58 3.01 2.00

Gas C h r o m a t o g r a p h y / M a s s S p e c t r o m e t r y (GC/MS) GC: column:

temp, program: carrier gas: injection:

MS-CI:

MS-EI:

GC 3400 (Varian, TX) equipped with DB5 fused silica capillary, 30 m x 0.32 mm i.d. x 0.25 jam film thickness, (J&W, Folsom, CA), head connected to a retention gap (3 m X 0.53 mm i.d., deactivated) 35 °C hold 2 min - ramp at 40 °C/min to 50 °C hold 2 min ramp at 6.0 °C/min to 230 °C hold 10 min. He, head pressure 6 psi, capillary flow 30 cm/s (230 °C) Direct cold on column injection of the sample (0.2 to 1.5 )LIL) using a Septum Programmable Injector (temp, program same as column) ITS40 (Finnigan, Atlanta, GA), Magnum-CI, methanol as reagent gas for chemical ionization, MeOH-CI 65-250 for Table 3; #12, 13, 14, 15; MeOH-CI 80-250 for all other numbers Mass selective detector (MSD 5970, Hewlett Packard), electron impact ionization at 70 eV for identification of synthesized isotope labeled standard compounds

93 5. R E S U L T S A N D D I S C U S S I O N 5.1. Comparison of SIDA and UIS In Table 2 the concentration values are listed for hexanal (#1), methional (#2), dimethyltrisulfide (#4), l-octen-3-one (#6), (E)-2-nonenal (#8) and (E,E)-2,4decadienal (#10) calculated from their corresponding deuterium labeled standards (SIDA). These data are regarded as the most accurate ones and are compared with the values derived from calculation using the unlabeled standards, 4-heptanone or 4-decanone (UIS). UIS values are 80, 44, 84, 73 and 66 % lower than SIDA values. Hexanal could be quantified only by SIDA (from 3 mL sample volume, see section 4.4.) as its quantity overloaded the GC/MS system when 4-heptanone was recorded at reasonable intensity (from 200 |aL sample volume). No other aldehyde or ketone (e.g. 4-octanone) standard eluting close to hexanal and applicable at suitable amounts for hexanal quantitation was found since saturated and unsaturated aldehydes derived from lipid peroxidation occurred naturally in the sample already. The concentration of methional determined by UIS is 80 % lower vs. the value from SIDA. Methional is obviously recovered at a much lower degree during sample preparation than the standard, 4-heptanone. An extremely low recovery of 1 % was reported for the unstable methional by Buttery (6), which caused difficulties in the quantitation of this important aroma impact compound without using SIDA (6, 11). Figure 4 demonstrates that SIDA allows accurate GC/MS quantitation of methional by extraction of the Cl-ion traces of methional (B) and ds-methional (C) from the total ion chromatogram (A) when an interfering compound (D) is present. pro SIDA: • enables quantitation of different compounds at wide concentration range in the same experiment • enables accurate quantitation of unstable compounds of low recovery con SIDA: • requires expensive or/and time consuming synthesis of stable isotope labeled standards pro UIS: • cheaper than SIDA as standards are commercially available con UIS: • without recovery factor determination analyte concentration values from UIS may deviate over an order of magnitude from SIDA values • recovery factor determination should be carried out with standards in a medium identical to that of the analysis sample to deliver accurate factor values, which may be difficult or impossible

94

W

CO

O

CO

^ o t4-l

CO IC CD Gi Oi

O

q

CO

o 1—1 CD 00

^

^

^

(M 00

(N (N 00

(N 00

CO CX)

o o 1—1

\o^

^

/-^^ ^N*-^

CO

CX)

^

N

/^> :<,

a

S

§

i-H

a d cd X CO

'Ti

CC X! CD

TH

r-\

t1—1 TH

00 1—1

o O l y-A i > CD

c6 d

lO t>

'^ i> t>

o o

oq Tf

o o

o CO

05 CD

CO CO

O

^ t>

d

CM

O t>

o

CD 00

1—1

CO

\o

t>

fH 1—1

TJH'

C^l

o

q

i> io

rH

CO UO

^ 1—1 ^

rH

^

o r-\

00

UO CO CD

Cd

4^

O 0 T3

of

H

0

Cd

Tt

? (M^

H

Cd C! 0

©^ rH

O

a o ad c o 0

1—1 y-i

^ ^ 4

• *

0

rH

0

CD

^

Cd

1—1 1—1

^

1—1

CM 1—1

pj

0

o

fl a o (M a Cd o H ^.^ 0 (M

^ 4

o:>

0

CM 0 0

(M CO

CO (M

lO

4*

T—1

y-*,

^

0 II! 0 S=!

0

B

Tj^

^

fl 0

rH

00

fl o fl

CD t >

c; o

h 0

CO

so

T^

0 o ^J ad o c o 4J ft rH

O CO

^

l>

t>

UO 0 0

I> CM

o

^

\o l > CM lO

^

1—1

o fl cd -u ft 0

4

LO

o ^UO

1—1

^

1—1

o o

d d

1—1 1—1

(M l> <M

d

^ 1—1

o o

00

^I C

o TH l> <M 1—1

CM UO CO 1—1

t> (M

1—1

rH

o

IC

o 1—1

TH

CD CM

CO CM

0

3 ^- M 0

Ti> i

0

O i 00 CNj I C t > Tt^*

^

Tt<

o

^

T—1

^'

lO

CM 00

lb

T*!

IC

T^ UO

0

4J

'^

0

o o fl cd

^

13 •

s0 BCO

*S iif\

TJ

0

^

s 3 ,_O_S,

CZ3

fi O

Tj^

^a

0

>i ri:^ 4J

s CM CO

I—I

CO

cd

a cd cd 5=1

0

"3 cd

I—H

P CO

r2

0

P5

a. o o

4-3

cd

13

•I-H

>>

'4-3

^3 fH cd J3 o^

«)

o

a

a

o a cd

4^

o u CN cd

cd 0o H (In

o

0)

o r—I

o

CO

0)

o

m

CO

CO cq

(M 1-1

0 0 CO t - 00

a

CD

CD -«* 05 '«^' rH 00

T-{ 0 0 CD 0 5

o o CO 00

(M 00

l> 00

^

05 CD

^

T H 00 C
CO lO

0

^ 00 CM i C CO Tj^'

05 Tj^

a>

P.

cd

d o cd X 0 ^ ^ 4

cd

a o CD 1 r d Tt<

F—(

d

cd fl cd

cd fl cd 4^ ;3

^

•+J

;5 ^

t-

'^

CO

|>^ 1-H O l > CD (M

(N

O

00

Tt^

UO CO t > (M UO i C

(M

o uo r>

O

i d 00 O CM rH

05

d

o CO I > (M i C

(N

CO UO

d

cd

d cd o

O

CD

^

rH

•^

CD

/-~s

O 00 1—i

00

O

CO

TH

o

CD Ol T—1

T^

a

cd

^

CD

W

CD

a

CD •rH Cd

Cd

?4 g ^ (M g

S ^

^

f>i

1—1

^

^

1—1

05 CO rH

^

05

CO !M rH

o 1—1 f-H T H

CD

W

lO CD rH

^

1—1 l O CO CD (M CD CO r H r H rH

rH r H

o

a

cn

T^ o c
00 IC

TO

O

m cd a CD

g '^ 4 1—1 cd

a O

w

00 05 O rH t-H C^

^

CD

Q

0)

u cd

d cd cd d

0) CD

rQ

"d d

•r-( 4-i

d cd d CO

d d o ft

a o o

o

4-S

0) d cd o H OH

95

96 (m/z) IBBx

(A) TOTAL ION CHRONATOGRAM

TOIH M M,i

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

T

1

1

1

I I

I

I


I

I

I

I

I

I

T—I—I—I—I—I—r-

(B) riETHIONAL

104 j

IBsl 4.58x1 187

(C) D3-nETHI0NAL

188 63>d

I

I

I

I

I

I

I

1

r

I

1

1

1

I

I

I

1

I

I

I

I — I

I

I

I

I

(D) UNKNOUN COMPOUND

97 114 I

338 5:38

I

I

I

I

335 5:35

I

I

r

348 5:48

I — I

358 5:58

355 5:55

time (min)

Figure 4. GC/MS chromatogram (cutting) for quantitation of methional by Stable Isotope Dilution Assay (SIDA) using 6.3-methional as internal standard

5.2. UIS of homologous structure Also analyte concentration values derived from the standards 4-heptanone or 4-decanone are compared with those derived from analytes in the sample which are homologous in chemical structure (see Table 3) and whose concentrations were determined by SIDA (see Table 2) in the same experiment. To facilitate response factor determination without loss of valuable SIDA standards, they were not directly used for quantitation of compounds homologous in structure. Using (E,E)-2,4-decadienal (#19) for the quantitation of (E)-2-nonenal results in a value much closer to the one determined by SIDA (#18) compared to the one using 4-decanone (#20). In all cases (#13, 15, 17, 20, 22) where the ketones are used as standards, the analyte (aldehyde) concentration values are 30 to 73 % lower than those where structurally homologous standards are used. The latter are obviously recovered at a lower degree, closer to that of the analyte compared to the ketones.

97

pro/con UIS: If analytes are quantified with standards of homologous structure in UIS methods without recovery factor determination, analyte concentration values may be obtained which are relatively close to SIDA values. However, those UIS standards often cannot be applied without a combination with SIDA as they occur naturally in the analysis sample. A combination of SIDA and UIS is then necessary. 6. SUMMARY A N D CONCLUSION Quantitation of aroma compounds via unlabeled internal standards (UIS) can be two to six times less accurate than via stable isotope labeled standards (SIDA). SIDA is recommended for one-experiment quantitation of compounds at different concentration range, for unstable compounds, and compounds of low recovery in general. In the SIDA experiment, some analytes whose concentrations were determined by SIDA, may function as unlabeled internal standards for quantitation of other compounds homologous in chemical structure. GC/MS response factors are then applied. This method may very much improve the quantitation accuracy compared to UIS using standards which are different in chemical structure to the analytes, and when no recovery factors are determined. Expenses for synthesis of stable isotope labeled standards may be minimized for quantitation of analytes which are homologous in chemical structure, if different stable isotope labeled standards are only used for different functional groups.

7. REFERENCES 1 2

3

4 5 6 7 8 9 10 11

W. Grosch, Flavour and Fragrance Journal, 91 (1994) 47 T.E. Acree, In: Flavor Science, Sensible Principles and Techniques, T.E. Acree, R. Teranishi, eds., ACS Professional Reference Book, ACS, Washington, DC, (1993), 1 A.A.P. da Silva, D.S. Lundahl and M.R. McDaniel, Trends In Flavour Research, H. Maarse, D.G. van der Heij, eds., Elsevier, Amsterdam-LondonNew York-Tokyo, (1994), 191 P. Schieberle and W. Grosch, J. Agric. Food Chem., 35 (1987) 252 H. Guth and W. Grosch, Lebensm. Wiss. Technol., 23 (1990) 513 R.G. Buttery, D.J. Stern and L.C. Ling, J. Agric. Food Chem., 42 (1994) 791 R.G. Buttery, L.C. Ling and D.J. Stern, J. Agric. Food Chem., 45 (1997) 837 M. Iwamoto, N. Kubota, Y. Tagaki and K. Hayashi, Agric. Biol. Chem., 46 (9) (1982) 2383 H. Guth and W. Grosch, Lebm. Wiss. Technol., 26 (1993) 171 A. Sen and W. Grosch, Zeitschrift fuer Lebensmittel-Untersuchung und Forschung, 192 (1991) 541 K.R. Cadwallader, Q. Tan, F. Chen and S.P. Meyers, J. Agric. Food Chem., 43 (1995) 2432

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

99

Simplification of Complex Flavor Mixtures via Micro Extraction Class Separation Thomas H. Parliment Kraft Foods Technical Center, 555 So. Broadway, Tarrytown, NY, 10591

Abstract Isolation and identification studies covering natural materials are hampered by the complexity of the systems under investigation. For example, roasted coffee contains more than 800 identified aromatics, while cocoa and grapes have 500 and 450 respectively. Although efficiency of gas chromatographic columns have improved in recent years, complete resolution of all components of a complex material is not yet possible. One solution to this problem to pre-simplify the mixture before chromatographic analysis. Although this is commonly done on a macro-scale certain advantages resuh when this is done on a micro scale. This study evaluates a sequential micro-procedure for simplifying complex mixtures. The sample aromatics are concentrated and the first analysis performed. Subsequently, the sample is backwashed with base, acid and 2,4-dinitrophenylhydrazine (2,4-DNPH), and re-analyzed. Each stage eliminates another layer of confounding peaks, revealing a new set of compounds. All extractions are performed on the same ethereal solution, and in the same micro-mixing device. This systematic approach is applied to a model system and a coffee sample. Odor assessing may be used as an adjunct to pinpoint compounds of critical importance.

1. INTRODUCTION Sample preparation techniques in the flavor field have been discussed in books since 1971 (1) to as recently as 1996 (2). Although many new procedures have been developed, the topic still engenders lively discussions and technical developments. The purpose of this chapter is to describe a new technique which has been developed in our laboratory for the isolation and simplification of samples prior to analysis by gas chromatography. This technique is easy to employ, requires a minimum of equipment and will produce reproducible, meaningfiil results. Samples produced by this technique are particularly amenable to organoleptic analysis. As has been described previously (3) sample preparation is complicated by a number of factors. 1.1. Concentration Level The levels of the aromatics is generally low, typically in the ppm, ppb or ppt level.

100 1.2. Matrix The sample frequently contains non-volatile components such as lipids, proteins and carbohydrates, which complicates the isolation process. These components may create problems of foaming and emulsification during isolation procedures, and will create artifacts if injected into a hot gas chromatographic injection port. 1.3. Complexities of Aromas The aromatic compositions of foods are frequently very complex. For example, coffee currently has 800 identified components and wine has more than 600. Complicating the picture is the fact that the classes of compounds present covers the range of polarities, solubilities and pHs. 1.4. Variation of Volatility The components possess boiling points ranging from well below room temperature to those which are solids 1.5. Instability Many components in an aroma are unstable and may be oxidized by air, degraded by heat or extremes of pH. 2. BACKGROUND One of the most common sample preparation techniques employed today involves steam distillation followed by solvent extraction. The distillation may be carried out at atmospheric pressure or under vacuum. While the primary advantage is that the distillation step separates the volatiles from the non-volatiles, distillation is also simple, reproducible, rapid, and can handle a large range of samples. Steam distillation works best for those compounds which are slightly volatile and water insoluble. In addition, those compounds which have a boiling point of less than lOO^'C will also pass over. The process described in this contribution expands upon a technique previously suggested by this author (4). The technique is particularly useful if the aqueous phase is limited in quantity and involves a set of sequential experiments. The sample is placed in an extractor, the pH made basic and the sample extracted with solvent. Sufficient sample is removed for gas chromatographic analysis, e.g. l|iL. The aqueous phase is made acidic and the sample re-extracted with the same diethyl ether and gas chromatographic analysis repeated. Finally the sample is treated with 2,4-dinitrophenylhydrazine (2,4-DNPH) and re-extracted. The ethereal phase is re-analyzed. In this fashion a series of different analyses can be made from the same sample in a short period of time and subjected to gc/ms and organoleptic analysis.

2.1. Direct Distillation The sample is normally placed in a flask and dispersed in water. The aqueous slurry can be heated directly (with continuous stirring) to carry over the steam distillable

101 components. Problems can be encountered due to scorching of the sample, and bumping may occur when the sample contains particulates. Stirring may prevent these problems. Foaming is another potential problem and while the addition of antifoams (e.g. DC polydimethyl siloxanes) may prevent this problem, these silicones usually end up in the distillate as evidenced by GC/MS peaks at m/z=73, 147, 207, 221, 281, and 341. 2.2. Indirect Steam Distillation Indirect steam distillation has many advantages over the direct technique. It is more rapid and less decomposition of the sample occurs because of indirect heating. The steam and volatiles are usually condensed in a series of traps cooled with a succession of coolants ranging from ice water to dry ice/ acetone or methanol. 2.3. Vacuum Steam Distillation If sample decomposition remains a concern, then the steam distillation may be operated under vacuum. In this case inert gas should be bled into the system to aid in agitation. A number of cooled traps should be in line to protect the pump from water vapor and the sample from pump oil vapors. Another simple method to generate a condensate under vacuum is by use of a rotary evaporator. Bumping is normally not a problem in this case. The higher boiling components do not distill as efficiently as they do under atmospheric distillation. 2.4. Extraction and Concentration Once the vapors have been condensed, it remains to extract the sample, which is normally very dilute. When relatively large amounts of aqueous samples are available, then separatory funnels or commercial liquid-liquid extractors may be employed. A large number of solvents have been summarized by Weurman (5) and reviewed by Sugisawa (6). The solvents most commonly used today are: diethyl ether; diethyl ether/pentane mixtures; hydrocarbons; Freons; methylene chloride. The latter two have the advantage of being non-flammable. Solvent selection is an important factor to consider and the current status has been summarized by Reineccius (7). Slow and carefiil distillation of the solvent will produce the aroma concentrate. 2.5. Analysis Gas chromatographic analysis is normally used to separate the sample. After separation the individual components may be detected by various GC detectors (FID; mass spec) or by sensory analysis. When, as is often the case, the gas chromatographic column does not resolve all components then fiirther refinements must be made. This particular problemfrequentlyoccurs when the sample is highly complex.

2.6. Sample Simplification One solution to the problem of a highly complex sample is to pre-fractionate the sample prior to gas chromatographic analysis. The general technique was first described in

102 a recent book covering techniques of sample preparation (2). This can be done by class separation based upon solvent solubility class. The purpose of this contribution is to describe a new technique which permits sequential removal of classes of compounds, revealing a progressively less complex sample. This procedure is carried out in one extractor, thus minimizing sample loss throughout the process. The sample is placed in a Mixxor*™ Chamber B, the pH adjusted to about 3 with acid and the sample extracted with diethyl ether. Sufficient sample is removed for gas chromatographic analysis, e.g. IjiL. The aqueous phase is made alkaline and the sample re-extracted with the same diethyl ether and gas chromatographic analysis repeated. Finally the sample is made neutral and saturated with a reagent to remove carbonyls and re-extracted. The ethereal phase is re-analyzed. In this case three different analyses can be made from the same sample in a short period of time and subjected to gc/ms, and organoleptic analysis. 3. EXPERIMENTAL 3.1. Manipulation of the Aqueous Phase Adjustment of the pH of the aqueous phase before extraction may accomplish two goals. First, emulsions may be broken permitting phase separation to take place rapidly. Second, class separation will take place which may simplify the gas chromatographic pattern. Frequently small peaks are concealed under larger ones and the smaller ones may be revealed for organoleptic evaluation or identification. This chemical manipulation of the aqueous phase can be carried even further. Many food aromatics contain carbonyl compounds. By adding sodium bisulfite to the aqueous phase it is possible to selectively remove the aldehydes and the methyl ketones by forming their water soluble bisulfite addition complexes. Alternatively, 2,4-dinitrophenylhydrazine may be used to produce a carbonyl-free sample. 3.2. Model System A system was prepared to represent a variety of classes of organic flavor compounds. Twenty mg each of 2-hexanone, 2-octanone, 3-octanone, 1-heptanol, heptanoic acid, limonene, octanal, 2-hexenal, methyl anthranilate, eugenol, anethol, 2-acetyl pyridine, and ethyl nonanoate were placed in 0.5mL methanol as a stock solution. Ten microliters of the above were placed in l.OmL ether to represent the flavor system. 3.3. R&G Coffee Sample lOOg of roasted and ground coffee was placed in IL of distilled water. This was atmospherically steam distilled using a Likens/Nickerson extractor for Ihr with 1:1 pentane:diethyl ether as the solvent. The solvent was carefully distilled to l.OmL. 3.4. 2,4-Dinitrophenylhydrazine reagent (2,4-DNPH) Three g of 2,4-DNPH was dissolved in 15mL of cone, sulfuric acid. Add this solution, with stirring, to a mixture of 20mL water and 70mL of 95% ethanol.

103 3.5. Extraction Use of the Mixxor''^ (New Biology Systems Ltd., Israel) in sample preparation has been described by Parliment (8) and its value described in a number of publications (9,10). Such a device is shown in Figure 1. These extractors are available with sample volumes ranging from 2mL to lOOmL. The lOmL capacity extractor is particularly convenient capacity for flavor research. Briefly, the aqueous phase is placed in receiver B and the whole assembly is cooled and a quantity of a low density immisible solvent containing the analyte is added. The solvent may contain an internal standard. The system is extracted by moving piston A up and down a number of times. After phase separation occurs, the solvent D, is forced into axial chamber C where it can be removed with a syringe for analysis. A less sophisticated alternative exits. The organic sample may be placed in a screw-capped centrifuge tube and a small amount of aqueous reagent added. After exhaustive shaking, the tube can be centrifuged to break the emulsion and separate the layers. The organic phase can be sampled with a syringe.

Figure 1

3.6. Analysis a. GC/MS Analysis of Fractionated Samples. The gas chromatographic analysis of the samples was performed on a Hewlett-Packard 5890 gas chromatograph interfaced to a HP 5972 MSD detector. The column was HP-5 (5% phenyl methyl silicon liquid phase), measuring 30m x 0.25mm with a 0.25|im film thickness with a helium flow rate of ImL/min. The oven temperature was held 2min @40°C, then increased in a linear rate of 7°C/min to 250°C. Final hold was lOmin. Normally IjiL of sample was injected using a split injector. b. Sensory Analysis of Fractionated Coffee Samples Three microliter samples of the various coffee samples were injected into a Hewlett-Packard 5890 gas chromatograph modified by DATU (Geneva, NY) for sensory evaluation of odors. The column was DB-5 (5%phenylmethyl silicon liquid phase), measuring 30m x 0.53mm with a 3|am film thickness with a helium flow rate of 3mL/min. The oven temperature was held 2min @40°C, then increased in a linear rate of 10°C/min to 250°C. Final hold was 4min. 3.7. General Fractionation Procedure a. Total Sample. One milliliter of the model system in diethyl ether was extracted with 1.5mL water in the Mixxor and the organic phase analyzed by gc/ms.

104 b. Removal of Acids. Concentrated sodium hydroxide was added to make the aqueous phase alkaline to phenolphthalein and the sample re-extracted with the same ether in the same system. c. Removal of Bases. The same system from b. was taken and the aqueous phase made acid (to UI paper) with sulfuric acid. The extraction was repeated. d. Removal of Carbonyls. The ethereal phase from c. was taken and combined with O.SmL of 2,4-DNPH solution. After a 10 min reaction period, water was added and two phases formed. The organic phase was analyzed. 3.8. Fractionation Procedure for Coffee Sample The procedure followed was similar to that described in the previous section. The primary difference was that after each extraction in the Mixxor^^ and analysis of the organic phase, the aqueous phase was removed. New reagent (dilute base; dilute acid; 2,4DNPH) was added for each step. This was necessitated since the coffee sample is a highly complex one.

4. RESULTS and DISCUSSION 4.1. Model System The four chromatograms representing the four stages are presented in Figure 2. Table 1 gives the identifications and retention times of the components. Table 1. Composition and Retention Times of Model System Compound 2-hexanone 2-hexenal 3-octanone 1-heptanol 2-octanone octanal limonene 2-acetyl pyridine heptanoic acid anethol ethyl nonanoate methyl anthranilate eugenol

Retention Time, min 4.5 5.9 7.9 8.6 9.1 9.4 10.0 10.1 11.3 15.5 15.6 16.7 16.9

105

[Abundance 300000 TOTAL SAMPLE

4.00

6.00

8.00

10.00

12.00

14.00

16.00

IL

18.00

20.00

22>00

24.00

ACroS REMOVED

4.00

Jh~i

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

[Abundance 250000 BASES REMOVED

200000 150000 100000 50000

4.00

6.00

00

10.00

•4-r-A12.00 14.00

' I ' ' •'• '' I ' ' ' ' I ' ' ' '' I ' ' 18.00 20.00 22.00 24.00

16.00

500000CARBONYLS REMOVED 4000003000002000001000000-

L.

4.00

i'

T-V^^

6.00

8.00

..A

10.00

12.00

14.00

li

1

16.00

I 18.00

20.00

22.00

Figure 2. Chromatogram of model system showing stages of simpilfication

24.00

106 Figure 2a is the pattern for the original mixture. The next Figure (2b) shows the elimination of heptanoic acid at a retention time (RT) of 11.3 min when the sample is treated with base. Figure 2c represents the removal of the bases. In this case, the acetyl pyridine is eliminated. The final figure (2d) shows the result of removing the carbonyls and the bases. All carbonyls were removed, except for 3-octanone, which was markedly reduced. The 1-heptanol, heptanoic acid, limonene, methyl anthranilate, eugenol, anethol, and ethyl nonanoate remained. The fact that the sample became progressively simplified led to an experiment on a real-world sample. 4.2. Coffee Sample The L/N extract of R&G coffee was concentrated to about ImL. Since coffee is a much more complex sample, a somewhat different procedure was followed. In this case, the aqueous phasefi-omeach extraction was removed, and the original organic phase was fiirther worked up. thus each extraction produced a progressively simpler sample. The first analysis was performed on the intact sample to give the complex chromatogram in Figure 3 a. The organic phase was taken and extracted with ca ImL of 0.025N NaOH in a 5mL Mixxor^^. The organic phase, now fi'eed of the acids, was analyzed as Figure 3b. In the RT region of 6.5 min 2-and 3-methyl butanoic acids elute. They are cleanly eliminated by the alkaline extraction. The aqueous alkaline phase was removed and the organic phase was extracted with dilute sulfuric acid to remove bases. The resulting chromatogram is shown in Figure 3 c. The group of two carbon-substituted (i.e. ethyl and dimethyl) pyrazines which were located at RT 7.0 to 7.5 min have been eliminated. Now revealed at RT 7.2 min is acetyl fijran; in addition, the very important coffee flavor compound fiirfiiryl mercaptan is now readily evident at RT 7.17 min. Also lost is methyl pyrazine at RT 5.1 min. The aqueous phase was removed and the organic was treated with 2,4-DNPH and then partitioned against water. The chromatogram of the residual organic solvent is presented in Figure 3d. At RT 5.4 the compound fiirfural has been removed, revealing under it methoxy methyl fiiran and benzene methanol. In addition, acetyl fiiran (RT 7.2) has been removed andfiirfiirylmercaptan is much more clearly evident. It is clear that this procedure sequentially removes classes of compounds. If the removed compounds are of interest, then the aqueous phase may be retained and reanalyzed. For example, if the alkaline phase (0.025N NaOH) fi-om the earlier step is acidified and re-extracted with ether, the acid fi-action may be analyzed. If the dilute sulfiiric acid is made alkaline and re-extracted then bases such as the pyridines and pyrazines are available for qualitative analysis.

107

TOTAL SAMPLE

abundance 1500000ACroS REMOVED 1000000-

500000-

0-

hr^

4.00

.,i,,.

6.00

8.00

10,00

12.00

14.00

^w^^Ayyv^p^ T^r" r i- -f • i • f • i f i i -i i r i • i i16.00 18.00 20.00 22.00 24.00

abundance

BASES REMOVED

4.00

ji

6.00

8.00

|^JUY^..p.J^.•..>^AA^^^ 10.00 12.00

14.00

16.00

18.00

20.00

22.00

24.00

?\bundance 1500000CARBONYLS REMOVED 1000000-

500000-

•

^'1 ' ' 4.00

. ^6.00

1 ,,,

'i*

8.00

1

1 • 1

10.00

1

12.00

14.00

-4;^

A I , . ..^ 18.00 16.00

20.00

22.00

Figure 3. Chromatogram of coffee sample showing stages of simpilfication

24.00

108 4.3. Sensory Evaluation of Coffee Sample The samples were odor evaluated by a trained flavorist. An appended 3 min version of the evaluation is presented in Table 2. A number of observations can be made. At 9.0 min a fatty acid elutes; this character is removed by the base extraction revealing a fruity character. At 9.8 min an interesting cheese aroma elutes. This aroma remains through acid and base extraction; it is eliminated by the 2,4-DNPH. Thus this component may be a carbonyl compound. The initial impression of the peak at RT 10.6 was unimpressive. Only after the acids and bases were removed was a distinct coffee aroma evident. Thus this sample would be the choice for identification studies. A pleasant roasted peanut aroma was observed at RT 10.8 min. This character disappeared when the sample was acid extracted. Thus it is probably a nitrogen heterocyclic compound. It would be possible to take the aqueous acid solution and make it basic and re-extract. This would produce a basic fraction, less complex in character, more amenable to odor and mass spectral analysis. The chromatograms representing the odor assessment work are presented in Figure 4. It is apparent how each stage of extraction produces a simpler sample. Table 2. Odor Assessment of Coffee Fractions Retention Time, Intact Sample Base Extracted min 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12

Acid Extracted 2,4-DNPH Treated

Sulfury

Nice green

Skunk rubber

Skunk

Skunky

Skunk

Sulfur

Valeric acid Cheese acid Cheese acid Sharp green Cheesy Skunk Sweaty Potato Sulfury Roasted nut SI sharp Sweaty Potato Sweaty Cucumber Green

Fruity

SI fruity

Green pungent Cheesy Sulfury

Sharp green

Sharp green Cheesy Sulfury Sharp green Potato Potato Sulfury, phenolic Coffee Peanut Sour Sour

Skunk, rubber Solvent Potato Coffee

Sour, almond Green, almond Green herbaceous Sulfur cabbage Green Cucumber Cucumber Green

'"i J' 'i S S 'i 'i 'i '^ '^ ^ r'"^' 'i 'i 'i 'i 'i ^ ^ ^ ^'

109

a o •5b a o

O

\-* o

I CI. <1>

3,

I B

I

I E

no 5. REFERENCES 1. R. Teranishi, I. Homstein, P. Issenberg and E. Wick, Flavor Research: Principles and Techniques, Marcel Dekker, New York, 1971 2. R. Marsili, Techniques for Analyzing Food Aroma, Marcel Dekker, New York, 1996 3. T.H. Parliment In: Biogeneration ofAromas; T.H. Parliment and R. Croteau, Eds. ACS Symposium Series #317; American Chemical Society, Washington, DC, 1986; pp 34-52 4. T.H. Parliment, In: Techniques for Analyzing Food Aroma, R. Marsili, Ed. Marcel Dekker, New York, 1996, pp 1-26. 5. C. Weurman, J. Agric. Food Chem., 17 (1969) 370 6. R. Teranishi, R. Flath and H. Sugisawa, In Flavor Research, Recent Advances, Marcel Dekker, New York, 1981, pp 27-31 7. M. Leahy and G. Reineccius, In: Analysis of Volatiles. Methods, Applications., P. Schreier, Ed., de Gruyter, NY, 1984, ppl9-48 8. T.H. Parliment, Perf. Flav. 1 (1986) 1 9. T.H. Parliment and H.D. Stahl, In: Sulfur Compounds in Foods, C. Mussinan and M. Keelan, Eds. ACS Symposium series #564 ; American Chemical Society, Washington; DC, 1994, pp 160-170 10. T. Parliment and H. Stahl, In: Developments in Food Science V37A Food Flavors: Generation, Analysis and Process Influence. G. Charalambous Ed. Elsevier, New York, 1995, pp 805-813

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

111

A simulated mouth to study flavor release from alcoholic beverages S. J. Withers, J. M. Conner, J. R. Piggott and A. Paterson University of Strathclyde, Department of Bioscience and Biotechnology, 204 George Street, Glasgow G l IXW, United Kingdom

Abstract Static headspace techniques have contributed much to our understanding of the interactions of Scotch malt whisky solutions. Such methods, however, do not account for the numerous and changing conditions of the mouth. The Buccal Headspace Technique addresses such effects by sampling air directly from the mouth as the whisky is warmed and mixed with saliva. The complexities of Scotch malt whisky can be more fully imderstood by creating simple model systems in the form of whisky analogues. These analogues, which may not be suitable for human consumption, are analysed using a Simulated Mouth, the conditions of which were set using data from the Buccal Headspace technique. The Simulated Mouth may provide a useful tool in the understanding of Scotch malt whisky flavor.

1. INTRODUCTION

Sensory and chemical techniques have made important contributions to flavor research. For the past twenty years such methods have been used by this laboratory to study the flavor characteristics of Scotch whisky (1). Aided by statistical techniques such as Partial Least Squares Regression analysis (2) an overall impression of the characteristics of Scotch whisky has been established. However, flavor perception is a dynamic system which assesses aroma and taste simultaneously through a complex series of reactions. When food or drink are introduced to the buccal cavity the non-volatiles are detected by the receptors for the four basic taste qualities; these are located throughout the surface of the tongue. As the food is warmed in the mouth and mixed with saliva, the volatiles from the material are released and passed through the retronasal passage where they are detected. Therefore, to obtain an accurate assessment of flavor we need to take account of a number of factors such as the mouth warming of the food towards the physiological temperature of 37°C; the saliva interactions with the food throughout the period of consumption and frictional forces contributed by the tongue and the teeth. A method which accounts for all of these factors is Buccal Headspace Analysis

112

(3). This technique involves the measurement of volatiles in the headspace directly above a food or drink in the buccal cavity. Scotch whisky is chemically a very complex system with many different reactions. It is often simpler to break down the reactions into smaller component parts by creating model systems. However, panellists are unwilling to sample solutions of alcohols and pure chemicals. Therefore we found that it was neccessary to develop a simulated mouth system. Simulated mouths have been constructed by other investigators (4,5and 6) but these systems were more concerned with mastication. We wanted to create a simple system capable of sampling both real and model systems, with the following attributes: an artificial saliva, constant temperature (37°C), agitation and frictional forces working within the artificial buccal cavity.

2. METHOD A series of experiments were conducted to measure the effect of mouth warming on the volatiles of model whisky systems. The model solution consisted of ethyl decanoate dissolved in 23% v/v alcohol. The headspace volatiles of the model solution were compared at 25°C and 37°C. The same comparison was made with the addition of wood extract. To study the effect of temperature increase in a real system we decided to use Buccal Headspace Analysis (3) The apparatus for this technique, which is illustrated in Figure 1, consisted of teflon nosepieces which were inserted into the nostrils of the panellist. The nosepieces were attached via PTFE tubing to a Tenax trap. The air from the buccal cavity was drawn through this apparatus using a pump. The Tenax trap was thermally desorbed using a Purge and Trap Injector Control unit. The desorbed volatiles were analysed by gas chromatography mass spectrometry (GC-MS) with a Finnegan -MAT ITS-40. The apparatus for the Simulated Mouth apparatus is illustrated in Figure 2. It consisted of a glass flask containing 8.4mL of whisky(23% v/v), 3.3mL of artificial saliva and thirty-two glass beads, to contribute a frictional force to the system. The flask was contained in a shaking water bath heated to 37°C. Hydrated air was passed over the headspace of the flask. The headspace of the whisky and saliva mixture was sampled using a Tenax trap and sampled by the GC-MS as in the previous experiment.

3. RESULTS AND DISCUSSION Our initial experiment indicated that the activity coefficient of ethyl decanoate decreased in the model solution at 37°C (Figure 1 ) . So in effect the flavor release of ethyl decanoate from the model solution was reduced upon heating . The effect of wood extract addition to the solution is illustrated in Figure 2.

113

Teflon nose pieces

Figure 1. Apparatus for Buccal Headspace Analysis

Tenax Trap

Hydrated Air /

Shaker Water Bath r a t 37°C Whisky(23%v/v)+Artificial Saliva+ 32 Glass Beads Figure 2. Strathclyde's simulated mouth.

114

Again the activity coefficient of the ethyl decanoate in the headspace was reduced, but to an even greater extent. The release of volatile compounds from alcoholic beverages in the mouth appears to be limited by the formation of ethanol agglomerates. The presence of ethanol agglomerates was suggested from reductions in the activity coefficient of hydrophobic ethyl decanoate. In wood maturations, increasing concentrations of short and medium chain organic acids decreased the critical aggregation concentration of ethanol resulting in decreased activity coefficients from 5 to 40% (v/v) ethanol. On the basis of these results the Buccal Headspace Analysis was carried out. Unfortunately a number of problems were encountered with this methodology: reproducibility, as everyone has a unique breathing and eating pattern. Over a long period of time this technique can be uncomfortable and for reasons of safety, panellists are unable to participate in more than two whisky sessions per day. It was thought that our Simulated Mouth would solve the panel effect we found with Buccal Headspace Analysis. However, reproducibility was again a problem and measurements of air flow and pressure proved unreliable.

6 -r

10

15

20

25

Ethanol concentration (% v/v)

Figure 3. The effect of temperature on the activity coefficient of ethyl decanoate at different ethanol concentrations.

115 6 -r

5.5

+

5 + -4—25 -C -•— + wood ext 25 'C 4.5 + Hi— + wood ext 37 °C

10

15

20

25

30

35

40

Ethanol concentration (% v/v) Figure 4. The effect of changing ethanol concentration on the activity coefficient of ethyl decanoate in different model systems.

4. CONCLUSION

Our initial experiments showed a decrease in the flavor release of ethyl decanoate in an alcohol and water solution at 37°C, and a further decrease with the addition of wood extract. By using Buccal Headspace Analysis and our Simulated Mouth system we hoped to examine these effects in greater detail. However, our trapping and sampling method proved to be unreliable and for the moment our findings remain inconclusive.

116 Acknowledgements:

The UK Biotechnology and Biological Sciences Research Council (BBSRC) and The Chivas and Glenlivet Group provided financial support and technical assistance for this work.

References: 1 S.J. Withers, J.R. Piggott, J.M. Conner and A. Paterson, Journal of the Institute of Brewing, 1995, Vol 101, pp359-364. 2 M. Martens and H. Martens. In: Statistical Procedures in Food Research (J. R. Piggott, ed.), Elsevier Applied Science, London, 1989, p293. 3 C. M. Delahunty, J. R. Piggott, J. M. Conner and A. Paterson, Journal of the Food and Agriculture, 1996, Vol 71, No 3 pp273-281.

Science of

4 W.E. Lee, Journal of Food Science, 1986, Vol 51, No 1 pp249-250. 5 S.M. Van Ruth, J.P. Roozen and J.L. Cozijnsen, Chemical Senses, 1995, Vol 20 Nol ppl46-149. 6 D.D. Roberts and T.E. Acree, Chemical Senses, 1995, Vol. 20, No.6, pp246-249

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

Comparisons of volatile compounds released during consumption Cheddar cheeses by different consumers

117

of

CM. Delahunty, P.J. O'Riordan, E.M. Sheehan and P.A. Morrissey Department of Nutrition, University College, Cork, Ireland

Abstract Methods exist for measuring volatile compounds released in the mouth during food consumption, however little work has compared the volatile compounds released during consumption by different consumers or related individual differences to consumers' chewing patterns and saliva production rates. In this work, eight consumers were chosen and each consumed six Cheddar cheeses during Buccal Headspace Analysis (BHA). Released volatile compounds were measured for each cheese and for each consumer. Electromyography was used to record each consumers chewing style, and their saliva production rate was also measured. It was found that although there were differences in consumers' chewing styles and saliva production rates, the volatile profiles obtained by BHA, for each individual, were similar for each cheese when compared with the other cheeses examined.

1. INTRODUCTION It is the volatile compounds of a food, released in sufficient concentration during consumption, which stimulate the olfactory epithelium and induce perceived odor. Recent flavor research has emphasised the importance of volatile release from a food matrix and shown how volatile release is related to consumer flavor perception. This work is often driven by a food industry which must reduce costs or meet the demands of diet conscious, but discerning, consumers who wish to reduce fat and salt intake. Substitution for these ingredients is necessary to restore removed flavor and regulate flavor release. However, understanding of flavor release is made difficult by the complexity of the interactions between foods and consumers. Each volatile compound has different physiochemical properties and its release is influenced by interactions with other food matrix variables such as moisture, fat, protein, carbohydrate, and other soluble (salt, sugars) and non-soluble materials. In addition, food breakdown and mixing with saliva during consumption, respiratory air flow over and around the food and temperature and pH changes occurring in the consumers mouth will influence volatile release and subsequent flavor perception. It is also known that individual consumers expression of flavor differs as a result of physiological, psychological and social differences [1,2]. Therefore an underlying question which remains unanswered is; what part of flavor differences between foods result from volatile release dynamics from the food matrix, and what part result from differences between consumers? Conclusions reached in response to this question have been mixed.

118 There are model systems which measure volatile compounds released while mimicking conditions in the mouth [3-5]. Other methods measure volatile release directly during consumption using mass spectrometry of breath [6,7] and indirectly by trapping volatiles on adsorbents, such as Tenax, before analysis [8-10]. Soeting and Heidema [6] showed thirtyfold differences in the relative quantities of 2-pentanone which was measured directly from the breath of different consumers. Van Ruth et al [10] also found subject specific volatile profiles were released during consumption of vegetables. Taylor et al [11] trapped volatiles released from mint sweets during consumption and also found differences between subjects in terms of the quantities of volatiles released. However, they concluded that there were similarities between the relative concentrations of volatiles released for each subject. Delahunty et al [12], who analyzed Buccal Headspace Analysis [BHA;13] data using Principal Components Analysis [PCA;14] to examine the volatile profiles released during consumption of cheeses by three different consumers, found product specific volatile release was most important. However, three consumers were too few to draw any firm conclusions. Workers studying food texture have developed methods such as electromyography [15] which measure muscle activity during mastication of a food matrix. From these measurements they have shown mastication patterns and can calculate the amount of work done by a consumer during consumption. These methods, which show considerable differences between consumers' mastication characteristics, have recently been related to differences between consumers' temporal perception of flavor intensity measured by timeintensity sensory analysis [16]. Other physiological parameters, such as the influence of saliva [4] and air flow through the mouth [17] have also been investigated. The present study was carried out to investigate discrepancies in the literature relating to the differences between consumers' interactions with foods and the relationships found between physiological measures during chewing and individuals' differences in flavor perception. In order to achieve this, similar varieties of a complex food were chosen and a multivariate technique, PCA, was used to examine the volatile profiles released.

2. EXPERIMENTAL 2.1. Samples and consumers Six Cheddar cheeses, in 5kg blocks, of equal age (6-8 months) were obtained from 4 different producers. Eight consumers, 3 female and 5 male, aged between 22 and 28 were used for all studies. 2.2. Buccal Headspace Analysis Buccal Headspace Analysis of each cheese was carried out for each consumer in triplicate. For this method a 50 g cheese sample was consumed in 10 x 5g pieces in a normal way, allowing 30 s for the consumption of each piece. During the entire consumption time (5 min) volatile compounds released were displaced through the nose by vacuum and trapped on a Tenax-TA trap. The order of sample analysis was balanced for consumers, cheeses and day of consumption [18]. A blank buccal headspace sample was taken each day for each consumer. Traps were thermally desorbed using a Teckmar Purge and Trap 3000 concentrator (Teckmar, Cincinnati, OH, USA). Desorbed volatiles were identified and quantified using gas chromatography-mass spectrometry (GC-MS) with a Varian Saturn GC-3400CX

119 incorporating a Varian Saturn 3 GC/MS detector (Varian chromatography systems, Mitchell drive, Walnut Creek, CA, USA).The column was a DB-5ms, 30m x 0.257mm fused silica capillary column, with a film thickness of 0.25 |Lim (J & W scientific, Folsom, CA, USA) 2.3 Mastication behaviour The activity of the consumers left and right masseter muscles during chewing was recorded by Electromyography [15]. The electromyograph record was measured for 1 cheese over a period of 5 min (10 x 30 s for each 5g piece ), in triplicate, for each of the eight consumers. Each individuals electromyogram was integrated using a poly VIEW data acquisition and analysis system (Grass instrument division, Astro-Med Inc., East Greenwich Avenue, West Warwick, UK) 2.4 Saliva production Consumers unstimulated saliva production was measured by allowing their saliva to drip into a beaker for a 5 min period. The consumers swallowed immediately before collection and forcefully spat out at the end [19]. The stimulated saliva production was measured by dividing the volume of saliva produced by each consumer in response to 50g of cheese (10 x 5g) by the chewing time required by the consumer for that cheese [19]. Each measurement was repeated four times. 2.5 Data Analysis Buccal headspace data was analyzed by PC A, using the Unscrambler v 6.0 (CAMO AS, N-7041 Trondheim, Norway), of the log transformed peak areas of volatile compounds. Electromyography data was analyzed by Analysis of Variance (ANOVA) using SPSS v 6.1 (SPSS Inc. Chicago, IL 60611, USA) of the totals for chew number, chew time, chew rate and chew work. Saliva production data was analyzed by ANOVA of the unstimulated and stimulated saliva flow rates. Differences between cheeses and between subjects were investigated using ANOVA. Relationships between data sets were investigated by linear and Partial Least Squares regression [PLS;20], using the Unscrambler v 6.0.

3. RESULTS AND DISCUSSION In the present study the quantities and balance of volatile compounds released during consumption of a food, by different consumers, was compared. For this purpose Cheddar cheese was chosen as this represents a complex protein matrix containing fat and moisture. To minimize product related compositional differences, and therefore to maximize the influence of consumer related differences to volatile release from one food type, cheeses of equal age were chosen. Eighteen volatile compounds were selected from chromatograms of BHA of all cheeses and the amounts of each present were quantified. Both Figures 1 and 2 depict two PCA's. The first (in italics) was calculated using individual consumers' headspace data (triplicates averaged) and the second using the average of the 8 consumers.

120 Figure 1. PC A scores on PC's 1 and 3 for 6 cheeses assessed by BHA using 8 subjects (A-H) (see text for explanation).The pooled SD for the analysis is represented by an ellipse on cheese 1. 4 T

3B 4C

13H ^^

c o a.

4G

-4

5C

"^ fr ^^ Ic ,2iP2f 4H 2F 3G

5F

6E IG

-im

4A

IB

2C

2 6D

OH

5 6G

-4 -^

Principal component 1 {?>9Vo)(24%) Principal Components (PC) 1 and 3, which accounted for 39% and 15% of the explained variance, respectively, of the PC A of compound peak areas, showed significant differences {p = 0.017 and p = 0.021) between cheeses (Table 1 and Figure 1). Differences between consumers (p = 0.050), which accounted for \3% of the explained experimental variance, were found on PC3 (Table 1 and Figure 2). Table 1 ANOVA between cheeses (1 - 6 ) and between consumers ( subjects A • H)on Principal Components 1 - 4 of the PC A Principal component Cheese Subject PCI PC2 PC3 PC4

0.017 0.657 0.021 0.055

0.873 0.072 0.050 0.351

The volatile compounds which distinguished the cheeses from one another on these components are shown in the PC loadings plots (Figures 3 and 4).The differences found between cheeses on PCI, which was the most important as it contained the highest proportion of the experimental variance (39%), were caused mostly by the quantities of compounds released during consumption by each consumer rather than by their balance.

121 Figure 3. PC A loadings for 18 volatile compounds on PC's 1 and 3 for 6 cheeses assessed by BHA using 8 subjects (A-H). 0.5

T 2-heptanone

cyclohexanel cyclohexane2 cpdl3 cpdl4

o OH

-0.5

cpd3 cpd5

toluene

cpd2

dmds

cpdl7

0.5

heptane

(Eodecane

ethyl butyrate cpdl2

cpdl6 -0.5 -^

Principal component 1 (39%) However, differences between cheeses found on PC's 2 and 3 were caused mostly by differences in the balance of the compounds released. This can be determined from the relative positions of the volatile compounds in the loadings plots (Figure 4). In a previous study physiological differences between consumers have been related to differences in flavor perception [16]. In this study very significant differences were found between consumers mastication characteristics and also between their saliva production rates (p = 0.000 for all parameters apart from chew rate (p = 0.021) and chew work (p = 0.045)) (Table 2 and Figure 5). Using linear regression chew number and chew work were found to relate to saliva production rate during cheese consumption for 5 of the 8 subjects (r = 0.98 and r = 0.84, respectively). However by using PLS and linear regression, no significant relationships were found between the measured physiological characteristics and total volatile release. Sensory evaluation of the cheeses is not reported in this study and therefore no conclusions can be made with regard to consumers' expressions of flavor perception. Further work is also required to investigate the dynamics of volatile release during time of consumption.

4. CONCLUSION Some differences were found between the quantities of volatile compounds released during cheese consumption by different consumers. Very significant differences were found between consumers mastication characteristics and between saliva production rates during cheese consumption. Despite these differences, the distribution of experimental variance explained

122 Table 2 Mastication behaviour and saliva production rates of 8 consumers ( subjects A-H) Subject TCN

A B C D E F G H pooled SD P

224.33 138.67 348.00 324.33 228.67 215.67 184.33 282.33 22.84 0.000

Electromyography^ TCT CR

141.26 169.07 212.27 198.53 144.00 212.07 145.71 180.99 28.35 0.000

1.59 1.08 1.67 1.64 1.68 1.02 1.27 1.55 0.12 0.021

CW

2435.86 1011.45 3823.63 4497.6 2066.55 1194.73 866.71 3010.14 1403.50 0.045

Saliva flow rate'' Unstim. Stimulated

0.77 0.43 0.59 1.06 0.53 0.70 0.76 0.35 0.12 0.000

5.30 4.06 1.69 7.31 4.52 4.89 3.25 3.48 0.65 0.000

^ TCN = total chew number; TCT = total chew time (sec); CR = chew rate (chew / sec); CW = chew work b Unstim. = unstimulated saliva production rate (mL / min); Stimulated saliva production rate (mL / min)

Figure 5. Mastication behaviour of 3 consumers during consumption of one 5 g piece of cheese. CN = chew number; CT = chew time (sec); CR = chew rate (chew / sec); CW = chew work. Pat (A) CN = 23 CT= 14.66 CR=1.57 C W = 133.03

a

Chew Time (sec)

123 Figure 3. PCA scores on PC's 2 and 3 for 6 cheeses assessed by BHA using 8 subjects (A-H). The pooled SD for the analysis is represented by an ellipse on cheese 6. 4 T

4C 2A IG 6C

C

ID 3D

2B 2F

o

r^

4A 2C

OH

5E 6F 5D 6Bry

ex

ifP

5G

3F6D

,^

-4 -^

Principal component 2 (\3%)(]2%) Figure 4. PCA loadings of 18 volatile compounds on PC's 2 and 3 for 6 cheeses assessed by BHA using 8 subjects (A-H). 0.5 T 2-heptanone

cyclohexanel cycldhexane2 pctane cdd7 cpdl2 cpdl4

o a.

|cpd3

cpf

-0.5 dmds

dodecane

heptane

cpd2

0.5 toluene

cpdl7 ethylbutyrate cpdl2 -0.5 -^

cpdl6

Principal component 2 (13%)

124 by the PCA showed that Cheddar cheese of equal age could be identified by their product specific volatile release. Therefore, the volatile profile for a particular cheese at the end of consumption was found to be similar in all consumers.

5. ACKNOWLEDGEMENT This work was part funded by the Department of Agriculture, Food and Forestry, Ireland, under the Food Industry Sub-Programme of EU Structural Funds.

6. REFERENCES 1 D. Lancet, In: Sensory Transduction (D.P. Corey and S.D. Roper, eds.). Pp. 73, Rockefeller University, New York, 1992. 2 J.R. Piggott, Fd. Qual. Pref, 5 (1994) 167. 3 W.E. Lee III, J. Fd. Sci., 51 (1986) 249. 4 D.D. Roberts and T.E. Acree, J. Agric. Fd. Chem., 43 (1995) 2179. 5 K. Napi, F. Kropf and H. Klostermeyer, Z Lebensm Unters Forsch, 201 (1995) 62. 6 W.J. Soeting and J. Heidema, Chem. Senses, 13:4 (1988) 607. 7 R.S.T. Linforth, K.E. Ingham and A.J.Taylor, In: Flavour Science: Recent Developments (A.J. Taylor and D.S. Mottram, eds.). Pp. 361, Royal Society of Chemistry, Oxford, 1997. 8 R.S.T. Linforth and A.J.Taylor, Fd. Chem., 48 (1993) 115. 9 CM. Delahunty, J.R. Piggott, J.M. Conner and A. Paterson, In: Trends in Flavour Research (H. Maarse and D.G. van der Heij, eds.). Pp. 47, Elsevier Applied Science, Amsterdam, 1994. 10 S.M. Van Ruth, J.P. Roozen and J.L. Cozijnsen, Fd. Chem., 53 (1995) 15. 11 A.J. Taylor, R.S.T. Linforth, K.E. Ingham and A.R. Clawson, In: Bioflavour '95 (P. Etievant and P. Schreier, eds.). Pp. 45, INRA, Paris, 1995. 12 CM. Delahunty, F. Crowe and P.A. Morrissey, In: Flavour Science: Recent Developments (A.J. Taylor and D.S. Mottram, eds.). Pp. 339, Royal Society of Chemistry, Oxford, 1997. 13 CM. Delahunty, J.R. Piggott, J.M. Conner and A. Paterson, J. Sci. Fd. Agric, 71 (1996) 273. 14 J.R. Piggott and K. Sharman. In: Statistical Procedures in Food Research (J.R. Piggott, ed.) Pp. 181, Elsevier Applied Science, London, 1986. 15 M.M. Boyar and D. Kilcast, J. Fd. Sci., 51 (1986) 859. 16 W.E. Brown, C Dauchel and I. Wakeling, J. Texture Stud., 27 (1996) 433. 17 M. Harrison and B.P. Hills, Int. J. Fd. Sci. Tech., 32 (1997) 1. 18 H.J.H. MacFie, N. Bratchell, K. Greenhoff and I.V. ValHs, 1989. J. Sens. Stud., 4 (1989) 129. 19 S. Watanabe and C Dawes, Arch. Oral Biol., 33:1 (1988), 1. 20 M. Martens and H. Martens, In: Statistical Procedures in Food Research (J.R. Piggott, ed.) Pp. 293, Elsevier Applied Science, London, 1986.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

125

Effect of adsorbent particle size on the w a t e r - e t h a n o l separation by cellnlosic sidistrates G. Vareli, P. G. Demertzis, and K. Akrida-Demertzi Laboratory of Food Chemistry, Department of Chemistry, University of loannina, 45110 - loannina, Greece

Abstract Inverse gas chromatography was used to study the adsorption of water and ethanol on two fractions of wheat straw with two different particle size compositions (80-100 and 100-120 mesh), in the temperature range 50-90°C, before and after its regeneration by thermal treatment at 140°C for 24 h. From the chromatographic retention data it was possible to calculate the separation factor (s) of the two solutes and to obtain the values for Gibb's free energy (AGs) and enthalpy (AHs) of adsorption. The results showed that water was adsorbed more strongly than ethanol by both fractions, at all temperatures, both for the untreated and thermally treated wheat straw. In addition, it was found that adsorption of both solutes was more spontaneous at lower temperatures, at which the separation factor had the higher values. Adsorption of both solutes was slightly stronger on wheat straw of 100-120 mesh particle size, whereas values for the separation factor were similar for both fractions. Thermal treatment had no effect on the adsorption of ethanol on both fractions. On the other hand adsorption of water was stronger on the untreated wheat straw, thus leading to a decrease of the separation factor for the thermally treated material.

1- INTRODUCTION Ethanol, either alone or blended with other fuels, has been used as a motor fuel for many years. Brazil in particular operates a large fraction of its automobile fleet on ethanol or gasoline-ethanol blends ("gasohol"). Ethanol has a high octane number compared to gasoline, and burns more cleanly than gasoline, producing lower levels of carbon monoxide, oxides of nitrogen a n d total hydrocarbon emissions. The major disadvantage in blending gasoline and ethanol is that under certain conditions the alcohol may separate from the gasoline. This problem can be overcome by proper adjustment and maintenance of the engine [1].

126 Any material that contains sugar can potentially be fermented to produce several kinds of alcohols. Ethanol can be produced from either grain or biomass (energy crops, forestry and agriculture residues, municipal wastes, etc.) by first converting them to fermentable sugars [2-5]. The conversion of sugar using biotechnology, leads to a broth that contains 6 to 12% b.wt. ethanol with small amounts of aldehydes, ketones, amyl alcohols and methanol [6]. Recovery of ethanol from this fermentation broth by distillation seems to be the major problem in the use of ethanol as a liquid fuel since several years ago it was found that distillation consumes 50 to 80% of the overall energy used in a typical grain ethanol plant [7-9]. An alternative process has been proposed by Ladisch and Dyck: distillation of fermentation broth to 75 - 90% b.wt. ethanol, followed by adsorption of the remaining water in a variety of a d s o r b e n t s such as silica gel, barium oxide and biomass m a t e r i a l s (cornstarch, cellulose, etc.) [10-17]. The energy consumption of this combined process is about 3.9 MJ/kg compared with values in the range 6-9 MJ/kg for the distillation process. In this article, adsorption of water and ethanol on two fractions of wheat straw with two different particle size compositions (80-100 and 100-120 mesh) in the temperature range of 50-90^C, before and after its regeneration by thermal treatment at 140 °C for 24 h is reported. Wheat straw is a domestic product of Greece in abundance.

2 . MATERIALS A N D METHODS 2.1 Inverse gas chromatography IGC is widely used to investigate the interaction of volatile probes of known properties with a solid surface under investigation. The l a t t e r comprises the stationary phase of a gas chromatographic column. IGC has become a common surface characterisation technique, because it is a rapid, simple, and high precision method [18-21]. The time that elapses from the injection of the sample to the recording of the peak maximum is called retention time. The difference between the retention times of a solute and an unadsorbed indicator is the net retention time. In this work, air was used as the unadsorbed compound. The ratio of the net retention times of two solutes is the separation factor: _ tnl ^~tn2 where: s is the separation factor, tni is the net retention time of the first solute, and tn2 is the net retention time of the second solute.

127

From the chromatographic retention data it is also possible, through a series of equations, to obtain values for thermodynamic parameters such as Gibb's free energy (AGs) and enthalpy (AHs) of adsorption [22]. 2.2 Preparation of columns Wheat straw is a product of Greece. It was cut in a rotary knife cutter (Gurgens, Bauknecht, SKM4853). The 80-100 and 100420 mesh fractions were obtained and stored in the dessicator. They were dried in a vacuum oven at 50°C for 2h, then dessicated for 24 h. The samples prepared in this way were diluted with an inert support of the same particle size, Chromosorb WAW, DMCS, purchased from Serva Germany. The inert support was dried under the same conditions and stored in a dessicator. In order to make retention time measurements with water, the sample of 80-100 mesh particle size was diluted 7 to 93 parts of inert support, while the 100-120 mesh particle size sample, was diluted 5 to 95 parts of inert support. The dilution was necessary due to the very long water retention times on the cellulosic material which would otherwise make the experiments extremely time consuming. This proved to be a negligible source of error since water net retention times per gram on 100% Chromosorb WAW columns ranged from 0.073 min/g at 90°C to 0.081 min/g at 50^C, while water retention times on wheat straw ranged from 8.04 min/g at 90°C to 89 min/g at 50°C. In order to make ethanol retention time measurements, 50% wheat straw and 50% inert support columns were prepared. The greater percentage of wheat straw was necessary because of relatively small retention times on both inert support and wheat straw. The net retention time per gram for a 100% wheat straw column (tng/g ) was estimated from the following equation: tNT- tNA/g M A

tns/g -

yi^

where, tNT is the net retention time of the solute in the diluted sample column, tNA/g is the net retention time per gram of the solute in the 100% Chromosorb column at the same temperature as the diluted sample column. MA is the mass of the Chromosorb in the diluted sample column, Ms is the mass of the wheat straw in the diluted sample column. The densities of the two fractions of wheat straw were determined using a stereopycnometer by Quantachrome (USA), model SPY-3 and the average values of triplicate measurements were 1.48 g/mL for 80-100 mesh fraction, and 1.49 g/mL for 100-120 mesh fraction of wheat straw. Aluminum tubing was used for the construction of chromatographic columns with a 6.35-mm o.d. and a length of 1 m. Each analytical column was conditioned at least 12 h by passing helium carrier gas through it.

128

2.3 GC instminentatioii A gas chromatograph (Shimadzu, GC-8A, Japan), equipped with a thermal conductivity detector (TCD) was used to measure the retention times of air, water and ethanol. The thermal conductivity detector temperature was set at 200°C. The injection port was also set at the same temperature. Helium (high purity) was used as the carrier gas. Pressure was regulated with a twostage regulator and set at 6 atm. Pressure drops in the columns ranged from 0.22 atm to 0.5 atm depending on the flow rate of the carrier gas. The carrier gas velocities (flow rates) were measured with a soap bubble flow m e t e r attached to the thermal conductivity detector outlet and adjusted to 35 mL/ min. Flow rates were determined for each column at each temperature after attaining a steady baseline of the recorder indicative of equilibrium of the column. A 5^lL Hamilton syringe was used to inject 2|il of the sample into the chromatograph.

3 . RESULTS A N D DISCUSSION Figure 1 shows the net retention times per gram of wheat straw for water (a) and ethanol (b), in the temperature range 50-90°C, before and after regeneration by thermal treatment. It is clearly observed that the net retention times of water were significantly higher than those of ethanol at all temperatures and for both fractions of wheat straw, both for untreated and thermally treated wheat straw. 100-

• 80-100 mesh o 100-120 mesh

0.15• 80-100 meshl o 100-120 mesH 0.125 H

75 H • Untreated wheat straw

Untreated wheat straw

0.1 H vo 25 H

0

0.075

•Thermally""^: treated wheat straw

0.05

I

40

50

60

70

• Thermally treated wheat straw

80

90

Temperature, °C

100

40

50

60

70

80

90

100

Temperature, °C

(a) (b) Figure 1. Net retention time per gram of untreated (-) and thermally treated ( ••) wheat straw for water (a) and ethanol (b), on 80-100 (0) and 100-120 (o) mesh particle size.

129

The net retention times for both solutes, water and ethanol, decrease as the temperature increases. The decrease in the net retention times of water is, however, more pronounced than those of ethanol. For example, the net retention time of water for untreated wheat straw of 100-120 mesh decreased approximately sevenfold from 90.0 min/g at 50°C to 12.3 at 90°C. On the other hand, the net retention time of ethanol for the same substrate decreased approximately threefold from 0.14 min/g at 50°C to 0.05 min/g at 90°C. Furthermore, data in Fig 1 show that at all temperatures the retention times of water and ethanol are higher in the fraction of 100-120 mesh, however without significant variation from the 80-100 mesh fraction. For example, at 70°C the net retention time of water in wheat straw of 80-100 mesh is 31.6 min/g, while the respective one for wheat straw of 100-120 mesh is 32.8 min/g. The separation potential of water from ethanol can be further investigated by calculating the so-called separation factor, s, that is the ratio of the net retention time per gram of water to the net retention time per gram of ethanol. Figure 2 a shows the separation factors obtained for both fractions of wheat straw, at all temperatures, for untreated and for thermally treated material. It is observed that the separation factor follows in general the trend observed for the retention times in Fig 1., i.e. it increases with decreasing temperature. 7008 80-100 mesh - Untreated straw 600O 100-120 mesh 500-I

— Untreated wheat straw

400H

30oJ 200-1 Thermally

treated wheat straw

100

50

I

Thermally treated wheat straw

• 80-100 mesh o 100-120 mesh ethanol

— I —

40

water

100

0 2.7

2.8 2.9 1/T •lO^, K"l Temperature, °C (b) (a) Figure 2. (a) Separation factor (s) for water and ethanol on untreated (-) and thermally treated (•••) wheat straw of 80-100 (*) and 100-120 (o) mesh particle size. (b) InVg^vs 1/T*10^, for water and ethanol, on untreated (-) and thermally treated (••) wheat straw of 80-100 (•) and 100-120 (o) mesh particle size. There is no significant difference in the separation factors between the different particle size of wheat straw both for the untreated and the thermally treated straw. On the other hand, for both fractions, the separation factor is

130

lower for the thermally treated straw than for the untreated. For example, at 50°C the separation factor for untreated wheat straw of 100-120 mesh is 648 and after thermal treatment it decreases to 459. The major components of wheat straw are cellulose ('-40%), hemicellulose (-'28%), and lignin (14-20%). Cellulose is a linear, crystalline polymer of P-D-glucose units. Hemicellulose of wheat straw is mainly thought to be composed of p-1-4 linked D-xylopyranose units with side chains of various lengths containing L-arabinose, D-glucuronic acid or its 4-O-methyl ether, Dgalactose and possibly D-glucose, while much of the hemicellulosic fraction is of xylan type. Lignin is a polymer of phenylpropane units which form a threedimensional network. Within the plant, hemicelluloses are mostly connected to lignin by covalent bonds and are thus fixed in the fiber structure [23-24]. The decrease in water retention time on thermally treated straw may be attributed to structural changes of wheat straw's components. Thermal treatment probably affects the accessible hydroxyl groups of cellulose leading to partial loss of their hydrogen bonding capacity. It may also result in an increase of cellulose crystallinity. Furthermore, thermal treatment may also cause hemicellulose's partial transformation to more hydrophobic products [25-26], The fact that water retention times are quite similar for the two fractions, may be attributed to the errors induced in the apparent size of sieved particles due to the particle size distribution within each fraction and to the adherence of fine fibers to the surface of larger fibers. Table 1 presents values of free energies of adsorption of both solutes on both fractions of wheat straw. Table 1 Free energy of adsorption, AGs (Kcal/mol) 50 Water Untreated wheat straw 80-100 mesh -5.36 100-120 mesh -5.35 Thermally treated straw 80- 100 mesh -5.10 100-120 mesh -5.08 Ethanol Untreated wheat straw 80-100 mesh -1.21 100-120 mesh -1.20 Thermally treated straw 80-100 mesh -1.16 100-120 mesh -1.16

Temperature (°C) 60 _70

80

90

-5.20 -5.18

-5.00 -5.02

-4.82 -4.87

-4.62 -4.64

-4.94 -4.97

-4.76 -4.79

-4.56 -4.62

-4.38 -4.44

-1.02 -1.08

-0.90 -0.88

-0.87 -0.88

-0.76 -0.72

-1.02 -1.00

-0.89 -0.87

-0.87 -0.84

-0.87 -0.80

131

The higher negative values obtained for water on both fractions compared to those of ethanol confirm preferential adsorption of water. This can be attributed to the fact that the water molecule can form up to four hydrogen bonds because of its tetrahedral arrangement. Moreover, the dipole moment of water is 1.84 Debye units while that of ethanol is 1.68 Debye units at 28°C. Furthermore, the higher negative values of both solutes obtained at the lower t e m p e r a t u r e s used, indicate their stronger adsorption at lower temperatures. By plotting lnVg°versus 1/T (Fig. 2 b) straight lines with slope equal to -AHs/R are constructed. Values of enthalpies of adsorption are presented in Table 2. Table 2 Enthalpy of adsorption, AHs (Kcal/mol) Water

Ethanol

Untreated wheat straw 80-100 mesh 100-120 mesh

-11.9 -11.4

-5.3 -5.4

Thermally treated straw 80-100 mesh 100-120 mesh

-11.5 -10.8

-4.2 -4.7

Enthalpy of adsorption is a molar quantity directly related to the energy of interaction between sorbed solute molecules and sorption sites in the substrate, thus providing information on the exothermic or endothermic character of the interaction. The AHs values obtained are equal or somewhat higher than average physical adsorption values (3-9 KJ mol"-'^) but significantly smaller than typical average chemisorption values (20-40 KJ mol"-*^) [27]. 4- CONCLUSIONS Obtained results suggest that wheat straw can successfully be used as a biomass water-ethanol separation system through the preferential adsorption of water. Values for the separation factor were quite similar for both fractions either before or after regeneration. Thermal treatment had no effect on the adsorption of ethanol. On the other hand, adsorption of water was stronger on the untreated wheat straw, thus leading to a decrease of the separation factor for the thermally treated material.

132

5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

K.J. Lorenz and K. Kulp (eds.), Handbook of Cereal Science and Technology, Marcel Dekker, New York, 1991. N.P. Cheremisinoff, P.N. Cheremisinoff and F. Ellerbusch, B i o m a s s , Applications, Technology, and Production, Marcel Dekker, New York, 1980. M.R. Ladisch, Process Biochem., 14 (1979) 21. G.T. Tsao, M. Ladisch, T.A. Hsu, B. Dale and T. Chou, Ann. Rep. Ferment., 2(1978)1. M.R. Ladisch, CM. Ladisch and G.T. Tsao, Science, 201 (1978) 743. L.F. Hatch, Ethyl Alcohol, Enjay Chemical Company, New York, 1962. M.L. David, G.S. Hammaker, R.J. Buzenberg and J.P. Wanger, Gasohol Economic Feasibility Study (Development, Planning and Research Associates), Inc., Manhattan, Kan. 1978. T.K. Ghose and R.D. Tyagi, Biotechnol. Bioeng., 21 (1979) 1387. W.C. Buttows, C M . Hudson and M.L. Kaesser, N.A. Santer, Changing Portable Energy Sources - An Assessment, J. Deere Co, Moline,III, 1977. M.R. Ladisch and K.K. Dyck, Science, 205 (1979) 898. M.R. Ladisch, M. Voloch, J. Hong, P. Bienkowski, and G.T. Tsao, lEC Process Des. Develop., 23 (1984) 437. M. Voloch, J. Hong, and M.R. Ladisch, Second Chemical Congress of North American Continent, Las Vegas, NV, paper 43, 1980. J. Hong, M. Voloch, M.R. Ladisch and G.T. Tsao, Biotechnol. Bioeng., 24 (1982) 725. P.R. Bienkowski, A Barthe, M. Voloch, R.N. Neuman and M.R. Ladisch, Biotechnol. Bioeng., 27 (1986) 960. R. Neuman, M. Voloch, P. Bienkowski and M.R. Ladisch, lEC Fundam., 25 (1986) 422. A.A. Hassaballah and J.H. Hills, Biotechnol. Bioeng., 35 (1990) 598. V. Rebar, E.R. Fischbach, D. Apostolopoulos and J.L. Kokini, Biotechnol. Bioeng., 26 (1984) 513. R.J. Laub and R.L. Peesok (eds.), Physicochemical Applications of Gas Chromatography, Wiley, New York, 1978. R. L. Grob (ed.). Modern Practice of Gas Chromatography, Wiley, New York, 1977. V.G. Berezkin, V.R. Alishoyev and LB. Nemirovskaya (eds.). Gas Chromatography of Polymers, Elsevier, New York, 1977. V.G. Berezkin (eds.). Analytical Reaction Gas C h r o m a t o g r a p h y , Plenum, New York, 1982. G. Vareli, P.G. Demertzis and K. Akrida-Demertzi, Z. Lebensm. Unters. Forsch., 1997, in press. J. M. Lawther, R. Sun, and W.B. Banks, J. Agric. Food Chem., 43 (1995) 667. R. Bailey (ed.). Chemistry and Biochemistry of Herbage 1, London 1973. B. Kolin, and T.S. Janezic, Holzforschung, 50 (1996) 263. P.J. Weimer, J.M. Hackney and A.D. French, Biotechnol. and Bioeng., 48, (1995), 169. A.W. Adamson (ed.). Physical Chemistry of Surfaces, New York, 1976.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

133

Influence of extraction procedure on the aroma composition of Thymus zygis L. and Mentha pulegium L. M. Moldao-Martins*, R. Trigo, M.A. Nolasco*, M.G. Bernardo Gil** and M.L. Beirao da Costa*, *Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa CODEX, PORTUGAL ** Dep. Eng. Quimica, Av. Rovisco Pais, 1096 Lisboa CODEX, PORTUGAL

Abstract The present work compares the results of two extraction procedures (Clevenger distillation and compressed CO2 extraction) on the yield and composition of the aromatic extracts. A RSM was applied in order to determine the best extraction conditions by compressed CO2. The matrix was established for the following ranges: time 60-180 min, temperature 40-50°C and pressure 9-20 MPa. All the extracts were analysed by GC and GCMS. The results showed that the yields are generally higher for compressed CO2: 2.1% for Mentha pulegium and about 3% for Thymus, compared to a value of 0.9% and P/o by distillation. The main compound found in M pulegium is pulegeone. In T. zygis the main compounds are thymol, geraniol and geranyl acetate.

1. mTRODUCTION Some aromatic herbs may be interesting sources of aroma for use in the food, pharmaceutical and cosmetic industries. Many of them are not always available year round and therefore the production of extracts is an extremely convenient process. The Labiatae family includes a large group of aromatic herbs, specifically Thymus, Mentha and Origanum. The dependence of aromatic composition on environment conditions is a well-known phenomenon. The genus Thymus includes numerous species with quite different botanical characteristics and a broad chemical heterogeneousness [1]. Thymus zygis \.. essential oil is usually rich in phenols (thymol and/or carvacrol), in terpenic alcohols (linalool, terpineol, geraniol or mircenol) depending on the quemyotipe [2]. Mentha pulegium shows a very different aromatic profile with the main component being either pulegone or isomenthone [3]. Supercritical fluid extraction is a clean technology with very interesting applications in food products because it is free from solvent residues. In addition this is a non-polluting process. On the other hand, the essential oils and oleoresins produced by using compressed fluid extraction may provide high quality products [4-5]. Carbon dioxide is the preferred solvent in the food industry as it presents a low critical temperature and pressure, and is non-corrosive.

134 low cost, non-flammable and readily available [6-7]. In the literature many references have been found applying the extraction of natural aroma compounds by this procedure. Supercritical fluid extraction, however, also presents some disadvantages, specifically the extraction of undesirable compounds. These problems may not be completely eliminated even with complementary treatments, like fractionated collection. Response surface methodology (RSM) is a quite useful way to achieve process optimisation [8-10] The aim of this work is to determine the best SFE conditions for tow Labiatae (Mentha ptilegium and Thymus zygis), by using RSM and using a Clevenger distillation as a standard process.

2. MATERIAL AND METHODS 2.1. Material Blossoms and leaves of Thymus zygis L. spp sylvestris, Thymus capitatus L and Mentha pulegium L. (collective sample) were collected during the flowering period. Material was air dried in dark till 10-12% moisture. The carbon dioxide was 99.95% (w/w) pure from Air Liquido/Portugal. All other reagents are analytical grade and standards are GC grade. 2.2. Extraction methods Distillation on a modified Clevenger apparatus (CLEV) was conducted for 30 min at atmospheric pressure on about 100 g of composite sample. Time was measured from the falling of the first drop of distillate. For each sample two replications of each extraction were done. 3

Compressed CO2 extraction was performed in the apparatus equipped with a 0.003 dm tubular extractor [11]. To approach the extraction optimisation conditions a response surface methodology (RSM) was employed using a factorial matrix". The independent variables were pressure, temperature and extraction time. The dependent variables studied were yield and composition of the main compounds. The independent variables were tested for the following ranges: pressure (9 to 20MPa), temperature (40 to 50°C) and time (60 to ISOmin). The results were fitted to the second-order polynomial equations through a stepwise multiple regression analysis using Statistica version 5 software. 2.3. Analytical methods The essential oil yield was evaluated by gravimetric method and expressed in terms of w/w. The essential oil was analysed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS). GC analysis was performed on a Hewlett-Packard 5890 gas chromatograph equipped with a FID and an HP-5 column (cross-linked 5% biphenyl, 95% dimethylissiloxane) 50 m x 0.32 mm i.d., film thickness 0.17 ^im.

135 GC conditions. Oven temperature: programmed 60°C for 10 min, followed by a slope of 2°C/min to 180X; 10°C/min to 200°C and a plateau at 200T/30 min. Injector and detector temperatures were 200 and 250°C, respectively. Carrier gas, N2 was adjusted to a linear velocity of 1 ml/min. The samples were injected using the split mode (split ratio 1:8) being the injection volume 0.2 }il. The quantification of the components was made by internal standard methodology. GC-MS unit consisted of a Hewlett-Packard 5970 mass selective detector operating in the electron impact mode (70 eV) coupled to a Hewlett-Packard 5890 gas chromatograph. A capillary column Supelco Wax 10 of 30 m x 0.25 mm i.d., film thickness 0.25 |im was used. Analytical conditions. Oven temperature: programmed 80°C for 10 min, followed by a slope of 2°C/min to 180°C ; 10°C/min to 200 °C and a plateau at 200°C/30 min. Injector temperature was 200°C. Samples were injected using the split mode (split ratio 1:19) being the injection volume 0.2 [i\. Carrier gas, He, was adjusted to a linear velocity of 0.89 ml/min.

RESULTS AND DISCUSSION Table 1 shows the results of the yield produced both by Clevenger and SCF. The SCF yield is much larger than the Clevenger when working with T. zygis (1.0 and 3.1 respectively). However, these higher values are not related to the aroma compounds, but to other kind of compounds, such as waxes and pigments. The M polegiiim yields do not show such as big a difference possibly due to the lower level of waxes in this plant.

Table 1 Extraction yield (% w / w)

Yield

Thymus zygis Extraction method CLEV SCF 1.0 3.1

Menta pulegium Extraction method CLEV SCF 0.9 2.1

Table 2 shows the amounts of main volatile compounds identified on the essential oils of the studied herbs. It is interesting to note that the kinds of the main compounds identified in T. zygis are thymol (20.6%), geranyl acetate (16.3%), p-cymene (13.6%) and y-terpinene (13.6%). For the M. pidegium, pulegeone (39.5%)) and isopulegol (17.3%)), are the main compounds. When comparing supercritical extracts to essential oil (Figure 1) the main differences are observed in thymol and pulegeone, where the SCF yields are less.

136 Table 2 Chemical composition of essential oil and supercritical extracts of T, zygis and M puleghim Thymus mis Extraction method CLEV SCF Compound a-Thujene a-Pinene Camphene Sabinene p- Pinene Myrcene a-Phellandrene a-Terpinene Limonene p-Cymene Cineole-1,8 /-Terpinene Terpinen-l-ol Terpinolene Linalool Camphor Bomeol Terpinen-4-ol Menthone Menthol Isomenthone Mirtenol Nerol Neral Pulegeone Piperitone Geraniol Linalyl Acetate Bomyl Acetate Thymol Carvacrol Isopulegol Geranyl Acetate Anetol Piperitona )9-CaryophyIlene

1 |

Menta pulegium Extraction method CLEV SCF

1.0 1.0 1.4 0.3 0.3 1.4 1.2 0.1 1.6 13.6 0.6 13.6 0.4 0.1 2.4 0.3 2.1 0.1

0.5 0.4 1.4 0.1 0.3 0.8 0.1 0.2 0.7 15.0 0.1 7.0 0.3 0.0 2.4 0.3 3.0 0.2

0.2 0.34

0.1 0.1

-

-

0.8

0.1

-

-

-

-

5.7 1.0 0.6 0.3

0.6 0.3 0.3 0.4

0.2 0.3

0.1 0.6

-

-

-

-

12.6 1.1 0.1 20.6 1.3

14.0 0.1 0.2 11.0 1.8

39.5 1.7 0.4

11.3 1.0 0.5

-

-

17.3 0.2 0.3

18.9 0.8 0.3

-

-

16.3

17.5

-

-

3.5

2.0

-

CLEV - Distillation on a modified Clevenger apparatus. * 15 MPa/40T/90 min; **11.3 MPa/42°C/155 min. 1.7

137

Figure. 1. Main constituents of essential oil and supercritical extracts of T. zygi and M pideghim In a first approach to the response surface analysis it can be stated that the responses for the main dependent variables studied are in agreement to the statistic model used as the regression coefficients are always significant at the significance level (p) <0,05. Table 3 shows regression coefficients for same dependent variables on the M piilegium SFE. In the case of isopulegol the model is not significant. Table 3 Regression coefficients DViPuIegeone; DV:Menthone; DV;Piperitone; 1 DV:Myrcene; R-sqr=0.94731; R-sqr=0.94774; R-sqr=0.90534; R-sqr=0.98529; Adj: 0.84194 Adj: 0.95587 Adj: 0.84322 Adj: 0.71603 1 Factor Regressn coeff. J^egressn coeff. Regressn coeff. Regressn coeff. -1215.42** -126.335** -11.1750** 1 Mean/Interc. -50.7064* (1) Pressure (L) -.81 -.050 -.0348 -.0040* .00 .000 1 Pressure (Q) .0001 .0000* 5.888** 2.4104* 1 (2)Temperature (L) 57.67** .5200** -.065** -.02268* -.0057** Temperature (Q) -.64** -.035 -.0085 (3)Time (L) -.28 -.0020 ^000 ^000 1 Time (Q) Loo ^000

DV:MenthoI; 11 R-sqr=0.96042; Adj: 0.88126 Regressn coeff. -15.9685** -.0080 .0000 .7565** -.0084** -.0042

.0000 * p<0.05 Significant; ** 0.05> p >0.01 Highly significant; *** p<0.001 Very highly significant

Temperature is a higher significant factor for all dependent variable. Pressure is a significant factor in the case of the extraction of myrcene. Extraction time, in the studied condition, does not seem to be significant.

1

138

Figures 2, 3, 4, 5 and 6 show the surfaces of five dependent variable as a function of pressure and temperature and corresponding equations. The best conditions seems to be: 10 MPa / 4 5 T . As the extraction time is not significant, the extraction must be conducted at the lower time tested, 80 min. With regard to the extraction of T zygis, pressure seem to be the most important parameter, with 18 MPa being the most suitable condition, although non volatile compounds like waxes, pigments are extracted. Temperature did not significantly affect both the yield and extract composition. The most convenient temperature found was about 40-45X.

Wm -3.849 • • -2.276 • I -0.702 B i 0.871 (1112.444 IZU 4.017 WM 5.590 8 8 7.164 • • 8.737 wm 10.310 •Bi above

z=-1215.4180444108-.81398111979166*x+.0027041015625 V 2 +57.672555555576V.64122222222245V2-15.131429

Fig. 2 - Response surface for pulegeone relating the effect of temperature and pressure

4. CONCLUSIONS Response Surface methodology (RSM) is an effective methodology to optimise compressed CO2 extraction of aroma, when applied to aromatic herbs. The best extraction conditions for M piilegiiim seems to be 10 MPa / 45°C As the extraction time is not significant, the extraction may be conducted at the lower time test of «0 min. For T. zygis pressure is a more significant extraction parameter

139

• i -0.322 WM -0.191 • • -0.060 H i 0.072 IZU 0.203 EH] 0.334 W^ 0.466 • i 0.597 • i 0.728 WM 0.860 • • above

z=-126.33514290203-.049776855468751 *x+.000173583984375*x'^2 +5.8880555555577>.065361111111135V2-2.0888163

Fig. 3 - Response surface for mentone relating the effect of temperature and pressure

• i • I • 1 iBl {ZJ CD nm (ai •H

0.076 0.145 0.213 0.282 0.351 0.419 0.488 0.556 0.625

B i above

z=-50.706411411019-.03484318033854rx+.0001136474609375*x'^2 +2.4104166666675>.026791666666676 V2-.39926531

Fig. 4 - Response surface for piperitone relating the effect of temperature and pressure

140

• • 0.121 • 1 0.134 • 1 0.146

mm 0.159 CD cm B^l • i • 1 • 1 •B

0.171 0.183 0.196 0.208 0.221 0.233 above

z=-11.174982427692-.0040450032552084*x+.0000140380859375*x'^2 +.51997222222242 V.0057361111111133 V 2 - . 10114286

Fig. 5 - Response surface for myrcene relating the effect of temperature and pressure

• 1 0.112 • i 0.130 • i 0.147 H i 0.165 r ~ l 0.182 i 1 0.199 EB 0.217 B i 0.234 • I 0.252 g a 0.269 S S above

.=-15 968530511771 -.0080463053385417*x+.0000277099609375'iv^ + 7564722222225 V.0084027777777809y2-.24285714

Fig, 6 - Response surface for menthol relating the effect of temperature and pressure

141 5. REFERENCES 1. E., Stahl-Biskup, J. Ess. Oil Res. 3, (1991), 61-82. 2. D., Garcia-Martin and M., C, Garcia-Valejo, Ix^^ international Congress of Essential Oils, Singapore, 24 pp. (1979). 3. J. S., Carvalho, Silva Lusitana, 2, (1994), 193, 206. 4. W.G. Schultz and J.M. Randall, Food technology, 24, (1970), 1282-1286. 5. S.S.H. Rizvi, J.A. Daniels, A.L. Benado and J.A. Zollweg, Food Technology, Vol. (7), (1986), 57-64. 6. A.R. Bhaskar , S.S.Rizvi and J.W Sherbon.,. J. Food Science, 58, (1993), 748-752. 7. T. Vardag and P. Korner, Food Marketing & Technology, Feb., (1995), 42-47. 8. J. D.Floros and , M. S. Chinnan, J. FoodSci., 53 (1988) 631-638. 9. G. S. Mudahar, R. T. Toledo, J. D. Floros and J. J. Jen, J. FoodSci., 54, (1989), 714-719. 10. V. A.-E King and R. R. Zall, Food Research International, 25, (1992), 1-8. 11. M. Esquivel and M. G. Bernardo-Gil, The J. of Supercritical Fluids, 6, (1993), 91-94.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

143

Hypericin and hypericin-like substances: analytical problems. F. Tateo, S. Martello, E. Lubian a n d M . Bononi D.LF.C.A.-Sezione di Chimica Analitica Agroalimentare University of Milan, Via Celoria n.2, 20133 Milan, Italy

ed

Ambientale,

Abstract Hypericin is a substance derived from Hypericum perforatum L., a plant utilized in the production of extracts used to aromatize alcoholic beverages and soft drinks and Umited in food and beverages by the E.E.C. Directives on Flavouring. This paper deals with the H.P.L.C. method developed to cover the lack of official and recommended methods concerning hypericin. It also considers the effect of alcohoHc content on extraction by infusion of hj^ericin and testifies to the presence of hj^ericin-Hke substances in Hypericum perforatum L. extracts, inexpUcably not Umited by law.

1. INTRODUCTION Hypericum perforatum L. is a perennial herbaceous plant belonging to the family of the Hypericaceae widely found in Europe, Asia, North Africa and, for some time now, in the United States of America. In Europe it can easily be found in waste ground, as well as near roads or woodlands, or in the plain and on hillsides. The plant is considered to be "medicinal" and was also used in popular medicine, both for internal and external use. It is commonly known, not least to English people who grow it in their gardens, as Saint John's Wort. The plant and flowers, according to the Codex VegetabiH by Steinmetz (1), are held to contain active ingredients, which prove to be nervine, stimulant, digestive, cholagogus, diuretic and a uterine tonic; the oil extracts are credited with disinfectant properties. Hypericum perforatum L. is widely used today in phytotherapy, due to its numerous therapeutical and medicinal properties, in the form of infusions and tinctures (cicatrizing and antiphlogistic effects). The properties that have aroused the most recent interest are its anti-depressive and anti-viral activity (2). Hypericum perforatum L. is included in the monography of numerous pharmacopoeia. In the Blue Book of the European Council it is hsted in category

144

N2, which includes natural sources of aromas frequently consumed in small doses and found in normal diets (herbs, spices, or condiments) (3). The interest shown for this plant also stems from its use as aromatizer in the preparation of food, alcoholic drinks, and above all in bitters or "digestive" ones. This article is born from the consideration regarding the use of Hypericum perforatum L. in the aromatization of food and drinks. Mention is made, in particular, of the inclusion of the active agent "h5rpericin" in a list of substances whose use is restricted. Studies relating to the chemical composition oi Hypericum perforatum L. began in 1830, with the isolation of hypericin by Buchner, who called this substance Hypericum Red (4). In 1904, work was carried out to determine essential oil content, the tannin-like substances, and the colorants found in Hypericum perforatum L. (5,6). The essential oil content ranges between 0.1 and 0.35% depending on the harvesting period and the quality of the aerial portion of the plant. In 1911, the substance isolated by Buchner, i.e., "hypericin red", was once again isolated and renamed hypericin by Cerny (7) together with other components considered to be of like structure. The correct structure of hj^ericin was, however, not defined until 1953 (8). In addition to the vast number of components found in the vegetative portion of the plant, it is also important to examine and determine the chemical composition of its aerial portion. That of Hypericum perforatum L. has been shown to have important components such as numerous polyphenolic compounds belonging to the class of antrachinons and bioflavonoids of diterpenoid and n-alcans. While extracts of Hypericum perforatum. L. utilized in phytotherapy are standardized only in their content of h5^ericin and hypericin-like compounds, other components of biological importance have been isolated and shown to be endowed with antimicrobial activity, such as hyperforin (9), h5^eresin 1 and 2 (10), adhyperforin (11) and 1,3,6,7-tetrahydroxantone. The structures for the hj^ericin and other h5rpericin-like compounds may be seen in Figure 1 while the structure for hjrperforin, adhj^erforin, and for some of the flavonoids present in h5^eric are seen in Figure 2. Contents of some active agents in the flowers of the apical part of hyperic are reported by Holzl (12). The data are a result of the analyses of 50 plants chosen at random from a population of 250 individuals (seeds from an old botanical garden for medicinal plants in Marburg). The enactment and adoption of the European Community Directives 83/388 and 91/71 relating to flavours is destined to be employed in food products as well as in basic materials for their preparation. These Directives concern only the maximum quantity of some substances stemming from flavours and from alimentary ingredients endowed with aromatizing properties and present in finished alimentary products in which aromas were employed. A maximum quantity is indicated for hypericin: that is equal to 0.1 mg/kg in food-stuffs and in beverages, 1 mg/kg in sweets and 10 mg/kg in alcoholic beverages. No reference appears for other hypericin-like substances that may be present together with hj^ericin, sometimes in far from neghgible quantities.

145 As regards the aforementioned regulatory aspects, one would expect to be able to find documentation in these Directives of an "official" analytical method for the quantitative determination of hypericin. However, so far no "official" analytical method has been published to provide for the quantitative control of restricted substances in flavour and in food preparations. Therefore, whenever an analytical problem regarding conformance with official regulation arises, one is forced to seek a method in sources other than the "official" ones. While the I.O.F.I. (International Organization of the Flavour Industry - rue CharlesHumbert, 8 - Geneva, Switzerland) has "recommended analytical methods" for many compounds, it does not pronounce itself ^dth regard to hypericin. The primary purpose of this research was to develop a method capable of quantifying hypericin. The method uses H.P.L.C. for the determination of hypericin and other hyperic compounds and has a detectability Hmit (evaluated by examining of the H.P.L.C. profiles) equal to 0.27 mg/kg for an injection of only 20 |iL of a solution containing hjrpericin. This method can be performed in a shorter time than the method reported by Holzl (12) and also uses one shorter column and operates at a higher flow-rate (1.0 ml min^) than Holzl's (0.6 ml minO- Another reason for performing this research was to show the possibility of identifying not only hypericin, but also hj^ericin-like components present in the aromatizing extracts of hyperic. Examination of the biological effect of these components, together with the estabhshment of dose limitations, is critical and warrants greater attention, since most of the potential biological effects of various hj^ericin-like components are not known. The solubility and extractabihty of hypericin and hypericin-like substances from Hypericum perforatum L. is another area of research needing examination, since the variability in the level of these components in alcohoHc and non-alcoholic beverages can vary considerably, depending upon the alcoholic content of the medium of extraction from Hypericum perforatum L.

2. EXPERIMENTAL 2.1 Instrument The research made use SHIMADZU Corporation (Kyoto, Japan) H.P.L.C. (High Performance Liquid Chromatography) consisting of an SCL-lOA System Controller, a pumping-system made up of a two-unit LC-IOAS Liquid Chromatograph, a Diode Array Detector SPD-MIOA, and an HP Deskjet 660 C Printer. A reversed-phase Techsphere C-18 (15 cm x 4.6 mm i.d., 3 \xm particle size) column was used. 2.2 Standards and Reagents Standard h5^ericin (minimum 85%) was purchased from Sigma Chemical Co. (St Louis, USA). H.P.L.C. grade methanol, acetonitrile and water were obtained from Merck (Darmstadt, Germany). Phosphoric acid was purchased from Baker

146 Analyzed® (Deventer, Holland). Hypericum perforatum L. dried herb came from EMANS (Milan, Italy). 2.3 Standard solutions and analytical method Hypericin standard solutions (85, 8.5, 0.85 mg/L) were prepared in methanol and analyzed by H.P.L.C. under the following conditions: the standard solutions were injected with a 20 |iL sample loop into a Techsphere C-18 column (15 cm x 4,6 mm i.d., 3 |im particle size). The mobile phase consisted of two eluents: {A) acetonitrile/methanol/phosphoric acid (59:40:1) and (B) acetonitrile/water/phosphoric acid (19:80:1). Eluents A and B were mixed in accordance with the gradient given in Table 1. Flow-rate was set at 1.0 mL/min, detection of hypericin and hypericin-like substances being achieved at 254 nm and 590 nm.

Table 1 Mobile phase-gradient conditions used in the H.P.L.C. analyses of the standard solutions, beverages and Hypericum perforatum L. extracts. Time (minutes)

Solvent A (%)

Solvent B (%)

0 8 28 55 70

0 0 100 100 0

100 100 0 0 100

2.4 Sample preparation The beverages analyzed for their hypericin content were prepared as follows: - Alcoholic beverages: sample diluted 1:1 with ethanol. - Non-alcoholic beverages: about 100 g of beverages is weighed and concentrated to dryness on a rotary evaporator. Distillation is performed by repeatedly adding quantities of ethanol equal to 10 mL, in such a way as to facilitate elimination of the water in the form of azeotrope. The residue obtained is recovered with 5 mL of ethanol and filtered with a MiUipore filter (0 = 0.45 ^im pore size). The samples were analyzed under the operating conditions described in 2.3. 2.5 Hypericum perforatum L. extracts Dried plant material was extracted by immersion in hydroalcohoUc medium with an extraction ratio of 2:10 (weight of herb/volume of extracting medium) and with an alcohoUc content of the extracting medium equal to 10%, 20%, 30%,

147 40%, 60%, 80%, 96% (% ethanol, v/v). The extraction was carried out under static conditions for 3 days (with shaking every six hours). The resulting infusions were analyzed under the operating conditions described in section 2.3.

3. RESULTS AND DISCUSSION Evaluation of the detectable hypericin concentration was carried out using the conditions described above. Figure 3 shows the plots obtained at 254 nm (Figure 3A) and at 590 nm (Figure 3B) after injection and H.P.L.C. analysis of a standard solution of hjrpericin at 85 mg/L of methanol. The result turns out to be conditioned by the value of A,, as is foreseeable according to what is detectable from spectrum examination within the 200-600 nm range and registered by the Diode Array Detector on the standard solution at concentration of 85 mg/L of methanol; the spectrum is shown in Figure 4. It is worth mentioning that a characteristic of hypericin and hypericin-like compounds is precisely this trend of the spectrum around 590 nm, a value in which other hyperic components show no absorption. By analysis of standard solutions it was possible to define a detectable Umit concentration of 0.27 mg/kg for hypericin. In the case of alcohoUc beverages, taking the preliminary dilution as 1:1, it is possible to detect the presence of hypericin and determine its content in the beverage only if it contains more than 0.5 mg/kg. The sensitivity Umit of the determination is more than sufficient, in that the acceptable value is 10 mg/kg for alcohoUc beverages. In the case of non-alcohoUc beverages, bearing in mind the preUminary treatment of concentration, it is possible to detect the presence of hypericin and determine its concentration only if its content is higher than 0.01 mg/kg. Even in this case the sensitivity Umit of the method is weU below the value of 0.1 mg/kg accepted for non-alcohoUc beverages. Verification of "yield" of hypericin and hypericin-Uke substances during extraction by infusion was performed in order to explain the "absence" of hypericin in bitter drinks found on the market. The data obtained are given in Figures 5 and 6. These figures clearly show that the extraction of hj^ericin is obtained decisively with an alcohoUc content of the order of 40% ethanol and more, being much higher with 60% ethanol and more. Nevertheless, pseudohypericin is already extracted with 30% ethanol and its signal at 590 nm (characteristic just Uke hypericin; see spectrum in Figure 7) turns out to be much higher than that of hypericin with 40% ethanol and more. Data show that the alcohoUc content of the extracts has a greater influence on hypericin extraction. However, Figure 8 compares hypericin extractabiUty over time and indicates that both time and alcohol concentration affect hypericin extraction. Analysis of the infusions points to the possibiUty of isolating some compounds in a concentration higher than that of hypericin. Identification of these

148

hypericin-like compounds is facilitated by the use of a Diode Array Detector which permits the identification of a large series of other hyperic components. Identification of hypercin-like substances, even in the absence of hypericin, makes it possible to deduce that hyperic was used in the preparation of the drink. Figure 9 shows an H.P.L.C. profile of a hyperic hydroalcohoHc infusion (80% EtOH, % v/v): the UV absorption spectra obtained with the Diode Array of hypericin, and hypericin-hke substances (probably including protohypericin and cyclopseudohypericin), hyperforin, and I 3' II 8 biapigenin are shown. Given the clear difference between the spectra, it is possible to distinguish between hypericin and hypericin-like substances.

4. CONCLUSIONS The method proposed for determining hypericin is suitable for checking both alcoholic and non-alcohoHc beverages, with a detectabihty limit of the order of 0.01 mg/kg. The analysis conditions appUed in this work made it possible to identify in the hyperic extracts various other characteristic components, including significant hypericin-Hke substances (pseudohypericins, cyclopseudohypericin and protohypericin). Determination of the hypericin-like substances, though not prescribed by current legislation, may turn out to be significant, since it has not so far been shown that the activity of hypericin-Hke substances is negligible compared with that of hypericin. This work has made it possible to show that alcoholic gradation is a limiting factor in hypericin extraction during the technological stage in the preparation of hyperic extracts. In particular, hypericin extraction has been found to be possible only as from 40*^ (EtOH%, v/v) on, unUke what happens in the case of other hypericin-hke substances.

5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12

Steinmetz, Codex Vegetabili, International Flavors & Fragrances E. BombardeUi, P.Morazzoni, Fitoterapia 66, 1 (1995) Flavouring substances and natural sources of flavourings, Council of Europe, 3^^ Edition, Strasbourg (1981) S. Buchner, Buchn. Repert. 34, 217 (1830) G. Haensel, Apoth. Z. 20, 145 (1905) E. R. MiUer, J. Am. Ph. A. 16, 824 (1927) C. Cerny, Z. Physiol. Chem. 73, 371 (1911) H. Brockman, W. Sanne, Naturwiss. 40, 509 (1953) A.I. Gurevich, M.N. Dobrynin, S.A. Kolosov, I D . Popravko, B.E. Aizenman, A.D. Garagulya., Antibiotiki 16, 510 (1971) K.N. Gaind, T.N. Ganjoo, Indian J. Pharm. 21, 172 (1959) P. Maisenbacher, A. Kovar, Planta Med, 58, 291 (1992) J. Holzl, E. Ostrowsky, Dtsch. Apoth. Ztg., 23, 1227 (1987)

149

HO

O

OH

HO

O

OH

Hypericin Pseudohypericin

HO

O

OH

HO

O

OH

HO

HO

R=H R=OH

OH

O

OH

Protohypericin R=H

HO

HO

Cyclopseudohypericin

O

O

OH

O

OH

Hyperico-dehydro-dianthranol OH

OH

OH

Emodinanthranol Figure 1. Structures of hypericin and hypericin-like compounds present in Hypericum perforatum L.

150

H,C

H,C

H3C

H3C

HO

HO

HX

H3C

Hyperforin

OH

Adhyperforin

O

Quercetin Quercitrin Isoquercetrin Rutin Hyperoside

R=H R=Rha R=Glu R=Rha-Glu R=Gal

OH

O

I 3^ II 8-Biapigenin

Figure 2. Structures of hyperforin, adhyperforin and of some flavonoids present in Hypericum perforatum L.

151

254 nm

250

20O

CI ^

150

< ^

10O

50

4

oJ (3

..__^___JL.^^j-u.—J 5

10

15

20

25

'

30

35

40

45

time

590 nm

250

B

20O

^

•rH

150

O

< ^

•PH

10O

K1

50

I

A

^r )

(

5

10

15

20

25

30

1 35

40

4

time

Figure 3. H.P.L.C. traces of hypericin standard solution at a concentration of 85 mgfkg of methanol at wave lenghts of 254 nm (A) and 590 nm (B). Operative conditions are described in section 2.3.

152

a

Figure 4. Absorption spectrum of hypericin obtained with Diode Array Detector.

^ >

o o kO

^ >

O o

O

utoTJecMjj LO

O

UTOTJcedXqopnesj

nvtti

i:^

I 1

!

i

O

1 j ^ ^

j UTOTJ[edXi{opneS(j ^

nv«i

CO

0 •iH

CD

O

o CO

o

7i

O

-a 2

to's^

Qj

o.S o

0;

CD

ri ^ ^.^

153

154

^ >

-M

o w o 5

>

o o CD

>

o

UTOijedXjj

5

-==

5

uTouedXnopnesj S

nyra

UTOTjadXjj

uToiJ[a(Mi[opn8S ^

nvra

~V

J c3

JUn

uiOTjedXjj

UTOTj[0(LCi[opnesj

nv™

s

a

o

a

O

o 00

o CD

O

0

5 ^

^

0

CD h l n

^^

o

0

•

CD

155

<

200

250

3O0

350

40io

450

500

sSo

600

nm Figure 7. Absorption spectrum of pseudohypericin obtained with Diode Array Detector.

156

1000 900 800 700 600

500

cd

a;

400 ^ 300

cd

a;

200

100

1 day

2 days

3 days

Time Figure 8. Effect of ethanol (% v/v) on the extractability of hypericin in infusions of Hypericum perforatum L.

157

Cyclopseudohypericin* P s e u d o h y p e r i c i n P r o t o h y p e r i c i n *

nm

I 3',II 8-Biapigenin Hyperforin\

200

nm

600 200

nm

600

\ Hypericin

600 200

nm

600

Figure 9. H.P.L.C. trace of an infusion at 80° (% EtOH v/v) at 254 nm limited around the zone of hypericin and hypericin-like compounds, with indication of the corresponding spectra (200-600 nm) of some of the components identified. The operative conditions of analysis are described in section 2.3. * Identity to be confirmed.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

159

Determination of the Cause of Off-Flavors in Milk by Dynamic Headspace GC/MS and Multivariate Data Analysis R. T. Marsili and N. Miller Research Dept., Dean Foods Technical Center, P.O. Box 7005, Rockford, IL 61125, U.S.A.

Abstract Milk is susceptible to off-flavor development by a variety of mechanisms. Multivariate analysis of GC data can be used to determine the cause of off-flavor development in customer complaint milk samples. Control samples of normal, good-tasting milk were analyzed by dynamic headspace GC/MS and a GC/FID technique that measures free fatty acids. The samples were then subjected to common abuse conditions (exposure to light, copper, and sanitizer) at various levels and re-analyzed. The abused samples provided the basis for KNN and SIMCA classification modeling. Results show that multivariate analysis can accurately predict the type of sample abuse responsible for off-flavors.

1. INTRODUCTION Milk is susceptible to formation of off-flavors by various mechanisms. Shipe et al. [1] have listed seven descriptors of off-flavor in milk based on causes: heated, lipolyzed, microbial, transmitted (from feed and weeds), light-induced, oxidized, and miscellaneous. Light-induced off-flavors, undoubtedly the most common flavor defect in milk, have two distinct components. Initially a burnt, activated sunlight flavor develops and predominates for about two or three days. Degradation of sulfur-containing amino acids of the serum (whey) proteins has been blamed for this reaction. The second component is attributed to lipid oxidation. This off-flavor, often characterized as metallic or cardboardy, usually develops after two days and does not dissipate. It has been estimated that exposure of milk in blow-mold plastic containers to fluorescent lights in supermarket dairy cases is responsible for the development of light-induced off-flavors in some 80% of store samples [2]. Two other common causes of off-flavor in milk are contamination with pro-oxidant metals (especially copper) and contamination by sanitizer (especially peroxyacetic acid-based sanitizer). Copper can be transmitted to milk from feed sources and leached from pipes and valves used in processing equipment. Sanitizer which hasn't been completely rinsed from processing lines after cleaning can contaminate milk. Peroxyacetic acid and other popular new robust sanitizers do an excellent job sanitizing processing equipment between runs, but because of their improved stability, they have a long lifetime in milk and can increase the risk of off-flavor development. These three mechanisms of off-flavor development can potentially generate off-flavors in

160 milk by degrading polyunsaturated fatty acids in milkfat triglycerides and phospholipid fractions and/or by degrading milk proteins. Each of these three types of off-flavor mechanisms can potentially generate similar oxidation byproducts (e.g., hexanal) in milk [3]. Deciding which mechanism is responsible for the off-flavor in a particular sample is impossible to do simply by tasting samples — even when trained organoleptic evaluators are used. The goals of this study were: (a) to determine if multivariate statistical analysis of gas chromatographic data can be used to classify milk with off-flavors by the type of abuse mechanism and by the level of abuse which has occurred (i.e., low, medium, high exposure levels); and (b) to use multivariate analysis to indicate which chemical byproducts are the best indicators of these three types of abuse mechanisms. Classification modeling can be conducted with multivariate analysis techniques and involves the computation and graphical display of class assignments based on multivariate similarity of one sample to others. One example of this technique is identification of bacteria based on fatty acid profiles of lipid extracts from bacterial cell walls [4]. Multivariate analysis has been applied to the study of a wide variety of food and beverage problems. A few examples include milk shelf-life prediction [5], chemotyping of essential oils [6], discriminating aromas of coffee samples [7], classifying wine samples [8], and characterizing peppermint oils [9]. Recently, Horimoto et al. used Principal Component Similarity Analysis (PCSA) for the classification of microbial defects in milk based on dynamic headspace GC data [10].

2. EXPERIMENTAL 2.1. Sampling for classification modeling One set of samples, consisting of homogenized whole-fat milk (3.3% fat), 2% fat milk, skim milk (<0.5% fat), and the raw milk from which they were made, was obtained from three different dairies (Chemung, IL; Albuquerque, NM; and Murray, KY). Milk was intentionally abused to provide samples that could be used to calibrate statistical prediction models. Each of the 12 samples was subjected to the following abuse conditions and assigned a classification code corresponding to one of the 10 class assignments indicated (in bold) below: Class A: Light abuse: • Class Al; Low level exposure (150 footcandles fluorescent light for 3 hr at 4°C; analyzed after 24 hr of storage at 4°C). • Class A2; Medium level exposure (150 footcandles fluorescent light for 8 hr at 4°C; analyzed after 24 hr of storage at 4°C). • Class A3; High level exposure (150 footcandles fluorescent light for 24 hr at 4°C and then analyzed). Class B: Copper abuse: • Class Bl; Low level contamination (5 ppm Cu added as cupric chloride, incubated 24 hr at 4°C and analyzed). • Class B2; Medium level contamination (50 ppm Cu added as cupric chloride, incubated 24 hr at 4°C and analyzed).

161 • Class B3; High level contamination (50 ppm Cu added as cupric chloride, incubated 72 hr at 4°C and analyzed). Class C: Sanitizer abuse. (Oxonia™ active sanitizer was used. It is a peroxyacetic acid sanitizer used extensively in the dairy industry and is the trademark of Ecolab, Inc., St. Paul, MN.): • Class CI; Low level contamination (400 ppm Oxonia active sanitizer, incubated at 4°C for 24 hr and analyzed). • Class C2; Medium level contamination (800 ppm Oxonia active sanitizer, incubated at 4°C for 24 hr and analyzed). • Class C3; High level of contamination (1200 ppm Oxonia active sanitizer, incubated at 4°C for 24 hr and analyzed). Class D: No abuse (fresh, control, good-tasting milk). The above sampling scheme generated a total of 120 samples to be used for classification modeling by KNN and SIMCA. GC analysis of these 120 samples generated approximately 80 different peaks for each sample, resulting in approximately 9,600 peak areas that were initially considered for use in classification modeling.

2.2. Gas chromatography methodologies 2.2.1. Dynamic headspace GC/MS methodology A milk sample (20 g) was heated to 40°C and purged with helium at a rate of 15 mL/min for 20 min using the Purge and Trap System from Scientific Instrument Services (S.I.S., Ringoes, NJ). The helium dry-purge line was set at a 10 mL/min flow rate. Volatiles were trapped on a Tenax-filled desorption cartridge. After the 20 min sampling period, the Tenax cartridge was removed from the S.I.S. system and placed in the desorption chamber of a CDS PeakMaster concentrator (CDS Analytical, Oxford, PA). Volatiles from the trap were desorbed by heating the cartridge to 185°C and purging with helium at a rate of 15 mL/min for 10 min. Volatiles were then trapped onto a second Tenax adsorbent trap in the CDS PeakMaster concentrator. After collection on this trap, an additional dry-purge cycle was used to eliminate any water vapor that may have accumulated on the trap during stripping. Dry purging was conducted at a temperature of 40°C for 4 min. The volatile components were then desorbed onto the analytical column from the second trap by heating to 200°C for 10 min and cryofocused at -100°C with liquid nitrogen prior to column injection. The analytical column used was a 30 m x 0.25-mm i.d. DB-5 fused-silica capillary column with a film thickness of 1 |Lim (J&W Scientific, Folsom, CA). The initial column temperature was 50°C for 1 min, then heated to 180°C at a rate of 6°C/min and held at 180°C for 4 min. The column was then heated to 240°C at 6°C/min and held at 240°C for 8 min. The performance of various types of capillary columns for the analysis of volatile chemicals in dairy products by dynamic headspace GC was evaluated by Imhof and Bosset [10]. This study showed best performance was obtained with a capillary column coated with a thick film of polydimethylsiloxane. GC/mass spectrometry was performed with a Varian Saturn 3 system from Varian Analytical Systems (San Fernando, CA).

162 2.2.2. Free fatty acid GC/FID methodology Milk samples were analyzed for free fatty acids by the method described by Deeth [12]. Approximately 10 g of milk and 5 mL of a C17 carboxylic acid internal standard solution (20 mg diluted to 100 mL with ethyl ether) were subjected to solid phase extraction with an alumina column. After washing with 2 x 5 mL portion of hexane/ethyl ether (1:1, v/v), the free fatty acids were eluted off the alumina with a 6% solution of formic acid in diisopropyl ether. The column used for FFA analysis was a 30 m x 0.25-mm i.d. FFAP fused-silica column with a film thickness of 0.25 |im (J&W Scientific, Folsom, CA). The column was heated to 140°C for 1 min, then heated to 240°C at a rate of 15°C/min and held at 240°C for 20 min. The injection volume was 2.0 |iL, and the split ratio was 30:1. The injector temperature was 250°C, and the flame ionization temperature was 260°C. This test measured acetic acid from the decomposition of peroxyacetic acid. The test can also be used to monitor free fatty off-flavors generated from active lipolytic enzymes reacting with milkfat and free fatty acids produced as psychrotrophic bacterial metabolites. 2.3. Multivariate classification methods Multivariate analysis was conducted with Pirouette™ multivariate data analysis software from Infometrix (Seattle, WA). Two types of classification techniques were investigated: KNearest Neighbors (KNN) and Soft Independent Modeling of Class Analogy (SIMCA). The KNN method is a similarity-based classification method which attempts to categorize unknown samples exclusively on their multivariate proximity to other samples of preassigned categories. In contrast to KNN, which is based simply on Euclidean distances among sample points, SIMCA develops principal component models for each class or category in the training set. When classifications are attempted for unknown samples, a comparison is made between the unknown's data and each class model. The class model which best fits the unknown, if any, is the class assigned to that sample. While KNN and SIMCA are both similarity-based techniques, their calculation approaches are distinctiy different. KNN is more appropriate to use for a sample-poor environment. KNN measures the Euclidean distance between the unknown sample and each of the known samples in the training set. The category assignment for the unknown is made by a plurality vote of the nearest neighbors, with an option of one to k neighbors considered. KNN is tolerant of sample-poor situations and is the only method which works well when categories are strongly subgrouped. Although SIMCA is not well suited for the case where there are only a few samples per category, SIMCA models the location and distribution of a category in the measurement space by constructing a principle component representation of this distribution for each category. Class assignments for unknown samples are based on their proximity to the nearest category model. SIMCA creates factor-based models of each category membership, with three possible outcomes: sample is a member of one category, sample is a member of more than one category, sample is not a member of any category. 2.3.1. Exploratory data analysis Peak area data for 80 different GC peaks from 120 milk samples were combined into a Pirouette spreadsheet. Peak areas were converted to peak area ratios by dividing the area of each peak by the area of an internal standard peak. 4-Methyl-2-pentanone was the internal

163 standard used for purge-and-trap analysis, and heptadecanoic acid was the internal standard used for free fatty acid analysis. A second internal standard, 2-ethylhexyl acetate, was also employed for dynamic headspace testing but was only utilized as a tool to help locate identical chemical peak components from chromatogram to chromatogram. Three-dimensional Principal Component Analysis (PCA) scores plots and PCA loadings plots were created with the Pirouette Exploratory algorithm and examined to see how well class groupings were accomplished and which independent variables (GC peak area ratios) contributed most to class differentiation. By using this technique to eliminate peaks that contributed little to sample classification assignments and by discarding peaks that were identified by mass spectrometry to be background noise peaks (e.g., chemicals from GC septa), the original number of independent variables was reduced from 80 to only 11. Also, inspection of dendograms created with the Hierarchical Cluster Analysis (HCA) algorithm helped to visualize sample class grouping and was useful in deciding which independent variables to eliminate in order to improve clustering of similarly abused samples. Class clustering in the PCA scores plot was significantly improved after performing two minor data transformations. Specifically, all peak area ratios for dimethyl disulfide were multiplied by 10, and all peak area ratios for acetic acid were multiplied by 100. These transformations increased the influence of dimethyl disulfide and acetic acid peak area ratios in determining class groupings.

V c s X C2,C3 • • - - - " > • 1C2

A2 X /

X

'^-

A3 ,

••••'">'

V*iii>5 C1 X ^ * - - - ' ^ ^ \ • IM ,.-••'

B2,B3f.i

\

)

\

Figure 1. PCA scores plot for all 120 samples using 11 independent variables (i.e., chromatographic peak area ratios). Plot shows clustering of samples according to abuse classification category where: Al, A2, and A3 represent low, medium, and high light abuse levels, respectively; Bl, B2, and B3 represent low, medium, and high copper abuse levels, respectively; CI, C2, and C3 represent low, medium, and high sanitizer abuse levels, respectively; and D represents control (non-abused) milk samples. Sample scores tend to fall primarily on principal component axes.

164 Performing these data transformations (and thereby giving dimethyl disulfide and acetic acid more significance) appeared justified for two reasons: (a) Acetic acid is a known degradation product of peroxyacetic acid, and dimethyl disulfide is a known photoxidation product of methionine, a sulfur-containing amino acid in milk proteins [13]; and (b) class clustering was significantly improved as a result of making these data transformations. When exploratory analysis was performed on the transformed data set (120 samples and 11 independent variables), the PCA scores plot showed that sample scores fall primarily on principal component axes (Figure 1). In fact, the three factors are largely associated with only one original variable, where the acetic acid peak is the only indicator of Oxonia sanitizer, the dimethyl disulfide peak is the most significant indicator of light abuse, and the hexanal peak is the primary indicator of copper abuse. 2.3.2. K-Nearest Neighbors After data reduction and transformation, a KNN model was created using no preprocessing and setting the k value at 5. The model was then saved to allow for predictions of abuse class assignments for unknown off-flavor samples based on peak area ratios for the 11 peaks used in the model. The optional value of k was set at 5 neighbors because the fewest number of misses occurred when k = 5. This means that to achieve the best prediction rate, the "votes" from the 5 closest samples to an unknown should be polled. Because only three independent variables (acetic acid, dimethyl disulfide, and hexanal) were shown to be the primary indicators of abuse type and level, a second KNN model was created using the 120 samples and only these three independent variables. The purpose for this was to determine if milk samples could be as accurately classified with only three chemical indicators as with 11 chemical indicators. 2.3.3. SIMCA modeling A SIMCA model was created using the 11 peak-ratio variables for each of the 120 samples. For SIMCA modeling. Preprocessing was set at None, and the Number of Components was set at 5. SIMCA classification modeling provided diagnostic information about which variables to use. For example, the Distance Object for diagnosing outliers is a plot of Mahalanobis distance vs. sample residual for each class assignment. Using this plot, six of the 120 samples were identified as outliers. Two SIMCA models were created: one model with all 120 samples (including Mahalanobis outliers) and the other with Mahalanobis outliers excluded. In addition, a third SIMCA model was created to see how few variables could be used for class prediction and how accurate predictions were with significandy less independent variables.

3. RESULTS AND DISCUSSION 3.1. Accuracy of KNN and SIMCA models in predicting abuse class 3.1.1. KNN prediction results The 120 abuse samples were treated as unknowns and tested by the KNN model to see how accurately abuse class assignments could be predicted. Two KNN models were tested:

165 one using 11 independent variables and one using only three independent variables (area ratio peaks for acetic acid, dimethyl disulfide, and hexanal). The model with the 11 variables correctly classified 103 of the 120 samples (86%) according to the 10 classification assignments. Examination of misclassified samples showed that when misclassification occurs, frequently it is not because samples are assigned to the wrong class based on type of abuse (none, light, copper, sanitizer); rather, the level of abuse is not properly estimated. Therefore, when class predictions were made based on only four categories (A = light abuse; B = copper abuse; C = sanitizer abuse; and D = no abuse) which ignore level of abuse, the 11-variable KNN model is able to correctly classify 93% of the 120 samples. When the samples in the 120-sample data set were treated as unknowns and analyzed for class assignments by the 3-variable KNN model, 101 of the 120 samples (84%) were correctly classified by both type and level of abuse, and 112 of the 120 samples (93%) were correctly classified by type of abuse but not necessarily level of abuse. Also, examination of the Misclassification Matrix revealed that the KNN model tends to classify class Al samples (low level of light abuse) as control (non-abused) samples. This type of misclassification is not unusual, since low level light exposure (150 footcandles for only 3 hrs) does not generate significant off-flavors in milk. For example, in one sensory taste panel experiment, only 2 of 12 people were able to detect a perceivable off-flavor in class Al homogenized whole milk, 2% fat milk, and skim milk samples. Furthermore, chromatograms of most of the class Al milk samples were nearly identical (both quantitatively and qualitatively) to class D chromatograms. With KNN modeling, accurate classification of samples can be accomplished with only three variables. However, the accuracy of classification indicated by the KNN models is misleading, since classifications were performed on the same samples used to develop the KNN model. In the future, abused samples not used for KNN modeling will be classified with the model to evaluate how well classification accuracy is performed. 3.1.2. SIMCA prediction results When the 120 abuse samples were examined by the SIMCA model, class predictions were less accurate than with the KNN model. The SIMCA model with all 120 abuse samples and 11 variables correctly classified (i.e., accurately predicted both type of abuse and extent of abuse) only 63% of the samples. With Pirouette software, the actual class assignments are presented in a tabular format. The first column provides the best ("Best") estimate of the class membership for the test samples, and the next column provides the next best ("NxtBstl") estimate of the class membership. When the next best estimates were examined, the model correctly classified 83% of the samples as the best or next best estimate of the class membership. This SIMCA model accurately classified 114 of the 120 samples (95%) by correct abuse type but not necessarily by correct abuse level. When the SIMCA model with the six sample outliers removed was used, the accuracy of predicted classes was slightly improved. The SIMCA model with the 114 samples correctly classified 67% of the samples as the best estimate, and 89% were correctly classed as the best or next best class estimate. This SIMCA model accurately classified 106 of the 114 samples (93%) by correct abuse type but not necessarily by correct abuse level. A SIMCA model was created with as few variables as possible. With this data set, the

166 Table 1 Accuracy of abuse class predictions made by KNN and SIMCA modeling I. KNN model: Correct Type But Correct Class Not Necessarily Level Sample Set Predicted of Abuse Predicted 120 Samples, 11 Variables 86% 93% 84% 120 Samples, 3 Variables 93% II. SIMCA model: Correct Type But Correct Predicted Not Necessarily Level Best or NxtBst Sample Set of Abuse Predicted 95% 83% 63% 120 Samples, 11 Variables 93% 89% 67% 114 Samples^, 11 Variables 86% 75% 48% 114 Samples^ 5 Variables^ ^Six Mahalanobis outiiers were identified in the data set and discarded in this model. ^The five variables were acetic acid, dimethyl disulfide, hexanal, heptanal, and nonanal. Correct Predicted as Best

Figure 2. PCA loadings plot for 120 milk samples using 11 variables (i.e., chromatographic peak area ratios). Hexanal (1), dimethyl disulfide (2), and acetic acid (3) are the key chemical indicators of sample abuse.

Figure 3. PCA loadings plot for control (nonabused) samples and all copper abused samples. Hexanal (1), heptanal (2), octanal (3), nonanal (4), oct-l-en-3-one (5), pentanal (6), and isopentanal (7) are the key copper abuse indicators.

Pirouette software would allow no fewer than five independent variables to be used for modeling. The independent variables included acetic acid, dimethyl disulfide, hexanal, heptanal, and nonanal. As indicated in Table 1, unlike KNN modeling, the accuracy of prediction significantly suffered when fewer independent variables were used for modeling. As in the case with KNN predictions, the accuracy of SIMCA predictions in Table 1 is

167 probably favorably biased because the same samples used to calibrate models were also used as "unknowns" to estimate the prediction accuracy. 3.2. Chemical markers as indicators of abuse mechanisms The PCA loadings plot shown in Figure 2 reveals which chemicals were the most significant contributors to determining class assignments when all 120 abuse samples and 11 variables were considered. These chemicals were dimethyl disulfide, hexanal, and acetic acid. By examining PCA loadings plots for each abuse class separately with all control (nonabused) samples, it was possible to determine the specific reaction byproducts that are the most important indicators for each type of abuse. For example, as determined by examination of PCA exploratory results, including the loadings plot (Figure 3), loadings values table, and eigenvalue table, the key chemical indicators of copper abuse are hexanal»>heptanal, octanal, nonanal>oct-l-en-3-one>pentanal>isopentanal. Of these chemicals, hexanal is produced in highest concentration, but oct-l-en-3-one is likely the chemical most responsible for the metallic off-flavor of the copper-abused samples [14]. The only significant marker for sanitizer abuse was acetic acid, a decomposition product of peroxyacetic acid, and the most significant indicators of light abuse were dimethyl disulfide and hexanal. While acetic acid was the only chemical marker revealed in this study, it is not the chemical responsible for the typical off-flavor noted with samples contaminated with peroxyacetic acid. These samples have a peroxide flavor, and the specific chemicals responsible could not be detected by the tests used in this study. Sample chromatograms of a fresh raw milk (non-abused) sample and a raw milk sample abused by light and copper are shown in Figure 4. This figure shows the 10 dynamic headspace chemicals (marked with an asterisk) that were used for classification modeling. An additional chemical, acetic acid, was quantitated by the free fatty acid GC test, and results for acetic acid were also included in the creation of the KNN and SIMCA models. 3.3. Unexpected results that impact flavor: ester degradation by exposure to light, copper, sanitizer, and heat During the early stages of data analysis, the number of independent variables (peak area ratios) was reduced firom 80 to 12. During the process involved in data reduction, a threedimensional PCA scores plot and a PCA loadings plot were created with 12 independent variables. This PCA loadings plot (Figure 5) shows that methyl butyrate has a significant influence on how sample groupings were made. Elimination of methyl butyrate from the data set significantly improved class groupings in the PCA scores plot, so it was not included in the KNN or SIMCA modeling. However, because the 12-variable PCA loadings plots showed that methyl butyrate demonstrated significant variance between samples, this peak was more closely scrutinized in sample chromatograms. Aqueous standard solutions of methyl butyrate, methyl caproate (observed and identified in several control raw milk samples), and ethyl butyrate along with 4-methyl-2-pentanone internal standard were analyzed to allow quantitation of these peaks in the samples. (Note: Although ethyl butyrate was not detected in any of the samples tested, it was included in the experiment in order to be sure the peak identified as methyl butyrate was methyl butyrate and not ethyl butyrate.) Quantitative results for these esters in some of the samples are shown in Table 2. These results show that ester concentrations are highest in fresh raw milk samples but are lost after

168

Control Raw Milk. No Abuse. Class P

-TIC

1. acetone

IS1

2. dimethylsulfide

3. 2-butanone 4. chloroform 5. dichloroethane

6. 3-methyl-2-butanone

13. limonene 14. nonanal* 15. decanal 151 « 4-methyl-2-pentanone 152 = 2-ethlyhexyl acetate B = chemical from GC septum

7. methyl isobutyrate 8. 2-pentanone

10

IS2

9. pentanal* 10. methyl butyrate 11. hexanal* 12. methyl caproate* B

6 5. 7

11

12 14 13

15

660

Light Abused Raw Milk. Class A3 16. dimethyl disulfide*

V JUJ

16

wUww T" T r Copper Abused Raw Milk. Class B2| 21. heptanal* 17. isopentanal* 22. 2-heptanone 18.1-p6ntanol(T)* 19. 4-methyl-1 -pentanol (T) 23. 2-heptenal (T) 20.1-hexanol(T) 24. oct-1-en-3-one* 25. octanal* 24 26. 2-oct6nal (T) 27. l-heptanol(T) 28. 2-nonenal (T) (T)=Tentative i.d. 25 by mass spec.

w Figure 4. Examples of dynamic headspace chromatograms of raw milk samples showing chemicals used for KNN and SIMCA modeling (* = chemicals used for modeling).

169 Table 2 Concentration of methyl butyrate (ppb), showing degradation effects of light, copper, sanitizer, and pasteurization.

Figure 5. PCA loadings plot for all 120 milk samples using 12 independent variables; methyl butyrate (1) included along with the 11 independent variables used in Figure 2. Hexanal (3) and acetic acid (2) also demonstrate significant betweensample variance.

No. 1^ No. 2 No. 3 Class Type 72 D Raw 71 7.3 49 Raw Al 77 7.1 — — 21 A2 Raw — Raw 4.3 5.9 A3 — 6.2 Raw Bl 50 12 Raw B2 5.5 5.5 1.4 8.2 Raw 4.6 B3 3.2 Raw 7.3 3.7 CI 3.2 3.4 Raw C2 7.3 4.1 2.3 Raw 7.3 C3 <0.2 <0.2 D 2.5 Homo 2% D 1.8 0.7 1.9 Skim 1.2 D <0.2 1.7 ^^o. 1, No. 2, and No. 3 refer to milk from three different processing plants.

exposure to light, copper, and sanitizer. Furthermore, when ester concentrations in homogenized whole fat, 2% fat, and skim milk samples made from these raw milk samples are examined, the data show that ester loss is even more significant. More than likely, heating during pasteurization is responsible for this loss. The implications of these findings are significant and may explain why raw milk is generally regarded as having a more desirable flavor than processed milk. A study by Moio et al. [15] concluded that esters of butanoic and hexanoic acid were the most important contributors to the flavor of bovine raw milk. In this work, the researchers used the Charm (Combined Hedonic Response Measurements) method to identify the odor-active components in bovine, ovine, caprine, and water buffalo milk. Dairy processors may be able to improve the flavor and market appeal of their dairy products by devising some way of maintaining/stabilizing ester levels during heat processing. Adding esters in an encapsulated form or adding them back to milk after pasteurization — but in such a way as not to compromise the microbiological integrity of products — could also be considered.

4. CONCLUSION Since all three abuse types studied involve oxidation reactions, prior to initiation of this study it was thought that each abuse mechanism would generate similar byproducts (predominantly aldehydes) and that the relationship between chemical peak areas generated would be multivariate in nature. However, results indicate the data is strongly univariate. Multivariate analysis is probably unwarranted for the purpose of distinguishing the three abuse types stud-

170 ied here, since the information can be generated by analysis of the three primary indicator peaks. Nonetheless, multivariate analysis with Pirouette proved to be a valuable tool for understanding the relationships between sample abuse treatments and peak area ratios. Advantages of using multivariate analysis for this work included: (a) PCA loadings plots and loadings values proved important in finding secondary reaction byproducts created by copper abuse and were useful in pointing out the ester degradation problem. (b) KNN and SIMCA classification techniques allow chemists with little experience in the interpretation of abuse sample chromatograms to accurately predict the cause of off-flavors in customer complaint samples when the cause is either Hght abuse, copper abuse, or sanitizer abuse. (c) As more types of abuse conditions are investigated, independent variable data may become more multivariate in nature, and results can be easily incorporated into the KNN and SIMCA models generated in this work to allow accurate predictions of abuse types. (d) Viewing sample data with PCA scores plots provides an excellent tool for comparing data in an historical database of customer complaint samples to see which types of abuses are occurring most often and from which dairy processing plants. (e) When unknown complaint samples are submitted and analyzed in the future, peak area ratios can be included in the Pirouette spreadsheet with the 120 samples, and residual plots can be examined with various Pirouette algorithms to see how the chemical profile of unknowns differs from the chemical profiles of the 120 samples in the database. Future studies will include investigation of additional mechanisms of abuse (e.g., heat, microbiological, etc.). Also, as data from more samples are collected, we will investigate making separate KNN and SIMCA models for each type of milk sample (i.e., a model for skim, a model for 2% fat milk, a model for homogenized whole fat milk, and a model for raw milk). This could significantly improve prediction rates, since peak areas for the apolar peaks tend to be lower as the fat content increases in the sample when samples are tested by dynamic headspace GC. When a KNN model was created with only skim samples, the model correctly classified 100% of the samples according to the appropriate abuse type and level when the skim samples were analyzed as unknowns. Similar results were obtained for raw, homogenized whole fat milk, and 2% fat milk samples when separate KNN models were made for these milk types.

5. REFERENCES 1 W.F. Shipe, R. Bassette, D.D. Deane, W.L. Dunkley, E.G. Hammond, W.J. Harper, D.H. Kleyn, M.E. Morgan, J.H. Nelson and R.A. Scanlan, J. Dairy Sci., 61 (1978) 855. 2 S.E. Bamard, J. Dairy Sci., 55 (1973) 134. 3 R.T. Marsili (ed.). Techniques for Analyzing Food Aroma, Marcel Dekker, Inc., New York, NY, p. 245. 4 L.S. Ramos, J. Chromatographic Sci., 32 (1994) 219. 5 B. Vallejo-Cordoba and S. Nakai, J. Agric. Food Chem., 42 (1994) 989 . 6 P. Ramanoelina, J. Viano, J. Bianchini and E.M. Gaydou, J. Agric. Food Chem. 42 (1994) 1177. 7 T. Aishima, J. Agric. Food Chem., 39 (1991) 753.

171 8 I. Moret, G. Scarponi and P. Cescon, J. Agric. Food Chem., 42 (1994) 1143. 9 F. Chialva, A. Ariozzi, D. Decastri, P. Manitto, S. Clementi and D. Bonelli, J. Agric. Food Chem., 41 (1993) 2028. 10 Y. Horimoto, K. Lee, and S. Nakai, J. Agric. Food Chem., 45 (1997) 733. 11 Rcn6 Imhof and Jacques O. Bosset, J. High Resolut. Chromatogr., 17 (1994) 25 . 12 H.C. Deeth, N. J. Dairy Sci. and Tech., 18 (1983) 13. 13 R.T. Marsili (ed.), Techniques for Analyzing Food Aroma, Marcel Dekker, Inc., New York, NY, p. 249. 14 W. Stark, D.A. Forss, J. Dairy Sci., 29 (1962) 173. 15 Luigi Moio, Dominique Langlois, Patrick Etievant, and Francesco Addeo, J. Dairy Research, 60 (1993) 215.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

173

Sensory properties of musty compounds in food E. Chambers IV^ E.G. Smith^ L. M. Seitz^ and D.B. Sauer^ ^The Sensory Analysis Center, Department of Foods and Nutrition, Kansas State University, Justin Hall, Manhattan, KS, United States of America ^Grain Marketing and Production Research Center, United States Department of Agriculture, Agricultural Research Service, 1515 College Avenue, Manhattan, KS, 66502. Mention of firm names or trade products does not constitute endorsement by the U. S. Department of Agriculture over others not mentioned.

Abstract Musty aroma and flavor is a major problem in a variety of foods and packaging materials. Some compounds such as geosmin and 2-methylisoborneol have been studied in relation to water, grains, fish, and potatoes, but a variety of other compounds can result in mustiness. Thirty-four chemical compounds, including chloroanisoles, p5n:azines, and aldehydes potentially related to mustiness in foods or packaging which were tested in this study. Six highly trained sensory panelists smelled each compound dissolved in propylene glycol (if soluble) at dilutions of 1000, 5000, and 10000 ppm, although some were tested at levels as low as 100 ppm or as high as 100000 ppm. A range of musty-type odors including earthy, moldy, fungal, damp basement-like, mushroomy, and fermented were found in at least some of the compounds. As expected, changes in concentration or slight modifications in functional groups resulted in large differences in character.

1. INTRODUCTION The sensory characteristic "musty" has been noted in research reports on a variety of products including water, grains, vegetables, edible fungi, dairy products, plastic packaging materials, and chemicals. A variety of chemicals have been reported as potentially producing mustiness in food and other products, and include many variations of borneol, anisole, octenols, and pyrazines. However, some of the chemical odors that have been cited as musty probably would not be considered musty by others, i.e. 3-chloroanisole was mentioned as musty, although it was described as predominantly wintergreen or minty. In water, trans-l,10-dimethyl-trans-9-decalol (geosmin), 2,3,6-trichloroanisole, 2-isopropyl-3-methoxypyTazine, 2-isobutyl-3-methoxypyrazine, and 2-

174 methylisoborneol were associated with earthy/musty characteristics (1-4)). It has been shown that 2-methyhsoborneol can be produced by several species of bacteria inhabiting water (5). In wheat, 3-methyl-l-butanol, l-octen-3-ol, and 3-octanone were identified as fungal odor compounds in bins containing hard red spring wheat. These compounds were associated with microflora on the kernals and high moisture content in both ventilated and non-ventilated bins containing wheat of 15.6 and 18.2% initial moisture content (6). 2-Methylisoborneol and geosmin also have been identified as compounds responsible for"musty-earthy" off-odor in wheat grains (7-8). Methylpyrazine, 2,6-dimethylpyrazine, methoxybenzaldehyde, and trimethoxybenzene were present in wheat samples considered "musty" (8). In grain with molds and bacteria, l-octen-3-ol was associated with fungal odor and 2-octen-lol was associated with musty-oily odor. Intense musty odor of wheat was associated with 2-methylisoborneol and geosmin (9). Six benzene derivatives were associated with distinct earthy, musty, potatobin-like aromas and volatiles in potatoes(lO). Musty odors also were attributed to geosmin, 2-methylisoborneol, and 2-isopropyl-3-methoxypyTazine (3), which some researchers thought resulted from metabolism of actinomycetes (1). In rice, chloroanisole, in concentrations greater than 5 ppb, was associated with a musty taint in rice stored in jute sacks (11). For fish, 2-methylenebornane, 2-methyl-2-bornene, and 2-methylisoborneol were detected in concentrations of 2 ppb (4). 2-methylenebornane and 2-methyl-2bornene were believed to be the cause of musty off-flavor. (3,12). In refrigerated meat, 2,4,6-trichloroanisole produced by microbial interaction with chlorophenol contained in refrigerator insulating material produced a musty odor (13). In chicken, 2,3,4,6-tetrachloroanisole, produced from microbial methylation of chlorophenols in wood shavings used as litter, was the major cause of musty taints in one study (14). Alkyl-methoxyp5n:azines were associated with bacteria that caused musty, earthy, bell-pepper or potato-like odors in many vegetables and plants (15). In dried fruit, chloroanisoles, a source of musty odor, probably originated from microbial activity on chlorophenol precursors within cardboard cartons used to transport packages of dried fruit (13). In mushrooms, a musty odor was associated with 2methylisoborneol (3) which was produced by several species of bacteria (actinomycetes) inhabiting soil (13). In dried beans, a musty odor due to geosmin, 2-methylisoborneol, and 2-isopropyl-3-methoxyp5a'azine also appeared to be associated with metabolism of actinomycetes (1). In beets, geosmin and 2methylisoborneol, although known to exist, were very difficult to quantify (1). These compounds were associated as metabolites of actinomycetes (3). In dairy products, 2,6-dimethyl-3-methoxypyrazine was responsible for production of musty/sour aromas by certain gram-negative bacteria (3). In cheese, P. roqueforti (responsible for some flavor characteristics of mold-ripened cheeses) and B. cineria bacteria, 2-methylisoborneol, 8-carbon alcohols and ketones were associated with overall musty-fruity odor (old spoiling fruit, cellar-like mustiness). 8-Carbon alcohols and ketones were associated with fresh mushroom-like aroma.

175 2-Methoxy-3-isopropylpyrazine was a compound with a potent earthy, snow pea podhke aroma (16). In other cheese molds, 2-methyhsoborneol was considered a musty/moldy component of aromas of PeniciUium cultures and 2-methoxy-3isopropylpyrazine exhibited intense earthy/raw potato aromas (17). 2-methylisoborneol; alkoxypyrazines - such as isopropylmethoxypyrazine; geosmin; mucidone (6-ethyl-3-isobutyl-2-p5rrone); l-phenyl-2-propanone and 2phenylethanol were listed as compounds with characteristic earthy/musty aromas in sweet corn and wild rice (3). In films, the acetal of propionaldehyde and dipropylene glycol ( a mixture 90% 2-ethyl-4,4-dimethyl-l,3,6-trioxane and 10% 2,5,8-trimethyl-l,4,7-trioxane) was identified as the musty odor constituent in polyols and polyurethane foams (18). 4,4,6-Trimethyl-l,3-dioxane was identified instrumentally as the musty odor compound of a plastic food packaging film. The origin of the compound was the cyclic ether precursor 2-methyl-2,4-pentanediol used as a solvent to help ink adhere to the film during printing (19). 2,4,6-Trichloroanisole and 2,3,4,6-tetrachloroanisole were identified as the components causing musty-flavored fruit after packaging in polyethylene film (20). Studies of chemicals indicated that (R)-(-)-l-octen-3-ol had a fruity mushroomlike characteristic, whereas (S)-(+)-l-octen-3-ol had a moldy, grassy note (21). 1Octen-3-ol was thought to be responsible for the characteristic musty-fungal odor of certain fungi, and 2-octen-l-ol was suggested as a useful chemical index of fungal growth because of its similarity to that odor (22). 2,3-Diethylpyrazine had an earthy, vegetable-like, musty, green grassy, and pungent odor (23). In the 2,6choloroanisole compounds, the predominant musty odor character of the chlorocompounds was less apparent when the chloro group was replace by methyl groups (24). Most of the reported research used individuals reporting qualitative, rather than quantitative, odor information and often were reports of individuals sniffing effluent from gas chromatographs or other instruments. Many of the reports simply described the overall odor or flavor as musty with little qualification of what type of mustiness was present and few reports used trained sensory panelists to describe the musty odor that was present. Therefore, the purpose of this research was to examine chemicals that had been associated with or described as "musty" in the literature and clearly define their odor characteristics at several intensity levels.

2. MATERIALS AND METHODS 2.1 Compounds The compounds selected for this study were chosen because of their citing frequency and availability from common chemical sources. Compounds known to be toxic were excluded from this study. The chemicals examined were: borneol; 2,3-dimethylpyrazine; 2-ethyl-l-hexanyl acetate; cis-3-hexen-l-ol; dihydrocarvone; cis,cis-2,6-dimethylcyclohexanol; 2-isobutyl-3-methoxypyrazine; 2-

176

isopropyl-3-methoxypyrazine; 3-octanone; l-octen-3-ol; 2-chloroanisole; 3chloroanisole; 4-chloroanisole; 2,3,4,5-tetrachloroanisole; 2,3,5,6-tetrachloroanisole; 2,3,4-trimethoxybenzaldehyde; 2,4,5-trimethoxybenzaldehyde; 2,4,6trimethoxybenzaldehyde; 3,4,5-trimethoxybenzaldehyde; 1,2,3-trimethoxybenzene (all obtained from Aldrich Chemical Company, Milwaukee, WI); o-anisaldehyde, manisaldehyde, p-anisaldehyde, fenchyl alcohol, 2-phenylethanol, 1,2,4trimethoxybenzene (obtained from Sigma, St. Louis, MO); and 2-methylisoborneol and geosmin (obtained from Wako Chemicals USA, Richmond, VA). 2.2 Dilutions All chemicals except the benzaldehyde compounds and the tetrachloroanisoles were dissolved in propylene glycol (most compounds) or water (geosmin and 2methylisoborneol). Typically, the dilutions were 10,000 ppm, 5,000 ppm, and 1,000 ppm although some were lower or higher depending on the amount available or the strength of the odors present. The solutions were stored in 120 mL glass bottles with Teflon covered lids. Prior to testing, a fragrance testing strip was dipped to the depth of 3mm in the chemical solution and then carefully placed in a coded, capped 20 mL glass tube. For the chemicals that were not soluble, either l.Og or O.lg, was placed in the capped glass tube and smelled without dilution (neat). 2.3 Testing Procedure The chemicals were profiled for odor by a 6-member trained descriptive panel consisting of 6 female panelists with a broad background in odor description. All panelists had completed 120 hours of sensory descriptive training, a 40 hour apprenticeship to check performance, and had over 2,000 hours of testing experience. Each chemical sample was presented individually, one at a time, with a three digit code, in random order. For the soluble compounds, each panelist removed the fragrance strip and waved it directly underneath her nose twice while taking quick, sharp sniffs. For the insoluble compounds, the cap was removed from the tube and the tube was waved under the nose twice. Each panelist evaluated the odor description of the compound, using descriptors determined in preliminary evaluations of the compounds and previous experience with musty foods. The term "musty" was not used as a descriptor, rather, terms were used that described the musty character, i.e. earthy, leather (new), moldy/damp, etc. were used. An intensity was given to each characteristic from 1 (just recognizable) to 10 (extremely intense). After scoring, panelists discussed the characteristics and intensities for the compound and a consensus score was determined. Most compounds were smelled multiple times, although none was sniffed more than twice in a single session. Approximately 20 minutes elapsed between sniffing different compounds to reduce carryover. Panelists stopped testing anytime carryover from one compound to another could not be avoided. Panelists never tested more that three compounds in one day. Fresh air and sniffs of distilled lime oil, which seemed to ameliorate odor carryover of the chemicals, was used to cleanse the nasal passages between samples.

177

3. RESULTS AND DISCUSSION Some chemicals were associated with mustiness in the hterature, but were not musty in this research at the levels tested (Table 1). Most chloroanisoles were not musty, but were wintergreen and sweet. Many pyrazines also were not musty, but had roasted or sweet, plant-like (e.g. minty) characteristics. None of the anisaldhydes were musty, but instead had sweet, medicinal, or cherry odors. Table 1 Chemicals that were not found to exhibit musty characteristics Chemical Characteristics Cherry, Floral, Sweet m-anisaldehye Medicinal, Brown/acrid, Ashy, Sweet o-anisaldehyde Sweet, Medicinal, Floral, Anise p-anisaldehye Floral, Sweet, Peppery, Minty, Rose 2-ethyl-l-hexanyl acetate Sweet, Floral, Rose, Honeysuckle, Lilac 2 -phenylethanol Green Cis-3-hexen-l-ol Medicinal, Wintergreen, Sweet, Cherry 2-chloroanisole^ Alcohol, Wintergreen, Sweet, Fruity, Pine 3-chloroanisole^ Sweet, Anise, Pine, Wintergreen, Floral 4-chloroanisole^ 2-4-5-trimethoxybenzaldehyde Sweet, Sour, Mustard, Smoky 2-4-6-trimethoxybenzaldehyde Cardboard, Acrid, Sour Roasted Peanut, Acetone, Hydrolyzed, Sweet 2-3-dimethylpyrazine Green, Pungent, Minty, Sweet 2-isobutyl-3-methoxypyrazine Many musty related sensory terms described the odor properties of the chemicals. Musty, as an individual term, was not used in the odor descriptions because it represents several different sensory attributes Table 2). Thus "musty" is not listed in the table; rather each term listed describes a type of mustiness. Table 2 Musty related sensory terminology found in the chemical profiles Sensory Term Dusty/Papery Earthy/humus Earthy/damp Earthy/potato Fermented Leather (new) Leather (old) Moldy/cheesy Moldy/damp Mushroomy

Description dry, musty, papery musty, sweet, decaying vegetation musty, damp, wet soil musty, dry soil, potato-like sweet, overripe, rotten, musty musty, new leather (like new shoes or purses) musty, old leather (like old book bindings) sour, musty, moldy musty, damp basement-like, earthy, moldy slightly musty, earthy

178

Table 3 lists chemicals that were associated with musty odor attributes. A profile for each chemical at each level tested is shown, unless the chemical was not soluble in propylene glycol or water; in that case only a single concentration is listed. Table 3 Aroma Profiles with Descriptions and Intensities for Chemicals Studied. 2,3,4,5-tetrachloroanisole (neat) 2,3,5,6-tetrachloroanisole (neat) Leather (old) - 6 Chemical-like - 6 Sweaty - 2 Moldy/Damp - 6 Leather (old) - 5 2,3,4-trimethoxybenzaldehyde 3,4,5-triniethoxybenzaldehyde (neat) (neat) Dusty/Paper - 4 Dusty/Paper - 3 Leather (old) - 3 Leather (old) - 3 Damp Basement - 3 2-isopropyl-3-inethoxypyrazine 5,000ppm l,000ppm 10,000ppm Earthy/Potato - 7 Earthy/Potato Earthy/Potato - 7 Sharp/Pungent - 5 Pea Pod - 4 Acetone - 6 Acetone - 5 Sharp/Pungent - 5 Pea Pod - 4 Brown roasted - 5 Brown Roasted - 4 Pea Pod - 4 1,2,4-trimethoxybenzene 50,000ppm 10,000ppm 100,000ppm Earthy/Potato - 4 Leather(old) - 3 Earthy/Potato - 7 GreeiiAVoody 4 Earthy/Potato - 1 Green/Woody - 5 Sweet - 3 Sweet - 1 Sweet - 3 2-6-Dimethylcyclohexanol l,000ppm 5,000ppm 10,000ppm Menthol - 7 Earthy/Humus - 7 Menthol - 9 Leather (new) - 6 Sharp/Pungent - 5 Camphor - 7 Camphor - 5 Leather (new) - 4 Alcohol - 6 Alcohol - 4 Medicinal - 4 Earthy/Humus - 6 Earthy/Humus - 4 l-octen-3-ol l,000ppm 10,000ppm 5,000ppm Mushroomy - 8 Mushroomy • Mushroomy - 8 Sweet - 1 Sweet - 3 Sweet - 3 Floral - 2 Floral - 2 3-octanone 5,000ppm 10,000ppm l,000ppm Fruity - 8 Fruity - 9 Moldy/cheesy - 7 Sweet - 8 Sweet - 9 Fruity - 5 Fermented - 7 Fermented - 8 Sweet - 5 Moldy/cheesy -7 Fermented - 4 Moldy/cheesy - 7 fenchyl alcohol l,000ppm 5,000ppm 10,000ppm Pine - 8 Pine - 7 Pine - 9 Earthy/Potato - 5 Earthy/Potato - 4 Earthy/Potato - 4 Sweet - 4 Sweet - 3 Sweet - 4

179 Table 3 cont. Dihydrocarvone 10,000ppm Moldy/Cheese - 6 Fruity - 6 Pungent - 5 Anise - 5 Geosmin l,000ppm Camphor - 7 Pine - 6 Earthy/damp - 4 Peppermint - 4 2-methylisoborneol l,000ppm Earthy/damp - 5 Camphor - 5 Eucalyptus - 3 Menthol - 3 Petroleum jelly - 2 Borneol 3,500ppm Menthol/Sweet - 5

5,000ppm Moldy/Cheese - 6 Fruity - 6 Pungent - 3 Anise - 2

l,000ppm Moldy/Cheese - 5 Fruity - 5 Pungent - 2 Anise - 1

lOOppm Earthy/damp - 3

lOOppm Earthy/damp - 3

1,2,3trimethoxybenzene (neat) Medicinal - 5 Leather (old) - 3 Sweet - 5 Iodine - 2

l,400ppm 700ppm Menthol/Sweet - 4 Menthol - 2 Humus - 4 Earthy/humus - 3 Intensity scale: 1 = just recognizable, 10 = extremely intense Generally, the main odor characteristics did not change, although the intensity decreased as the concentration was diluted. However, some odor properties that were present at very low intensities when the compound was at a high concentration disappeared when the compound was at lower concentrations. For example, l-octen-3-ol lost its floral characteristic as it was diluted. In most cases the musty-like sensory component of the aroma was higher at higher concentrations of the chemical compound. However, at the high levels, the musty component tended to compete with other odors for dominance. For example, geosmin was minty and camphoraceous as well as earthy/damp at 1000 ppm., but at 100 ppm geosmin was only earthy/damp. It is possible that with further dilution, the odor profile could have changed again. Musty is a characteristic that occurs in many food systems. Geosmin and 2methylisoborneol are the two chemicals most frequently cited in literature on mustiness. Clearly at the lower levels tested here, the earthy/damp odors are the main, if not the only odors, presented by those compounds. At higher levels, other odor notes became apparent and, especially in the case of geosmin, those other notes, including pine and camphoraceous, began to dominate the odor profile.

4. CONCLUSIONS Musty odors and flavors are present in many different foods and food related products such as packaging. The term has been used broadly to cover a range of

180 related, but different, odor properties including earthy, moldy, damp basement, fermented, and potato-like characteristics. This study identified compounds that have been reported to produce musty odors and characterized the musty and nonmusty odor properties. We note that some chemical compounds previously reported to be associated with mustiness are not musty at the levels we studied. Other compounds may produce var5dng mustiness depending on the concentration and may produce other odors at other concentrations. Chemical compounds often associated with mustiness, such as geosmin and 2-methylisoborneol were musty at all concentrations studied, but they had additional odor characteristics at high levels. 5. R E F E R E N C E S 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

N.N. Gerber, Develop. Indust. Micro. 20,(1979) 225. S. Lalezary, M. Pirbazari and M.J. McGuire, J. American Water Works Association. 78 (3) (1986) 62. J.A. Maga, Food Reviews Internl. 3 (1987) 269. C.I. Mashni and R.S. Safferman, Source of musty odor associated with mucidone. Environ. Res. Cent., Environ, Prot. Agency. Cincinnati, OH, 7 (1982) 369. M. Yagi and S. Nakashima, Water Supply, 7 (1989) 153. R.N. Sinha, D. Tuma, N.D. Abramson and W.E. Muir, Mycopathologia. 101 (1988) 53. S. Nitz, H. Kollmannsberger and F. Drawert, J. Chromatog. 471 (1989) 173. E. Wasowicz, E. Kaminski, H. Kollmannsberger, S. Nitz, R.G. Berger and F. Drawert, Chemie Mikrobiologie Technol. Lebensmittel. 11 (6) (1988) 161. S. Stawicki, E. Kaminski, A. Niewiarowicz, M. Trojan and E. Wasowicz, Technol. Agricole. 22(1973)449 G. Mazza and E.M. Pietrzak, Food Chem. 36 (1990) 97. H. Maarse, Perfumer & Flavorist. 12 (2) (1987) 45. J.F. Martin, T.H. Fisher and L.W. Bennett, J. Agric. Food Chem. 36 (1988) 1257. N.S. Davidson, Rural Res. No. 122 (1984) 11. N.M. Griffiths, Chem. Senses Flavor. 1 (1974) 187. A. Gallois and P.A.D. Grimont, Appl. Environ. Microbiol. 50 (1985) 1048. N.D. Harris, C. Karahadian and R.C. Lindsay, J. Food Protection. 49 (1986) 964. C. Karahadian, D.B. Josephson and R.C. Lindsay, J. Agric. Food Chem. 33 (1985) 339. S.H. Harris, P.E. Kreter and C.W. Policy, Polyurethanes. Proc. World Congr. FSK/SPL Technominic: Lancaster, PA. (1987) 848. R.J. McGorrin, T.R. Pofahl and W.R. Croasmun, Anal. Chem. 59 (1987) 1109A, F.B. Whitfield, T.H. Lynguyen, K.J. Shaw, J.H. Last, C.R. Tindale and G. Stanley, Chem. Ind. (London). (19) (1985) 661. A. Mosandl, G. Heusinger and M. Gessner, J. Agric. Food Chem. 34 (1986) 119. E. Kaminski, L.M. Libbey, S. Stawicki and E. Wasowicz, Appl. Microbiol. 24 (1972) 721. S.M. Fors and B.K. Olofsson, Chem. Senses 11 (1986) 65. N.M. Griffiths and G.R. Fenwick, Chem. Senses Flavor 1 (1977) 187.

Acknowledgement: This research was supported, in part, by USDA Grant #5430-44000005-04S, "Sensory Analysis of Grain Samples.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

181

Evaluation in score of the intensity of salty and umami tastes Rie Kuramitsu Akashi College of Technology, Uozumi, Akashi, Hyogo, 674, Japan Abstract It is important to know the balance of intensities of tastes in foods in order to analyze the tasting mechanism of foods. However, it is extremely difficult to compare intensity of different types of taste in a unified mode. This study compares the intensities of two different tastes, salty and umami, using a common scale based on their respective threshold values by making common scores. By employing these scores the tasting charac- teristics of soy sauces to which were added various saltiness-enhancing agents was studied.

1. INTRODUCTION Today, people's interest focuses on health, and particulariy the excessive intake of table salt has received tremendous attention resulting in an increased demand for low NaClcontaining foods. Soy sauce has a large amount of NaCl. In this study I attempted to decrease the NaCl content of the soy sauce without spoiling the flavor. Soy sauce contains 15 - 18% NaCl and 1 - 2% monosodium glutamate (MSG) and is a traditional seasoning with salty and u/77a/27/tastes as the core of the tasting characteristics. The low NaCl-containig soy sauce used in this study was prepared by using a pre-soy sauce with an NaCl content 0.5 time the NaCl content of conventional soy sauce obtained by the short term fermentation method developed by Muramatsu et al, [1] as the base material, and by adding a variety of peptidic saltinessenhancing substances which were developed by Tada et al. [2] and Tamura et al. [3] To objectively evaluate the quality of various low NaCl-containing soy sauces prepared by this method, I wanted to express the intensity of the salty and the umami tastes quantitatively on a common scale. For this purpose respective threshold values of NaCl, a standard substance for salty taste, and MSG, that of umamitaste, were used, and common scores were assigned for successively increased concentrations by fold of the threshold values.

2. MATERIALS AND METHODS 2.1. Soy sauce A soy sauce that was manufactured by Tenyo Takeda Co. was used as a reference for comparison. Its composition by analysis is shown in Table I. For the sake of differentiating this from the pre-soy sauce that is described in the next section, it is designated as the commercial soy sauce.

182

2.2. Pre-soy sauce The pre-soy sauce employed was a preparation made by the short fermentation process which was developed by Muramatsu et al.[l] Essentially, commercial soy sauce and pre-soy sauce are not different except in the two following points. The pre-soy sauce contained 8.13% NaCl, which was about half of NaCl concentration in the commercial soy sauce. For asceptic purposes, ethanol was added in at approximately twice that of the commercial soy sauce (Table 1). Table 1 Composition of commercial soy sauce and pre-soy sauce Contentes

NaCl Total nitrogen Formol nitrogen Reducing sugar Ethanol Lactic acid Acetic acid pH

Commercial soy sauce (wt/v %) 16.26 1.67 0.78 2.60 2.74 0.660 0.127 4.79

Pre-soy sauce (wt/v %) 8.13 1.54 0.82 2.26 5.34 0.550 0.088 4.85

2.3. Saltiness-enhancing substance In addition to KCl which has been used as a typical additive, omithyltaurine hydrochloride (Om-Tau- HCl), glycine ethyl ester hydrochloride (Gly-OEt* HCl) and lysine hydrochloride (Lys" HCl) were also used as saltiness enhancing additives.

2.4. Preparation of low NaCl-containing soy sauce The preparation of a 50% KCl-addtion was obtained by the following manner: To a presoy sauce containing 8.13% of NaCl, KCl was added to 8.13% in the sample preparation. This sample contained a sum of 16.26%, consisting of equal amount of NaCl and KCl , which was equal to the concentration of NaCl in the commercial soy sauce for comparison. Other samples were prepared in a similar way. These sample solutions of low NaClcontaining soy sauce were diluted serially to make the sum of the concentrations of each of the additives plus NaCl 0.75, 0.625, 0.50, 0.375, 0.25 and 0.125% for sensory testing.

2.5. Sensory analysis Taste intensity of samples were evaluated by a panel of 5 people using Table 2.

183

3. RESULTS AND DISCUSSION 3.1. Score of the intensity of salty and umami tastes NaCl was employed as the standard substance for salty taste. The threshold value of NaCl was estimated to be 0.063%. In daily life we encounter NaCl solutions in soups. Miso soup, a traditional Japanese food, contains about 1 % NaCl. Other foods that contain about 1 % NaCl include hamburgers, steaks and meuniere of fish. Samples of foods that contain about 0.5% NaCl are soups, fries with no coating and fried rice. These NaCl concentrations are popular Japanese foods. Accordingly, the threshold value of 0.063% was made a Score of 1 NaCl and the concentrations of NaCl between the threshold value and 1% were expressed in several grades. Thus, a 2-fold concentration of the threshold value (0.125%) was a Score of 2, a 4fold concentration (0.25%) a Score of 3 and an 8-fold concentration (0.5%) a Score of 5. An intermediate concentration (0.375%) of Scores 3 and 5 was made a Score of 4. A two-fold concentration of Score 5 (1.0%) was made Score 9, and concentrations between Scores 5 and 9 were divided into 4 making Score 6 (0.625%), Score 7 (0.75%) and Score 8 (0.875%). Score 10 corresponded to 1.2% NaCl. MSG was used as the standard substance of umami taste. The threshold value of MSG was estimated to be 0.025%. Scores for the intensity of umami taste were made similar to those for salty taste. The results are shown in Table 2.

Table 2 Scores of the intensity of salty and umami tastes Salty taste NaCl (%)

Score

1.2 1.0 0.875 0.75 0.625 0.5 0.375 0.25 0.125 0.063

10 9 8 7 6 5 4 3 2 1

Umami taste MSG (%) 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.025

3.2. Comparison of intensities of salty and umami tastes in low NaCl-containing soy sauce Previously I have reported that the low NaCl-containing soy sauces with the addition of saltiness enhancing substances at a ratio of 50%, had a satisfactory salty taste [4]. However, these soy sauces also produced tastes different than that of ordinary soy sauce, probably due to extensive changes in food composition caused by the addition of large amounts of the substitute substances. I reported that the change in taste was dependent on the intensity of umamitastCy but this judgement was merely subjective. In the present studies the intensities of salty and umami tastes in the low NaCl-containing soy sauces and the commercial soy sauce were represented quantitatively by using scores shown in Table 2, and the results are shown in

184

o o

0.75%

0.625%

0.50%

0.375%

0.2.5%

0.125%

Om -Tau-HCl •

lj»iii^l 0.75%

0.625%

0.50%

0.375%

0.25%

0.125*/

0.75%

0.625%

0J50%

0.375%

0.25%

0.125%

0.75%

0.625%

0.50%

0.375%

0.25%

O O

0.75%

0.6-25%

0.50%

0.375%

0.25%

ii^BLj 0.125%

0.125%

Concentration as NaCl (%) Intensity of salty taste H Intensity of a/77a/77; taste Figure 1. Comparison of intensity of salty and umam tastes in soy sauce by 50% adding saltiness-substituting substances

185 Figure 1. The scores of both tastes were represented on a common scale by the distances from respective threshold values in successively increased concentrations by folding. It is noted that the intensities of salty and umami tastes were in good agreement with those of commercial soy sauce. Except for 0.125%, the commercial soy sauce showed the same scores for both tastes; i.e., Score 7 for 0.75% and Score 6 for 0.625%. The results revealed that in this soy sauce, intensities of both tastes were the same distance from their respective threshold values and were well balanced for human taste evaluation. For the 0.125% soy sauce, the salty taste was stronger than umami taste by 1 score. With Om-Tau" HCl intensities of salty and umami tastes were considerably similar to those of the commercial soy sauce except at low concentrations (0.25, 0.125%). With Gly-OEt- HCl, the salty taste was stronger than the «/rzam/taste at higher concentrations (0.75 - 0.50%). With Lys- HCl, contrarily, the salty taste was weaker than the umami taste at higher concentrations. By contrast to these peptide replacers KCl demonstrated a very different effect. Over the entire range of concentrations of added KCl, the umami taste was more prominent, and the difference from salty taste was more distinct at higher concentrations. This effect is thought to be undesirable for the use of KCl in foods as a NaCl substitute. In addition to its bitter taste, which has been known from long ago this effect should work to reduce salty taste in foods when KCl is used.

4. CONCLUSION Based on the respective threshold values, intensities of salty and umami tastes were represented quantitatively in scores (Table 2). By using the scores in Table 2 the tasting characteristics of low NaCl-containing soy sauces to which were added various saltiness-enhancing substances were compared. The results revealed that commercial soy sauce was an ideal food in that it possessed well balanced intensities of salty and umami tastes. The balance of salty and [i/nam/tastes in the Om-TauHCl-added low NaCl-containing soy sauce was close to that of the commercial soy sauce. The low NaCl-containing soy sauce to which KCl, a long used substitute of NaCl, was added showed too strong of a umami taste that killed the effect of salty taste.

5. ACKNOWLEDGMENTS I thank Tenyo Takeda Co. for generous supply of commercial soy sauce and pre-soy sauce that were used in the present experiments.

6. REFERENCES 1 2 3 4

S. Muramatsu, Y. Sano, and Y. Uzuka, ACS Symposium Series 528, A. M. Spanier, H. Okai, and M. Tamum, (eds.), 200-210 (1993). M. Tada, I. Shinoda, and H. Okai, /. Agric, Food Chem., 32 (1984), 992-996. M. Tamura, T. Seki, Y. Kawasaki, M. Tada, E. Kikuchi, and H. Okai, Agric. Biol. Chem., 53 (1989), 1625-1633. D. Segawa, K. Nakamura, R. Kuramitsu, S. Muramatsu, Y. Sano, Y. Uzuka, M. Tamura, and H. Okai, Biosci. Biotech. Biochem., 59 (1995), 35-39.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

187

Sensory characteristics of chemical compounds potentially associated with smoky aroma in foods D. H. Chambers^ E. Chambers IV^ L. M. Seitz^ D. B. Sauer^ K. Robinson^ and A.A. Allison^ ^The Sensory Analysis Center, Department of Foods and Nutrition, Kansas State University, Justin Hall, Manhattan, KS, 66506-1407, United States of America ^Grain Marketing and Production Research Center, United States Department of Agriculture, Agricultural Research Service, 1515 College Avenue, Manhattan, KS, 66502. Mention of firm names or trade products does not constitute endorsement by the U. S. Department of Agriculture over others not mentioned. Abstract Smoky aroma can either enhance sensory quality of foods (e.g. meat or cheese) or be detrimental to the product (e.g. raw or processed grains and grain products). Twenty-one chemical compounds suggested from the literature or from gas chromatographic headspace analysis of raw grains as potentially responsible for smoky aroma were studied. Five highly trained sensory panelists smelled each compound dissolved in propylene glycol (if soluble) at a dilution of 5,000 to 20,000 ppm depending on the aroma strength of the compound. Approximately 60% of the compounds were found to have some smoke character, although the intensity of smokiness often was low. Some compounds such as benzyl disulfide and 3,7-dimethyl-6-octenoic acid were predominately smoky, but had other aroma characteristics that were lower in intensity. Most compounds had other aroma characteristics that were more intense than smoke (e.g. 2-ethylpyridine) and some compounds, such as 1-methylpyrrole were not smoky at all.

1. INTRODUCTION Smoke has been used as a preservation method for over 90,000 years (1). But in the modern "refrigeration" era, smoked foodstuffs, such as meat, fish, and cheese are demanded by consumers not for their preserved states, but for their unique and characteristic flavor, including smoky flavor. Because of this demand, smoked products retain their success with consumers. However, smokiness is not always a positive characteristic. Undesirable smoke flavors in grain have been identified by Smith et al. (2), who referenced carvacrol to describe the smoke/ash flavor found in grains such as corn, sorghum, and soybeans. The grains had a characteristic

described as a "black, charred aromatic, associated with ground-in residue of unwashed ashtrays." Although the number of chemical components that could result in smokiness is a continuous source of debate, over 200 compounds have been identified in smoke (3). There may be as many as 500 compounds (or more) that are linked to smoke flavor (4). Generally, smoke-related compounds can be placed into six categories: carbonyls, organic acids, phenols, organic bases, alcohols, and hydrocarbons, which include the potentially carcinogenic polycyclic aromatics (3,5). The phenolic fraction often has been cited as the main source of flavor and aroma in smoke and smoked products (3, 6, 7). The phenol fraction is complex, consisting of at least 30 components with both carbonyl and phenolic functional groups (8). Phenolic esters, such as guaiacol (2-methoxyphenol) and syringol (2,6dimethoxyphenol) and their derivatives, can be formed from the pyrolysis of lignin, which constitutes almost 25% of wood (1,4,9). The amount of phenolic compounds present in smoke varies with wood variety (1,10), recovery technique (8), and smoke generation methods (11). Bratzler et al. (6) correlated phenolic content to overall smoke intensity in bologna, concluding that the phenols had a greater contribution to smoke flavor than either the carbonyl or acid components. Lantz and Vaisey (10) also concluded that phenols had the greatest effect on smoke flavor in smoked Whitefish. Other researchers have reached similar conclusions (3). Three major phenolic compounds have been examined extensively and have been described sensorially by researchers because of their influence on overall smoke flavor: guaiacol (2-methoxyphenol), 4-methylguaiacol (creosol), and 2,6dimethoxyphenol (syringol) (1,9,12). Guaiacol, found predominantly from softwood smoke, has smoky, aromatic, sharp, sweet, phenolish, spicy and smoked-sausagelike odors and flavors (1,4,7,9). Creosol has been described as sweet, vanilla-like, fruity, cinnamon, smoky, sharp, caramel-like, and burnt (1,7,9). Syringol was described similarly to guaiacol as smoky, spicy, smoked-sausage-like, sharp, sweet, phenolish, smoky, whiskey, dry, and sharp (1,7). Other phenolic compounds also have been evaluated sensorially by Baltes et al. (1) including eugenol (4-allyl-2methoxyphenol), o-cresol, and dimethylphenol. Eugenol is an example of a phenol that has been found in both smoke and traditionally non-smoked products such as spices and grains. Phenol, cresol, eugenol, and guaiacol are found in cinnamon. Cloves, marjoram, nutmeg, and pepper also are spices with eugenol (1). Early research showed that carbonyl compounds also have been found to contain flavor characteristics, but are more likely to contribute to the smoky brown color that is characteristic of smoked products (13). Kim et al. (7) found that carbonyl and lactone components in addition to phenolic compounds were important flavor components, though carbonyl compounds such as 2-cyclopentenone and 2methyl-2-cyclopentenone had a grassy, bitter odor. The authors also found that alkyl substituted 1,2-cyclopentadione and 2-buterolide (internal lactone from 4hydroxy-2-butenoic acid) homologues (non-carbonyl neutral fraction) had considerable sweet, burnt and caramel-like flavors, possibly showing smoky flavors

189 contributed by non-phenolic compounds (7). In addition, research by Maga and Fapojuwo (14) showed that the carbonyl fraction is the primary contributor to the smoke aroma for most woods and recommended that because of the potential health hazards, phenolic compounds might be removed from smoke sources without eliminating smoke aroma intensity. Toth and Potthast (4) indicated that a number of carcinogenic polycyclic aromatic hydrocarbons (PAH) have been identified in smoke condensates and smoked foods. However, in commercial liquid smoke, a purified phenolic fraction can be produced without PAHs. Many researchers have used vocabulary describing potentially smoky chemicals, but the descriptors are poorly defined and may not represent smoky aromas at all (1,7). Profiles provided by Dravnieks (15) gave a wider variety of smoke related descriptors, but did not define the words. Work in our laboratories on meat, cheese, grains, and other products has led us to believe "smoky" is not an individual characteristic but is comprised of various smoky odor/flavors. Because smoke flavor and aroma compounds have been found in foods other than smoked meat and cheese products, further investigation into the sensory properties of potentially smoky chemicals is needed. Therefore, the objective of this study was to determine the sensory properties of a variety of chemical compounds that potentially had a smoky or smoke related character.

2. MATERIALS AND METHODS 2.1 Compounds The compounds selected for this study were chosen because of their citing frequency, because they had been described using smoky related terms (15), or because they had been found in the headspace of smoky grains. Availability also was taken into account. Compounds known to be toxic were excluded from this study. The chemicals examined were: ethyl 3-hydroxyhexanoate; guaiacyl phenylacetate; methylcyclopentenolone; 1-methylpyrrole; 2-ethylhexanoic acid; 3,7dimethyl-6-octenoic acid; furfuryl mercaptan; 1,2 cyclohexanedione; cyclohexanol; 2,6-dimethoxyphenol; 4-ethylguaiacol; D-xylose; benzyl disulfide; thymol; guaiacol; carvacrol; amylbenzene; 2-methylbenzofuran; 2,3-benzofuran; 2-ethylp5n:idine; and 1-phenyl hexane (hexylbenzene). 2.2 Dilutions All chemicals were dispersed in propylene glycol. Typically, the concentrations were 20,000 ppm, 10,000 ppm and 5,000ppm, although other levels were used. The determination of the level tested was made by serially diluting the chemical and having the sensory panelists determine if the dilution was smoky. The highest level of compound that was reasonably, but not overwhelmingly, smoky was used. If no level was smoky, a concentration that represented the odor character of the material was used. The propylene glycol solutions were stored in

190 120 ml glass bottles with Teflon covered lids. Prior to testing, a fragrance testing strip was dipped to the depth of 3mm in the chemical solution and then carefully placed in a 20 ml coded, capped glass tube. 2.3 Testing Procedure The chemicals were profiled for odor by a highly trained descriptive panel consisting of 5 female panelists with a broad background of experience in odor description. All panelists had completed 120 hours of sensory descriptive training, a 40 hour apprenticeship to check their performance, and then had over 2,000 hours of testing experience. Each chemical sample was presented monadically, with a three digit code, in random order. Each panelist removed the fragrance strip and waved it directly underneath her nose twice while taking quick, sharp sniffs. Each panelist evaluated the odor description of the compound, using descriptors determined in preliminary evaluations of the compounds and previous experience with smoky foods. An intensity was given to each characteristic from 1 (just recognizable) to 15 (extremely intense). After scoring, panelists discussed the characteristics and intensities for the compound and a consensus score was determined. Most compounds had to be smelled multiple times, although no compound was sniffed more than two times in a single session. Approximately 20 min elapsed between sniffing different compounds to reduce carryover. The panelists were instructed to stop testing anytime they believed carryover from one compound to another could not be avoided. Fresh air and sniffs of distilled lime oil, which seemed to ameliorate odor carryover of the chemicals, was used to cleanse the nasal passages between samples. 3. RESULTS AND DISCUSSION Table 1 lists the chemicals that were associated with smoky characteristics in the literature, but were not found to be smoky in this research. No particular chemical class of compounds was not smoky; compounds from many chemical classses were tested that the panel found not to be smoky. Commonly, the nonsmokey compounds exhibited sweet, fruity, medicinal characteristics that potentially could be related to smoky odors in the presence of other compounds. Some of these same odor notes were found in smoky compounds. However, in these chemicals the *T3lend" did not provide any overall smokiness. Many smoky related terms were found to describe the odor properties of these chemicals. Table 2 gives those terms that were found to contribute to the distinctive smoky character in the compounds tested. On the surface, many of the odor notes do not have an obvious smokiness. However, they were part of the smoky character of the chemical and provide the uniqueness to the smoke character in many of these compounds. The smokiness of the compounds tested was not simply "smoky," but was represented by many "types" of smoky characters, all of which had an underl5dng smokiness.

191 Table 1 Chemicals that did not exhibit smoky characteristics Characteristics Chemical ethyl 3-hydroxyhexanoate Sweet, Anise, Almond, Citrus Peel methyl cyclopentenolone (also Sweet, Maple, Caramelized, Woody containing some ketotautomer) Sweet, Medicinal, Brown/Spicy guaiacyl phenylacetate Petroleum, Chemical, Pungent, Sulfur 1-methylpyrrole Anise, Heated Vegetable Oil, Citrus Peel amylbenzene Shoe Glue, Ether 2,3-benzofuran Floral, Anise, Heated Vegetable Oil, Weedy 1-phenyl hexane Mothball, Resinous, Medicinal 2-methylbenzofuran

Table 2 Smoky related sensory terminology found in the chemical profiles Sensory Term Ashy Brown spice Burnt Burnt Sugar Clove Creosote Cured Maple Sulphur Woody

Description dry, dusty, burnt, charred sweet, dark brown spices sharp, acrid, charred dark brown, caramelized sweet, brown spice, minty, alcohol tar-like preserved meat, sweet brown sweet, caramelized, woody, vanilla pungent, rotten egg-like, sewer gas bark-like

Table 3 lists chemicals that were associated with smoky odor. A profile for each chemical is shown and in most cases the "smoky" character of the compound is less intense than some other sensory attributes of the chemical. For example, 3,7-dimethyl-6-octenoic acid is more waxy and fruity than smoky. Similarly, cyclohexanol is more resinous and medicinal than woody-smoky. Some compounds, like 4-ethyl guaiacol, had moderate smoke characteristics and also had other notes that would contribute additional odors to foods it might be present in. In foods containing these compounds it may be that only low smoky odors are noted (or desired) in those food classes. However, other smoky compounds also may be present and may boost the smoke character of compounds such as guaiacol. A few chemicals, such as 2,6-dimethoxyphenol and benzyl disulfide, are essentially nothing but smoky. Those two compounds illustrate the differences noted in smoky odors. The dimethoxyphenol compound gave a sweet, woody, burnt

192

smokiness, but the benzyl disulfide was predominantly a burnt, ashy smoke. Based on this data, it would be difficult to interchange the two compounds, although both are smoky. Similarly, although one of those compounds might provide a positive flavor character to a specific food class, it could result in a negative odor or flavor property to another food class. Table 3 Aroma profiles with sensory descriptors and intensities for smoky related chemicals studied. 2-ethylhexanoic acid Smoke - 1.5 Ether - 5.5 Alcohol - 5.0 Ashy - 3.0 Sweet - 2.0 furfuryl mercaptan Smoke - 4.5 Skunky - 9.0 Pungent - 8.0 Sulfur - 2.0

3,7-dimethyl-6-octenoic acid Smoke - 3.0 Waxy - 6.0 Ashy - 4.0 Fruity/pineapple - 3.5 Metallic - 2.5 Sweet - 2.5 2-ethylpyridine Smoke - 2.0 Weedy - 12.0 Chemical - 10.0

1,2 cyclohexanedione (containing some enol-tautomer) Smoke - 3.0 Burnt Sugar - 6.0 Sweet - 5.0 Celery - 4.5 Maple - 3.5 2,6 -dimethoxyphenol Smoke - 4.5 Cured - 4.5 Woody - 3.5 Sweet - 3.5 Burnt/Spice - 3.0

cyclohexanol Sweet - 2.0 Smoky/Burnt/Spicy - 2.0 Resinous - 6.0 Medicinal - 4.0 Woody - 3.5 Alcohol - 3.0 d-xylose Smoke - 2.0 Sulfur - 4.5 Wet Wool - 5.5 Sweet -1.0

4-ethylguaiacol Smoke - 5.0 Clove - 8.5 Sweet - 6.5 Medicinal - 6.5 Brown/Spicy - 6.0 Woody - 4.0 Pungent

benzyl disulfide Smoke - 3.5 Sulfur - 3.5 Burnt - 4.5 Ashy - 4.0

193 Table 3. Cont. thymol (2-isopropyl-5methylphenol) Smoke - 2.5 Pencil Shavings - 6.0 Woody - 4.5 Rubbery - 4.0

guaiacol (2-niethoxyphenol) Smoke - 1.5 Clove - 5.0 Medicinal - 5.5 Brown/Spice - 3.0 Cured - 2.0

c a r v a c r o l (5-isopropyl-2methylphenol) Smoke - 3.0 Creosote - 3.5 Chemical - 3.0 Smoke/Ashy - 3/5 Pungent - 2.5 Scale: 1 - just recognizable to 15 - extremely intense

4. CONCLUSIONS Some compounds previously thought to be associated with smoky odors are not smoky by themselves, at levels examined in this study. Many smoky compounds had different types of smoky characteristics indicating they may impart a unique "smoky" character that may be difficult to duplicate using other chemicals. Smoky compounds also may contribute other odor properties to the food products in which they are present.

5. REFERENCES 1. W. Baltes, R. Wittkowski, I. Sochtig, H. Block and L. Toth, The Quality of Foods And Beverages - Ch. 1. Academic Press, Inc., NY, 1981. 2. E.A. Smith, E. IV Chambers and S. CoUey, Cereal Foods World. 39 (1994) 495. 3. H. Daun, Food Technol. 41 (5) (1979) 66. 4. L. Toth and K. Potthast, Advances in Food Research. (29) Academic Press (1979) 87. 5. J. Gilbert and M.E. Knowles, J. Food Technol. 10 (1975) 24. 6. L.J. Bratzler, M.E. Spooner, J.B. Weatherspoon and J.A. Maxey, J. Food Sci. 34 (1969) 146. 7. K. Kim, T. Kurata and M. Fujimaki, Agr. Biol. Chem. 38 (1974): 53-63. 8. P. Issenberg, M.R. Korneich and A.O. Lustre, J. Food Sci. 36 (1971) 107. 9. J.A. Maga, Smoke In Food Processing. CRC Press: Boca Raton, FL, 1988. 10. A.W. Lance and M. Vaisey, J. Fisheries Res. Board Canada. 27 (1970) 1201.

194

11. D.J. Tilgner and H. Daiin, Lebensmittel Wissenschaft u. Technologie. 3 (1970) 77. 11. A.E. Wasserman, J. Food Sci. 33 (1966) 1005. 13. CM. Holenbeck, Pure & Appl. Chem. 49 (1977) 1687. 14. J.A Maga and 0 . 0 . Fapojuwo, J. Sensory Stud. 1 (1986) 9. 15. A. Dravnieks, Atlas of Odor Character Profiles (Data Series 61) American Society for Testing and Materials, Philadelphia, PA, 1985. Acknowledgment: This research was funded, in part, by USDA grant #58-5430-5124, "Grain Odor"

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

195

Identification of tasty compounds of cooked cured ham : physicochemical and sensory approaches J. Valentin", A.S. GuiUard^ C. Septier', C. Salles' and J.L. Le Quere' " Laboratoire de Recherches Sur les Aromes, INRA, 17 rue Sully, BV 1540, F-21034 Dijon Cedex, France ^ Centre Technique de la Salaison, de la Charcuterie et des conserves de Viande, 7 avenue du G"^ De Gaulle, F-94704 Maison-Alfort Cedex, France.

Abstract Tasty compounds from meat have not been studied in depth. The aim of this work was to isolate, identify and quantify tasty compounds from cooked cured ham and to link them to the sensory evaluations of the fractions from which they are extracted. The extraction of the water-soluble fraction from ham was done by an hydraulic press. The crude extract was ultrafiltered and both gel filtration and nanofiltration allowed to obtain edible fractions. Some links between the physicochemical and sensory data have been found, in particular, the umami taste was related to the presence of IMP and monosodium glutamate. The direct influence of proteolytic peptides on this taste is discussed. Moreover, the nanofiltration appears useful to remove salts from the extract but this technique needs to be optimized for a particular study of ham peptides.

1. INTRODUCTION Until today, numerous works have been carried out on food aroma but the number of studies on the compounds responsible for food taste is relatively low. However, taste takes an important part in the organoleptic properties and the acceptance of food, and all the components that contribute to it are not well known yet. The majority of the previous studies was made on cheeses [1] and it has been shown that most of the taste molecules are nonvolatile, polar and have a molecular weight lower than 1000 Da. The compounds originating from proteolysis were supposed to be active for taste because some synthetic small peptides have aheady been found tasty [1] but only some bitter peptides have been already identified [2]. In the case of meat, very few works were done on taste compounds. Aristoy and Toldra [3] have studied a water-soluble extract of dry cured ham and found that fractions containing peptides, amino acids and salts were bitter, acid and umami. Spanier et al [4] and Wang et al [5] have described the potential umami taste of an

196 octapeptide (Beefy meaty peptide) identified in a beef meat hydrolysate. However, the watersoluble fraction where all these compounds are found is very complex and their respective role on taste is unknown. The objective of this work was to obtain a representative water-soluble extract of cooked cured ham and to fractionate it in order to evaluate the taste of the fractions by sensory evaluation and to link the sensory data to their composition.

2. MATERIALS AND METHODS 2.1. Procedure for preparing the cooked cured ham The cooked cured ham was prepared at the Centre Technique de la Salaison, de la Charcuterie et des Conserves de Viande (Maison - Alfort, France). It was cooked in brine. The cooking was stopped when the heart of the ham reached 65°C. Slides or small sticks were made and frozen at -20°C until use. 2.2. Chemical products The quality of all the reagents were analytical grade. The pure water was obtained from a milliQ system® (Millipore, Bedford, MA). 2.3 Extraction of the water-soluble fraction Mixing with water - The grated frozen ham (300 g) was mixed with water (1/2, WA^). The mixture was centrifuged at 4000 g at 4°C for 30 min. This treatment was repeated with the pellet; The supematants were freeze-dried and redissolved in 75 ml water. This solution was ultracentrifiiged at 105 000 g for 30 min at 4°C. The supernatant was collected. This method has been currently used for the water-soluble cheeses extraction [6] Hydraulic press - 1200 g cooked cured ham was grated and mixed with 1380 g sand (150-200 jim; Prolabo, Paris, France). The mixture was pressed progressively until a pressure of 42 bars for 3 hours with an hydraulic press (Trans Hydro, Morlaix, France), according to Salvat-Brunaud et al [7]. The water phase collected was frozen at - 20°C until use. French press - This press (Bioblock, Illkirch, France) leads to the destruction of the muscle cells. A pressure of 1700 bars applied on a cell (50 ml) containing 75 g - aliquots of 200 g grinded cooked cured ham mixed with 37,5 ml pure water allowed to obtain a paste. This one was ultracentrifuged at 100 500 g at 4°C for 30 min and the supernatant was collected. 2.4. Purification by ultrafiltration The water-soluble extract was ultrafiltered by frontal filtration under stirring in a cell (V = 400 ml, d = 76 nmi; Millipore Corp). The cellulose ester membranes used had 10 000 and 1000 Da molecular weight cut-offs. The temperature and the nitrogen pressure were respectively 4°C and 4 bars. 2.5. Purification by gel filtration The ultrafiltered water-soluble fraction (MW < 1000 Da) was diluted 3 times and chromatographied on Toyopearl HW-40S (Tosoh Corp., Tokyo, Japan). Five milliliters of this fraction were applied on a Superformance column (2.6 x 60 cm ; Merck, Darmstadt,

197 Germany). The elution was made with pure water (2 ml/min) at 4°C, using a Eldex B lOO-S-2 pump (Bioblock, nikirch, France), leading to a pressure around 12 bars. The absorbance of the eluent at 214 nm was measured by a detector UV-VIS 118 (Gilson Medical Electronics Inc, Middleton, WI). The fractions (3.5 ml) were collected with a FC 204 fraction collector (Gilson) and were pooled according to the profile obtained. These pooled fractions were freeze-dried and dissolved in 5 ml pure water [8]. 2.6. Purification by nanonitration The water-soluble fraction of 250 g cooked cured ham was extracted by pure water. The clear extract obtained was ultrafiltered with a 1000 Da molecular weight cut-off membrane. The permeate was diluted in 2 1 pure water and adjusted to pH 5 with 1 N nitric acid. This solution was nanofiltered on a spiral membrane Nanomax 50 (area : 0.37 m^ ; Millipore) by tangential filtration on a pilot apparatus (MSP 006239 Prolab, Millipore Corp.) [9]. The temperature was 25°C and the pressure was 4 bars. The total duration was 10 min. The nanofiltration in the pilot apparatus was stopped when the volume of retentate in the loop was around 2 fold of the apparatus void volume above the membrane which was around 1 1. At this state, the volume concentration factor was around 3. The retentate was nanofiltered in a cell by frontal filtration with the same kind of membrane (area : 1.2 10"^ m^) in order to prepare a peptidic fraction for tasting during the sensory evaluation experiment. The temperature was 4°C, the nitrogen pressure was 5 bars and the total duration was 72 hours. 2.7. Analytical methods The dry matter values were obtained by evaporation of the water of each fraction with a centrifuge evaporator until constant weight (P = 0.5 10"^ bar, T = 60°C). Total nitrogen and total carbon were measured by elementary analysis (NC 2500, Carlo Erba Instruments, Milan, Italy). The glucose was quantified by enzymatic reaction with the glucose oxydase and peroxydase. The lactic acid was quantified by HPLC with an Aminex column (ion exchange HPX87H, 300 X 7.8 mm, Hewlett-Packard, Walbronn, Germany) warmed at 65°C. The eluent was 5 mM sulfuric acid. The flow-rate was 0.8 ml/min. The detection was done by refractometry. Sodium, potassium and calcium concentrations were evaluated with a flame ionization spectrophotometer Eldex 6361 (Eppendorff, Hambourg, Germany). Magnesium and iron concentrations were evaluated with a spectra 100 atomic absorption spectrophotometer (Varian, Les Ulis, France). Inorganic phosphorus and chloride ions were estimated by diagnostic kits (Sigma, St Quentin Fallavier, franee). The nucleotides were quantified by HPLC with a Synchropack AX anion exchange column (250 x 4.6 mm) (Synchrom Inc, Lafayette, IN). The compounds were eluted by a gradient made with two buffers - A : 15 mM KH2PO4 + 0.1% HCl pH 4.1 -B : 0.5 M KH2PO4 pH 4.5. The pH was adjusted with concentrated KOH solution. The gradient was made as follow : 0 - 7 min : 100 % A ; 7 - 17 min : from 100 % A to 100 % B , 17 - 35 min : 100 % B , 35 - 40 min : from 100 % B to 100 % A , 40 - 60 min : 100 % A. The flow-rate was 1.5 ml/min. The UV detection was done at 254 nm.

198 The amino acid analyses were done before and after acid hydrolysis (6N HCl, 110*^C, 24h). The amino acid compositions were determined on a LC 5000 amino acid analyzer (Biotronik, Maintal, Germany). 2.8. Sensory evaluation Before starting the work, the extracts were diluted with pure water to have the same total taste intensity as the crude cooked cured ham. This was done by four trained paneUsts. After a consensus between the four panelists have been obtained, the dilution factors affected were 5 for the water extract, 3 for the extract obtained by hydraulic press and 2 for the extract obtained by French press. Fourteen panelists were trained to recognize the basic taste (salty, bitter, acid, sweet, umami) with pure solutions (respectively : 2 g/1 NaCl, 5 g/1 L-Leucine, 0.165 % lactic acid, 12 g/1 glucose and 0.6 g/1 monosodium glutamate) and for each taste, to classify different concentrations of these pure solutions according to their taste intensity. The sensory evaluations were performed in an air-conditioned room (20.5°C) under red light. The paneUsts had their nostrils pinched to suppress olfactive sensations. The session was repeated twice. The panel had to evaluate the taste of the presented samples according to a sequential monadic presentation. For each sample to taste, each panelist had two volumes of 5 ml. They were instructed to taste once the total 5 ml and they could taste again an other time if necessary. They had to mark each taste intensity for each presented solution on a scale where 0 was pure water. Other reference solutions were given for each taste. Their concentrations were determined to have a taste intensity similar to the crude cooked cured ham. The acid, sweet and umami references were positioned at 70 % and the bitter and salty references were only positioned at 50 % on the scale because bitterness and saltiness are more intense in the extracts than in the crude cooked cured ham. The samples which were compared between them were: (i) the extracts obtained from the 3 different techniques, (ii) the permeate of the selected extract obtained by ultrafiltration respectively on membranes with a molecular weight cut-off of 10 000 Da and 1000 Da, (iii) the fractions Fl, F2, F3, F4 and N obtained by gel filtration and nanofiltration. The data were processed with the SAS statistical package (SAS Institute Inc., Gary, NG). Analyses of variance were conducted and a multiple comparison test was made with the Student-Newmann-Keuls procedure to compare the sample for each discriminent taste descriptor. For the sessions concerning the extraction and ultrafiltration methods, the "Means" procedure (SAS) was used to test the differences between the individual scores of the samples and the references for each descriptor.

3. RESULTS AND DISCUSSION 3.1. Extraction and purification The Figure 1 represents the extraction and purification pathways. The extraction of the water-soluble fraction is generally done with mixing the product with pure water. Its main disadvantage is that it leads to an important dilution of the hydrosoluble compounds. So, we have tested other extractive techniques, the hydraulic press, which was already successfully

199 used for hard paste cheeses [6], and the French press. The three extracts were compared to the crude cooked cured ham by sensory analysis. Concerning taste, it appears that the extract obtained by pressing with the hydraulic press is the more representative of the original product [10].

{M miration Croyo|)eai:lBW-4DS» Fronts n^iidSltr^idoii

® Sens^^iy « - ? i ^ B | | | |

Figure 1. Extraction and purification pathway The mass range of the taste compounds contained in this extract were respectively determined by ultrafiltration on membranes with a MWCO of 10000 and 1000 Da. No significant difference between these two permeates is observed by the panel. This indicates that most of the taste compounds extracted have a molecular weight lower than 1000 Da. So, we have used this fraction (permeate 1000 Da) for this study.

200

60

120

180

240

tiiiie(min)

Figure 2. Toyopearl HW-40S chromatography of the UWSE of the cooked cured ham (Extraction and elution with pure water) To purify this extract, the gel filtration technique was used because this technique alone can lead to separations with pure water as eluent. This constrain was imposed by the sensory evaluations of the fractions obtained. The profile is presented in Figure 2. Only four fractions were made because of the relatively bad reproducibility and the high number (60) of injections on the column necessary to have enough material for the sensory analysis experiments. 3.2. Physicochemical analysis Table 1 Composition of the fractions : salts, glucose, lactic acid, nucleotides (mg/100 mg dry matter UWSE) Fractions Dry matter Total N Total C Lactic acid Glucose IMP Amino acids Peptides Sodium Potassium Calcium Magnesium Iron Chloride Inorg. phosphorus

Fl 42.2 1 9 16.4 0.1 1.4 1.6 1.5 9.3 5.1 0.05 0.2 0.08 0.1 3.2

F2 48.1 3.6 9.2 2.1 4.7 0.2 4.1 0.8 7.5 2.4 0.05 0.2 8.7 -_

F3 2.7 0.6 1 0.5 0.2 -_

F4 0.2 0.2 0.03 2

The physicochemical analysis of the fractions are reported in Tables 1 and 2. Table 1 shows that the two first fractions contain the majority of the dry matter of the extract. The majority of mineral salts, lactic acid, MP, 58 % of total peptides and polar amino acids are in the fraction 1. Fraction 2 contains less mineral salts but an important part of chlorides, the

201 majority of glucose and 63 % free amino acids which are rather non polar. The other fractions contain only some hydrophobic amino acids. Table 2 Composition of the fractions : free amino acids (A) and peptides (P) (|ig/100 mg dry matter UWSE) _

_

25.5 41.5 198 122 Asp 45.4 Thr 49.3 148.2 41.6 Ser 52.9 201.9 74.4 X Asn 156 Glu 440.8 425.1 130.9 Gin 91.3 53.6 118.9 Pro - 318.5 254.4 . 73.4 Gly 45.1 51.9 541.4 Ala X Cit 146.8 53.1 0.8 191.9 78.9 Val 78.5 Cys 89.9 61.3 Met - 179.6 33.1 lie - 264.0 50.6 Leu Tyr Phe 14 10.4 5.6 Gaba 24.9 Orn 12.5 71.5 85.8 Lys 125.6 94.7 - 1010.4 - 1531.4 His X 1635.0 Cre* 745.5 X Arg 23.6 108.6 78.6 30.3 Total 1656.8 1452.2 4056 772.2 %A 25 63 %P 58 31 * Creatin

_ 101.2 126.0 7.9 29.6 255.3 520.1 9

— ~ 186.5 X

1.16 304.5 X

230.7 9

17.4 165.8 183.2 3

-

73.4

17.6 28.4 14.9 21.7 152.7 45.9 14.5 17.7 34.6

59.7 6.4 53.8 66.4 28.4 31.9 4.3 3 84.5 33.2 117.8 869.4 58.1 27.2 1647.1 78.6 2

106.1 35.9 41.2 X

183.8 X

60.8 154.4 40.1 27.2 18.7 22.7 11.1 14.8 8 X

9.5 3.6 48.5 581.4 X

22.2 448.6 21.4

The gel filtration technique only leads to a partial separation of the compounds which may participate to the taste, so, we have tested the nanofiltration technique with Nanomax 50 membrane [9] to try to improve at least the separation of organic and mineral compounds. After nanofiltration of the solution on the pilot containing the membrane Nanomax 50, the volume concentration factor is around 3. The rate of each compound present in the retentate and in the permeate at this time is reported in Figure 3.

202

R 4

T E N T A T E

r

1 '. ' 1

C

^ K

80

1 Peptides • Amino acids JMP ^ ^ ^ Lactic acid '•iiili^ Glucose P ^i^^HiCI

h• • 1

^^L

N Mag

^ ^ H^ ^ H Ca Total carbon Total nitrogen P Dyymfitter

0

p E R M E A T E

60

Figure 3. Rate of compounds present in the fractions obtained by nanofiltration of the UWSE on the pilot apparatus (%) During this filtration, 27 % dry matter are eliminated, corresponding respectively to 16 % and 15 % total carbon and total nitrogen. Around 50 % minerals and lactic acid are eliminated, except inorganic phosphorus which is more retained by the membrane. The rate of glucose eUminated in the permeate is rather low, what is rather surprising for a non ionic molecule. IMP is almost totally retained. Free amino acids seem better eliminated than peptides, apart for glutamic acid. The retentate previously obtained is nanofiltrated in a cell with the same kind of membrane. The composition of the retentate thus collected is indicated in Table 3. Its amino acid and peptide composition is reported in Table 2. Table 3 Composition of the retentate obtained by nanofiltration (mg/lOOmg dry matter UWSE)

12

Total nitrogen (% dry matter) 1.6(13%)

IMP 5%

Glucose 29%

Dry matter

amino acids 28%

Peptides 18%

Total carbon (% dry matter) 3.7(31%) lactic acid 45% Glutamate 15%

This fraction contains less dry matter than the gel filtration fractions 1 and 2. Compared to these two fractions, the retentate contains less mineral salts, lactic acid, glucose and peptides. The IMP concentrations of the retentate and of the fraction 1 are equivalent, as the glutamic acid concentration of the retentate and of the fraction 2.

203

3.3. Sensory evaluation The taste of the four fractions obtained by gel filtration and of the retentate obtained by nanofiltration (N) has been studied by sensory analysis. The results of the analysis of variance for each descriptor are reported in Table 4. Table 4 Analysis of variance for each taste descriptor Taste Umami Bitter Salty Acid Sweet

F 42.16 13.52 13.30 6.53 1.99

ProbabiUty 0.0001 0.0001 0.0001 0.0002 0.1094

The descriptors umami, bitter, salty and acid are discriminent (P < 0.05). The multiple comparison of the mean scores for each taste is reported in Table 5. Table 5 Taste mean scores of the fractions for each taste (The means with the same letter (abc) are not significantly different at the level of 5 %). Fraction Fl F2 F3 F4 N

Umami 6.65 a 5.08 b 0.72 c 0.53 c 6.77 a

Bitter 1.07 b 0.72 b 4.48 a 4.41a 1.52 b

Salty 3.25 a 4.45 a 0.96 b 0.47 b 1.56 b

Acid 0.40 b 0.70 b 0.56 b 0.64 b 2.26 a

Sweet 2.49 a 1.97 a 1.04 a 1.16a 1.85 a

3.4. Correlations between chemical and sensory analysis The fraction 1 and N have the most intense umami taste. The fraction Fl contains the totality of IMP, 58 % total peptides and glutamate at a concentration of 150 |ng/ml. The fraction N contains MP, glutamate and peptides. Glutamate is well-known for its umami taste [11] but it is present in these two fractions at a concentration lower than its threshold value which is 300 }ig/ml [12]. However, the concentration of IMP is higher than its threshold value (0.14 mg/ml [13]) in these fractions and the main property of this taste enhancer is to decrease significantly the threshold value of the glutamate by synergistic effect for the umami taste [13]. Thus, glutamate and IMP seem responsible for the umami taste of these fractions. Concerning peptides, it is difficult to give conclusions but the fact that their concentration is three times lower in the fraction N than in the fraction 1, though the intensity of the umami taste is similar for the two fractions, seems to minimize their impact on the umami taste. Though, we observed that the fraction 2 had a high intense umami taste, too, with a very low concentration of IMP and a lower concentration of glutamate and peptides. We notice that these fractions contained sodium chloride which can have synergistic effect on

204

peptide taste [5], as glutamate. However, these observations would have to be confirmed by studies with model mixtures. The bitterness of the fractions 3 and 4 is probably due to the presence of hydrophobic amino acids [12]. Their concentration is lower than their respective threshold value but some additive effects may occur. The salty taste observed for the fractions 1 and 2 is mainly linked to the presence of mineral salts in these fractions, the concentrations of which are higher than their threshold value (sodium and potassium chlorides : 1 to 3 mg/ml [14-15]). The low salty taste intensity of the fraction N showed that an important part of the salts are eliminated from the retentate of nanofiltration. The acid taste intensity of the fractions obtained by gel filtration is low. However, the fraction 1 contains 5.5 mg/ml lactic acid which is around 20 times higher than its threshold value (between 0.04 and 0.15 mg/ml [14]). That could be explained by the important quantity of matter present in this fraction which could mask the acid taste of lactic acid. The fi-action N is acid and contains 0.3 mg/ml lactic acid. Its perception may be due to the loss of the molecules responsible for the masking of the acid taste in the fraction 1. The sweet taste intensity is low for all the fractions and no significant differences were observed between them. The glucose concentration is lower than its threshold value in all the fractions. As the fractions obtained by gel filtration and that obtained by nanofiltration come from the UWSE made by different extraction methods, it is impossible to compare the performances of these two separation techniques. We can only compare the concentrations measured and the tastes perceived. The nanofiltration leads to the concentration of organic matter and the thinning of mineral matter. The consequences are a decrease of the salty taste intensity while the umami taste intensity is not affected. So, this method seems interesting to isolate the compounds responsible for the umami taste of this food. Concerning peptides, we have applied the same conditions as used for a study on goat cheese. It seems that this technique has to be optimized for each kind of product analyzed because the performances of the membrane depend on the composition of the medium. The physico-chemical environment of the membrane seems important for its selectivity. However, the mechanisms of solvent and solution transfer are not clear yet [9].

4. CONCLUSION The extraction of the water-soluble fraction of cooked cured ham by hydraulic press allowed the obtainment of a fraction representative of the taste of the raw food. Gel filtration and nanofiltration of this extract showed that IMP and sodium glutamate are mainly implied in the umami taste of some fractions. The role of the small peptides has to be clarified. It is necessary to optimize and improve the nanofiltration for the analysis of this product to isolate the small peptides with a good yield. Some studies with model mixtures should lead to the evaluation of the relative impact of each kind of compounds on the taste of the cooked cured ham.

205 Acknowledgments We gratefully acknowledge D. Le Bars (Laboratoire de biochimie et structure des proteines, INRA, Jouy-en-Josas, France) for the amino acids analysis, F. Michel for the use of the hydraulic press and A. Garem for the nanofiltration with the pilot apparatus (Laboratoire de Recherches en Technologic Laitiere, INRA, Rennes, France), I. Lesschaeve and S. Issanchou for their assistance in sensory analysis (Laboratoire de Recherches Sur les Aromes, INRA, Dijon, France).

5. REFERENCES 1 2 3 4

5 6 7 8 9

10

11 12

13 14 15

F. Roudot-Algaron, Lait, 76 (1996) 313. L. Lemieux and R.E. Simard, Lait, 72 (1992) 335. M.C. Aristoy, and F. Toldra, G. Charalambous (ed.). Food flavors: generation, analysis and process influence, Elsevier Science B. V., New York, (1995) 1323. A.M. Spanier, A.M., J.M. Bland, J.A. MiUer, J. Glinka, W. Wasz, and T. Duggins, Food flavors: generation, analysis and process influence, G. Charalambous, (ed.) Elsevier Science B, V., New York, (1995) 1365. K. Wang, J.A. Maga, and P.J. Bechtel, J. Food Sci., 61 (1996) 837. C. Salles, C. Septier, F. Roudot-algaron, A. Guillot, and P.X. Etievant, J. Agric. Food Chem., 43 (1995) 1659. D. Salvat-Brunaud, J.L. Maubois, Y. Le Graet, M. Piot, M.B. Maillard, C. Corre, and A. Thierry, Lait. 75 (1995) 239. C. Herve, C. Septier , I. Lesschaeve, S. Issanchou, J.L. Le Quere, and C. Salles, Lait, (1997) submitted. A. Garem, Applications de la nanofiltration au fractionnement des hydrolysats peptidiques. Thesis of doctorate, Ecole Nationale Superieure Agronomique de Rennes, France, 1995. A.S. Guillard, Etude des caracteristiques organoleptiques du jambon cuit, relations avec les matieres premieres et le traitement technologique. Thesis of doctorate, Universite Paris XI Orsay, France, 1997. K. Dceda, J. Tokyo Chem. Soc, 30 (1909) 820. H. Kato, M.R. Rhue, T. Nishimura,. Flavor chemistry. Trends and developments, R.T. Teranishi, R.G. Buttery and F. Shahidi, (eds.), American Chemichal Society, Washington, DC, (1989) 158. J.A. Maga, CRC Crit. Rev. Food Sci. Nutr., 18 (1983) 231. W.H. Stahl, (ed;). Compilation of odor and taste threshold values data, American Society for Testing and Materials, Philadelphia, USA, 1973. S. Yamaguchi, Umami: a basic taste, Y. Kawamura and M.R. Kare (eds.). Marcel Dekker, New York, (1987) 41.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

207

Isolation of a peptidic fraction from the goat cheese water-soluble extract by nanofiltration for sensory evaluation studies N. Somme^e^^ A. Garem^, D. Molle\ C. Septier", J.L. Le Quere^ and C. Salles' ^ Laboratoire de Recherches Sur les Aromes, INRA, 17 rue Sully, BV 1540, 21034 Dijon Cedex, France ^ Laboratoire de Recherches en Technologic Laitiere, E^IRA, 65 rue de St Brieuc, 35042 Rennes Cedex, France

Abstract The effect of the small water-soluble peptides on the taste of cheeses has not been clarified yet because of the high difficulties to isolate them from the other water-soluble compounds. A new filtration method, nanofiltration, using ionizable membranes, allowed us to purify a peptidic fraction prepared from the permeate obtained by ultrafiltration (MWCO=1000) of a goat cheese water-soluble extract. A large proportion of mineral salts and a part of amino acids were eliminated from the nanofiltration retentate where a majority of small peptides were concentrated. The peptidic fraction was incorporated in a cheese model with known synergistic effectors such as mineral salts and amino acids. Sensory evaluation was done by some omission tests. The results showed that the small peptides fraction had no effect on the taste of the cheese and no synergistic effects were observed. The nanofiltration technique showed its interest and should be applied to other cheeses and foodstuffs after optimization.

1. INTRODUCTION The taste of synthetic small peptides was studied in the past and it was shown that a lot of them can have one of each basic taste. These studies were reviewed by Roudot-Algaron, [1]. They have a large range of threshold values according to their structure, size, polarity, position of the amino acid residues and nature of the medium where they are dissolved, including possible synergistic effects with salts or amino acids [2]. However, in food, apart from the bitter peptides of cheeses formed during proteolysis [3], few tasty small peptides and no bitter ones have been formerly found and their exact role in food taste has not been clearly elucidated yet. In cheese, it has been shown that the water-soluble extract, and more particularly the fraction containing compounds with a molecular weight inferior to 1000 Da, has a strong

208

aroma and taste [4]. In the case of the goat cheese under investigation in this study, the dry matter of low molecular-weight fraction contains around 30 % of these small peptides, the other main compounds being salts and free amino acids. The aim of this work was to isolate these small peptides from the other main compounds such as amino acids, salts and organic acids, and to evaluate their importance in the taste of the cheese by incorporation of this peptidic fraction in a cheese model [5].

2. MATERIALS AND METHODS 2.1. Materials The pure water was obtained from a milliQ system® (MilUpore, Bedford, MA). All the reagents were analytical grade. The goat cheeses called "Bouton de culotte"® were bought at the Lycee Agricole of Davaye (Macon, France). Its ripening period was 3 weeks. 2.2. Extraction of the water-soluble extract The grated cheese was extracted by pure water [6]. The clear extract obtained was ultrafiltered by frontal filtration under stirring in a cell (V = 400 ml, d = 76 mm ; Millipore Corp). The cellulose ester membranes used were 1000 Da molecular weight cut-off. The temperature and the nitrogen pressure were respectively 4°C and 4 bars. The permeate was called UWSE (Ultrafiltered Water-Soluble Extract). 2.3. NanoHItration The conditions of nanofiltration are indicated in Table 1. Tablel Nanofiltration conditions

PH Temperature (°C) Volume (1) Pressure (bar) Duration Mode Nanomax 50 membrane Area (m^)

Pilot apparatus 7 and 5 25 2 4 lOmin Tangential Spiral

filtration cell 7 and 5 4 0,1 5 24 h Frontal Planar

0.37

1.2 10"^

The equivalent of 400 ml of water-soluble extract was diluted in 21 of water and adjusted to the right pH with 5 N NaOH. The membrane used was Nanomax 50 (Millipore Corp.). The nanofiltration in a pilot apparatus (MSP 006239 Prolab, Millipore Corp.) was stopped when the volume of retentate in the loop was around 2 fold the apparatus void volume which was around 1 1. At this stage, the volume concentration factor was around 3. In order to prepare

209 the peptidic fraction for tasting during the sensory evaluation experiment, the nanofiltration of the retentate was followed by frontal filtration in a cell with the same kind of membrane until the maximum of water was eliminated. 2.4. Analytical methods The dry matter values were obtained by evaporation of the water of each fraction with a centrifuge evaporator until constant weight (P = 0.5 10"^ bar, T = 60°C). Total nitrogen and carbon were measured by elementary analysis (NC 2500, Carlo Erba Instruments, Milan, Italy). The glucose was quantified by enzymatic reaction with the glucose oxydase and peroxydase. The lactic acid was quantified by HPLC with an Aminex column (ion exchange HPX87H, 300 X 7.8 mm, Hewlett-Packard, Walbronn, Germany) warmed at 65°C. The eluent was 5 mM sulfuric acid. The flow-rate was 0.8 ml/min. The detection was done by refractometry. Sodium, potassium and calcium concentrations were evaluated with a flame ionization spectrophotometer Eldex 6361 (Eppendorff, Hambourg, Germany). Magnesium concentration was evaluated with a Spectra 100 atomic absorption spectrophotometer (Varian, Les Ulis, France). Inorganic phosphorus and chloride ions were estimated by diagnostic kits (Sigma, St Quentin Fallavier, franee). The amino acid analysis was done before and after acid hydrolysis (6N HCl, 110°C, 24h). The amino acid compositions were determined on a LC 5000 amino acid analyser (Biotronik, Maintal, Germany). 2.5. HPLC and HPLC-MS HPLC analysis - 20 jil sample were injected on a ODS Hypersyl RP18 column (200 x 2.1 mm, 5 |nm ; Hewlet-Packard, Valbronn, Germany). The pump and the detector were model 1050 (Hewlet-Packard). The detection was monitored at 214 nm. The gradient was made with two eluents. The eluent A was 0.1 % trifluoroacetic acid (TFA) in pure water and eluent B was 0.1 % TFA in 70 % acetonitrile. Separations were conducted at a flow-rate of 0.3 ml/min with a linear gradient from 0 to 100 % of eluent B in 50 min. Preparative HPLC - The separation were carried out on a preparative ODS Intersil RP18 column (250 x 10 mm, 10 jam ; Interchim, Montlugon, France). The same apparatus and eluents (A and B) as above were used. The volume of the injection loop was 1 ml. Around 5 mg dry matter were injected each time. The detection was monitored at 214 nm. Separations were conducted at a flow-rate of 3 ml/min. From 0 to 5 min, the eluent was 100 % A ; from 5 to 15 min, a Unear gradient reached 100 % B and decrease to 100 % A in 10 min. The first large peak eluted at the end of the void volume and containing a large quantity of mineral salts was discarded. The rest of the eluate (until no more peak appeared) was collected in a flask, concentrated under vacuum and redissolved in 0.02 % TFA in water. HPLC-MS - Twenty microliters were injected on a ODS Nucleosil 50-5 RP18ec column (200 X 2.1 mm, 5 \xm ; Macherey-Nagel, Hoerdt, France) kept at 40°C. The flow-rate was 0.25 ml/min. Separations were carried out using a binary gradient. The two eluent solutions were : A - 0.106 % TFA in pure water, B - 0.1 % TFA in 80 % acetonitrile. The elution was made with pure eluent A for 5 min then the gradient was formed by linearly increasing the concentration of eluent B to reach 44 % B 45 min after the injection, 88 % B from 55 min to

210 57 min and 100 % A from 59 min to 65 min. The first peak was discarded and the eluent was introduced in the mass spectrometer (SCBEX API-HI ; Thomill, Canada) equipped with an electrospray ionization source at a flow-rate around 30 jid/min (split: 1/8). 2.6. Sensory evaluations The panel was composed of 13 panelists trained to recognize the bitterness, acidity, saltiness, umami and astringency. The evaluation were conducted under red light and the panelists nostrils were pinched to suppress olfactive sensations. During the tests, they could take, if necessary, some taste reference solutions to recall each taste. Some comparison tests were carried out between a reference and samples containing some model mixtures made with compounds present in the original cheese such as small peptides, salts and amino acids. The panel was instructed to mark the intensity of each taste: bitter, salty, acid, umami and astringency for each sample on a scale, in comparison with the reference sample which is marked at 50% for each descriptor. The model mixtures were incorporated in a cheese model for tasting these molecules in a medium similar to the food matrix. The procedure for preparing the cheese model was established in our laboratory [5]. Five samples, where one or several possible taste active components were omitted, were compared with a reference sample containing the retentate, free amino acids and salts (NaCl, KCl, CaCl2). The samples were : amino acids + salts, retentate + amino acids, retentate -h salts, retentate, cheese model without incorporation (used as a blank). These compounds were incorporated in the same concentration as quantified in the crude cheese [5,7] apart from salts. The concentration of salts to be incorporated was divided by 4 in order to obtain the same taste intensity as in the original cheese. The results were processed with the SAS statistical package version 6, 4th edition (SAS Institute Inc., Gary, NC). A comparison of the mean deviations from the reference for each descriptor was conducted by a Dunnett procedure.

3. RESULTS AND DISCUSSION 3.1. Interest of nanofiltration The ultrafiltered water-soluble extract (UWSE) obtained for goat cheese is very complex. It particularly contains mineral salts, amino acids, small peptides and organic acids which are difficult to separate. There are two possible ways to achieve the study of the taste of the small peptides present in this extract: (i) the first is to reconstitute a model mixture of peptides. That would imply the separation of the peptides, their identification, their quantification and chemical synthesis. This solution appears tedious and rather expensive owing to the numerous peptides present in the fraction; (ii) the second solution, that we have chosen for this study, consists in the isolation of a relatively pure peptidic fraction from the extract. Because of the sensory evaluation experiments which are to be used, buffers or solvents are prohibited in the separation procedure. Only two kinds of techniques appeared possible : gel filtration with water as eluent or filtration techniques. Until today, we have studied the UWSE of cheeses by chromatography of the extract by gel filtration on Toyopearl HW-40S with pure water [6,7]. The resolution was relatively poor. The first fractions contained small peptides but some of them were coeluted with salts, free

211 amino acids and organic acids. This is explained by the small mass differences between all these molecules and by the fact that water is not a good eluent for ionic molecules. Moreover, the obtainment of a large quantity of peptides necessary for sensory evaluations implied a lot of tedious repetitive injections on the colunrn. Nanofiltration appeared to be an alternative to the gel filtration technique. It is a membrane separation process which is intermediate between ultrafiltration and reverse osmosis. The range of molecular weight cut-off is between 200 and 1000 Da. The membrane we have chosen is Nanomax 50 because of its separation properties for small molecules [8,9]. The molecular weight cut-off of this membrane is 500 Da. It is made of a macroporous polysulfone support which gives good mechanical properties and high permeation flow rates. The active layer is made of polyamide. Its thickness is inferior to the micrometer and the diameter of the pores is around the nanometer. More generally, these nanofiltration membranes can be compared to porous areas of ion exchange so, the exclusion of molecules is not only due to their molecular weight but also to their charge. The separation of compounds depends essentially on the difference of charge between the solute and the membrane. This may lead to the selective separation of compounds according to the pH and the composition of the medium. 3.2. Performances of nanonitration In our experiment, we have compared the results obtained for nanofiltration experiments at pH 5 and at pH 7, using a tangential filtration pilot. The zero point charge of the membrane surface being around 4.5, the membrane was barely charged at pH 5 and strongly negatively charged at pH 7. Physicochemical analyses were done to compare the performances obtained at these two pH values. The transfer of the solutes by nanofiltration with the pilot apparatus was characterized by their retentate elimination rate (RER = 100 (CoMo - CrMr) / CoMo). Co and Mo, Cr and Mr are respectively the concentrations and the total mass in the initial solution and in the retentate. It shows the proportion of compounds eliminated from the retentate compared to the original solution. The Figure 1 represents the RER observed at pH 5 and pH 7 for dry matter, nitrogen, carbon, some salts, lactate, free amino acids and peptides. More dry matter and nitrogen are eUminated at pH 7 than at pH 5. Concerning salts, sodium, potassium, calcium and chlorides are efficiently eliminated at the same rate at the two pH values. The elimination of magnesium and lactate is more efficient at pH 5 than at pH 7. We observed the same thing for phosphate which is almost totally retained at pH 7 because of the high negative charge density of the membrane at pH 7. For the free amino acids, the rate of elimination is rather high and there is no difference between the results obtained at pH 5 and 7. For peptides, we observed that they are more retained by the membrane than free amino acids and this retention is more effective at pH 5. It appeared that nanofiltration is more efficient at pH 5 than at pH 7 to eliminate mineral salts and lactate from the organic compounds, and that the nitrogen compounds were better retained in the retentate at pH 5. The HPLC analysis of each fraction, UWSE, retentate and permeate is presented in Figure 2. It confirms the results of the previous analysis in showing that the majority of the numerous peptides present in the crude extract is recovered in the retentate while the chromatogram of the permeate is relatively poor.

212

Peptides Amino acids Lactate Pi CI Mg Ca K Na Ct Nt Dry Matter

I

I

10

20

I

I

I

30 40 50 Retentate Elimination Rate (%)

h-

60

70

Figure 1. Retentate elimination rates of compounds measured after nanofiltration of the UWSE on the pilot at pH 5 and 7

The mass distributions obtained by HPLC-electrospray-MS confirmed that most of the peptides were retained in the retentate. We can observe in Figure 3 that the masses of most of these peptides lie between 400 and 1000 Da. Nearly 40 different masses were confirmed by both positive and negative ionization. The sequence of some of them has been obtained by the analysis of purified water-soluble fractions by tandem mass spectrometry in our laboratory (unpubUshed results). These fractions were purified by preparative HPLC on reversed phase octadecyl silane before these analyses to reduce the residual salts which can perturb the ESI mass spectrometry analysis. Similarly we have discarded the first large peak of RP-HPLC analyses even if it certainly contained small polar peptides. However it could not be analyzed by ESI/MS because of its large amount of residual salts. To sunmiarize on the performances of nanofiltration, we can state that nanofiltration is more efficient at pH 5 than at pH 7 for the isolation and purification of small peptides. The loss of peptides at this pH is only 13% whereas 50% of free amino acids, 70% of lactate and about 60% of minerals are eliminated from the retentate. These results led us to work at pH 5 for the following experiments. The composition of the UWSE and of the retentate resulting of its nanofiltration at pH 5 is presented in Figure 4. Compared to gel filtration (Table 2), this method has numerous advantages such as rapidity, the quantity of sample treated and the obtaining of a relatively pure peptidic fraction.

213

0.100

1.000 t

Retentate T—(

<

0.100

Permeate

<

f^UL 0.000

. 20

1— 30

Time (min.)

Figure 2. HPLC analysis of the UWSE, retentate and permeate obtained at pH 5

—f 50

214

200-400

400-600

600-800

800-1000

1000-1200

1200-1400

Mass

Figure 3. Mass distribution of compounds present in the retentate obtained at pH 5 (obtained by HPLC - electrospray - MS)

S^ 80 o o

s



70 60

•S o

50

w^

{V C3

B

40

b 30

T3

S E2

*»^ a a

a

20 10 0

Amino acids

+

4Peptides

Mineral salts

+

lactic acid

Figure 4. Composition of the UWSE and of the retentate resulting of its nanofiltration at pH 5 with Nanomax 50 membrane.

215 Table 2 Comparison between gel filtration and nanofiltration Preparative scale for Obtainment of a large quantity of peptides

nanofiltration gel filtration +

Rapidity of preparation

+

Efficiency to obtain a pure peptidic fraction

+

Selectivity Cost

size & charge

size

-

+

3.3. Sensory evaluation The retentate containing the small peptides of the goat cheese obtained by nanofiltration at pH 5 has been incorporated in a cheese model to taste it in a medium similar to the food matrix. In addition, we have studied the effect of mineral salts and amino acids because they are the main compounds contained in the tasty cheese extract. Moreover, Wang et al [2] have shown that monosodium glutamate and sodium chloride have some synergistic effects with a tasty peptide for decreasing its threshold value. Some omission tests [10,11] were carried out to evaluate the impact of the elimination of one or several compounds on each basic taste. The interest of these tests are their ability to point out additive or synergistic effects. The mean scores of each descriptor were compared to a reference containing all the compounds to test and the mean deviations from the reference are reported in Figure 5. For each taste descriptor, the omission of the peptidic fraction or of the amino acids had no effect. On another hand, the omission of salts led to a significant decrease of the salty taste intensity and the omission of both salts and amino acids led to a significant decrease of each taste intensity, indicating a possible additive or synergistic effect between these two kinds of compounds. However, it appeared that the small peptides isolated from the cheese are not tasty and have no effect on the taste of the cheese.

216

acid

bitter

astringent

salty

umanu

* significantly different from R at the level of 5 %

Figure 5. Mean deviations from the reference for each taste of the model mixtures incorporated in a cheese model (Reference : R = retentate + amino acids + salts ; samples : 1 = amino acids + salts, 2 = retentate + amino acids, 3 = retentate + salts, 4 = nothing, 5 = retentate).

4. CONCLUSION Nanofiltration using Nanomax 50 membrane appeared to be a good way to isolate an edible fraction of small peptides from goat cheese. It gave a peptidic fraction where a high quantity of mineral salts has been eliminated, and allowed us to obtain easily and quickly the large quantity of peptides necessary for sensory analysis. Though they are present at a high rate, small peptides seem to have no effect on the taste of the cheese and no additive and synergistic effects have been found between those peptides and salts or amino acids. Now that we are able to isolate a relatively pure peptidic fraction, we can think of doing the same study on a larger variety of food such as other cheeses or meat in order to investigate possible roles of small peptides. Current work on cooked cured ham is in progress in our laboratory.

217 Acknowledgments We gratefully acknowledge D. Le Bars (Laboratoire de Biochimie et Structure des Proteines, INRA, Jouy-en-Josas, France) for the amino acids analysis, I. Lesschaeve and S. Issanchou for their assistance in sensory analysis (Laboratoire de Recherches Sur les Aromes, INRA, Dijon, France).

5. REFERENCES 1 2 3 4 5 6 7 8 9

10 11

F. Roudot-Algaron, Lait, 76 (1996) 313. K. Wang, J.A. Maga, P.J. Bechtel, J. Food Sci., 61 (1996) 837. L. Lemieux and R.E. Simard, Lait, 72 (1992) 335. J.W. Aston and L.K. Creamer, N.Z.J. Dairy Sci. TechnoL, 21 (1986) 229. C. Salles, S. Dalmas, C. Septier, S. Issanchou, Y. Noel, P. Etievant, J.L. Le Quere, Lait, 75 (1995) 535. C. Salles, C. Septier, F. Roudot-algaron, A. Guillot, and P.X. Etievant, J. Agric. Food Chem., 43 (1995) 1659. C. Herve, C. Septier , I. Lesschaeve, S. Issanchou, J.L. Le Quere, and C. Salles, Lait, (1997) submitted. A. Garem, J. Leonil, G. Daufm, J.L. Maubois, Lait, 76 (1996) 267. A. Garem, Applications de la nanofiltration au fractionnement des hydrolysats peptidiques. Thesis of doctorate, Ecole Nationale Superieure Agronomique de Rennes, France, 1995. S. Fujimura, S. Kawano, H. Koga, H. Takeda, M. Kadowaki, T. Ishibashi, Anim. Sci. TechnoL, 66 (1994) 43. S. Fujimura, H. Koga, H. Takeda, N. Tone, M. Kadowaki, T. Ishibashi, Anim. Sci. TechnoL, 67 (1996) 423.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

219

Effect of distillation process factors on ouzo flavor examined by sensory evaluation A. Geronti, C. Spiliotis, G.N. Liadakisand C. Tzia National Technical University of Athens, Department of Chemical Engineering, Laboratory of Food Technology, 5 Iroon Polytechniou Str., Polytechnioupoli, Zografou 15780, Athens, Greece

Abstract The effect of the time of distillation for the production of the appropriate fraction and the soaking of the seeds on the alcohol titer, the anethole concentration, and the sensory characteristics of ouzo were studied. A scheme score for ouzo was established. Differences between samples produced using different processing conditions were examined. Conditions that lead to the most pleasant aroma and flavor are proposed.

1. INTRODUCTION Ouzo is a Greek traditional distilled spirit with anise, the production of which is protected by legislation. "Ouzo" is recognized exclusively as a Greek name by Regulation 1576/89 of the European Commission [1]. It is the most popular alcoholic drink consumed in Greece and has economical importance as both a domestic and export product. Annual production of ouzo in Greece totals 22-27x10^ kg, covering 75% of the total production of alcoholic drinks. About 1x10^ kg of ouzo are exported to European countries and 1.3x10^ kg to other countries [2]. While 40% of the produced amounts are in bulk, there is an increasing trend for bottling and consequently a need for standardizing quality characteristics. Ouzo is produced by distillation of the seeds: Illicum verum, Pimpinella anissum, Foeniculum vulgare, Coriandrum sativum and Pistacia Chia lentiscus (P.latifolia) with ethanol of agricultural origin [3,4]. Its production is different from that of other alcoholic drinks with anise; the distillation of aroma and flavor compounds of ouzo is conducted in water/ethanol solution (40-60% v/v), while for other alcoholic drinks the essential oils are added to the water/ethanol solution. Distillation of ouzo must be done in copper stills of 1 m^ [1]. Ethanol used for aroma extraction must be present at 20% of the ouzo alcohol titer; the distillate must contain between 55-80% alcohol. The first fraction of the distillation

220

(alcohol titer 80% vol.), called the "head", contains undesirable, very volatile substances; the intermediate fraction (up to alcohol titer 30% vol.), called the "heart", is the part that should be used in ouzo production; and the last fraction (alcohol titer 6-4% vol.), called the "tail", causes a milky hue due to the desolubilization of anethole. The distillate is mixed with water to fix the alcohol titer and with sucrose to increase the sweetness. Ouzo must have an alcohol content above 37% vol. and a sugar concentration lower than 50 g/L. Flavorants are then added to the distillate, the most common of which is anise seed oil, containing mainly anethole. The latter is insoluble in water and forms an emulsion. Ouzo is allowed to contain distillate at 20-100% [1]. Anise seeds, the dry seeds of Pimpinella anissum, contain 2-8% w/w essential oil with a 85-90% concentration of anethole [5]. The essential oil of the Foeniculum vulgare seeds contains 50% anethole and 20% fenchone, while the oil of the Coriandrum sativum seeds contains 70% d-linalool. Resinous liquid of Pistacia Chia lentiscus contains pinene. Many of the chemical compounds responsible for the characteristic aroma and flavor of ouzo have been identified [6]. Extracted anethole consists mainly of the trans isomer (99%) which has a sweet taste and characteristic odor; cis-anethole is a toxic substance. Addition of natural anethole ("fruit identical") is allowed. It may be purified from terpenes, improving its solubility, flavor and stability. Ouzo is characterized by the special taste and aroma (bouquet) attributed to anethole. Other compounds identified in anise seed oil include: acetone, camphene, acetaldehyde, dipentene, a-phellandrene, y^-pinene, d-fenchane [6]. Consumer acceptance is based on the sensory characteristics of the product which must be evaluated in the ouzo distillery. In the present work the effect of some distillation processing factors such as the time of selection of the intermediate fraction, the soaking of the seeds in water or in alcohol and the addition of anethole on the alcohol titer and on the sensory characteristics of ouzo were studied.

2. MATERIALS AND METHODS 2.1. Experimental procedure The following seeds were used in these experiments: 12 g Pimpinella anissum, 6 g Illicum verum, 2 g Foeniculum vulgare, and 2 g Coriandrum sativum. The fractional distillation is conducted in a glass apparatus with 350 mL ethanol (96% v/v) of agricultural origin and 400 mL water. If soaking of the seeds was done before distillation, they were immersed and kept in 350 mL ethanol or 400 mL water for 3 h and then 400 mL water or 350 mL ethanol were added respectively. The three fractions, head, heart and tail were selected based on the time of distillation. The distillation time was measured from the beginning of the distillation. The time for selection of the intermediate fraction began at: 3, 5, 7, 9 min and finished at: 60, 65, 70, 75 min. The heart fraction was used at 100%, 50% and 20% for ouzo preparation. Sugar at 2.5% w/v of the distillate and the

221 required amounts of water and alcohol with anethole were added so that the final product had an alcohol titer of 40% and a standard concentration of anethole. 2.2. Analytical determinations Alcohol titer was determined after distillation using an alcohol hydrometer at 15°C. Anethole was determined by measurement of the absorption at 259 nm with a Varian BMS 80 UV-VIS spectrophotometer [7]. 2.3. S e n s o r y evaluation Sensory evaluation was carried out by 7 trained assessors using Quantitative Descriptive Profile Analysis (QDP). The assessors scored separately 16 sensory characteristics of appearance, taste, odor and flavor using a 10-point scale. From these values the total score was calculated. Also, a pleasure scoring for the overall acceptance of the samples was carried out using a 5-point scale. Differences between samples were evaluated using differential sensory tests (triangle test, pair). The samples were diluted in transparent glasses 2:1 with water at 5°C. Between tests, tasters were allowed to wait 3 h. The training of assessors was done with commercial samples of ouzo [8,9]. 2.4. S t a t i s t i c a l a n a l y s i s Statistical analysis of chemical and sensory data was carried out using ANOVA at 0.05 significance level. The Duncan test was applied to check the significance of the differences between the values [10]. The results presented are mean values of two trials.

3. RESULTS AND DISCUSSION The quality of ouzo is influenced by various factors. The sensory characteristics are significant for consumer selection and acceptability. The quality of the seeds is a definite factor, especially the concentration of transanethole in the essential oil. The ratio of the various seeds and their ratio to the water/ethanol solution is also important. Therefore the length of time of collection of the intermediate fraction of the distillation, beginning at about 85% vol. alcohol and finishing at about 40-20% vol. alcohol, is critical for the quality, especially the sensory quality of ouzo. The beginning times of the distillation were selected at 2 min intervals, while the finishing times were based on the milky hue of the distillates. This enabled the selection of desired aroma compounds and the removal of unpleasant compounds. Soaldng the seeds in water or ethanol before distillation and using different percentages of distillate in the final product were examined following the industrial practice of ouzo producers, to improve the quality of the product. The effect of the beginning and finishing times of distillation and of the soaking of the seeds on the alcohol titer and on the anethole concentration of the intermediate distillation fraction was examined by ANOVA-3. The mean values

222

of the experimental results are presented in Table 1. The results showed that the beginning time of the distillation had no significant effect on the alcohol titer of ouzo. On the contrary, the finishing time of the distillation, as well as the soaking of the seeds had significant effects on it. The time of 60, 65 or 70 min resulted in a significantly increased alcohol titer. Also, ouzo with significantly higher alcohol titer was produced by seeds without soaking or after soaking in ethanol. The concentration of anethole in ouzo distillates was influenced significantly only by the soaking of the seeds. Soaking in water or no soaking resulted in higher anethole concentrations in the distillates. The mean values for alcohol titer and anethole concentration for the significant processing factors are presented in Table 2. Table 1 Values of alcohol titer and anethole concentration of the intermediate fraction of distillation. Beginning time

Finishing

(min)

(min)

none

water

ethanol

none

water

ethanol

3 5 7 9 3 5 7 9 3 5 7 9 3 5 7 9

75 75 75 75 70 70 70 70 65 65 65 65 60 60 60 60

70.24 76.08 72.92 75.00 74.52 73.08 74.56 74.88 79.68 77.00 76.88 76.07 80.80 78.20 82.64 77.00

74.60 67.40 73.68 67.44 74.76 71.04 73.20 65.84 74.88 76.72 68.80 75.08 76.72 77.36 77.36 75.16

74.60 74.20 71.84 72.92 75.16 73.72 83.60 77.68 76.56 79.28 74.60 73.88 78.96 77.64 73.88 77.64

0.59 0.94 0.84 1.07 0.45 0.88 0.79 0.84 1.66 2.00 1.35 1.15 1.56 1.36 0.78 0.79

1.42 0.96 0.88 0.58 1.49 1.47 1.86 1.17 1.34 0.53 1.42 1.02 1.24 1.03 1.84 0.84

1.03 0.68 0.31 1.40 1.15 0.34 1.22 1.07 0.19 0.33 0.94 1.15 0.33 0.48 0.40 1.84

time

anethole concentration (g/L) alcohol titer (% vol.) soaking medium

By combining the factors that provide increased alcohol titer and higher concentrations of anethole, it can be concluded that soaking is not necessary. This is the most favorable economic solution. The main sensory attributes of ouzo that were isolated, include appearance (turbidity), odor and various characteristics of taste, aroma or flavor. A scheme score of the ouzo was established so that the most significant sensory characteristics of the product were isolated and scored by different coefficients according to their significance to the overall ouzo flavor. The coefficients were

223

estimated based on literature [3] and consumer acceptability data from the marketing department of ouzo industries. The proposed scheme score is: Q sensory = [1.15 X (appearance) + 2.15 x (balanced) + 1.15 x (sweet) + 1.15 X (bitter) - 1.95 x (neutralized) + 1.35 x (aroma of fruits) + 2.15 X (aroma of spices) + 1.95 x (aroma of mastich) + 0.25 x (burned) - 0.95 X (spoilage) + 2.15 x (strong) + 1.95 x (density) - 1.35 x (dry) + 1.55 X (pleasant aftertaste) + 0.95 x (strong aftertaste) + 1.35x(odor)]:16 Table 2 Values of alcohol titer and anethole concentration of the intermediate fraction with different distillation finishing time and soaking of seeds. Finishing time (min) 60 65 70 75 alcohol titer (% vol.) 77.78^ 75.79ab 74.34^^ 72.58^ Soaking medium none water ethanol alcohol titer (% vol.) 76.22^ 73.13^ 76.01^ anethole (g/L) 1.07^ 1.19^ 0.80^ Values in the same row followed by a different superscript are significantly different at P<0.05, according to the Duncan test. The Quantitative Descriptive Profile Diagram used for ouzo scoring is presented in Figure 1.

rkj^« Odor

Total score ..--T--,,^

Balanced

Strong aftertastePleasaint aftertaste/

Aroma of fruits

Density*^

' Aroma of spices Bl"ied

Aroma of ^^gy^h

Figure 1. Quantitative Descriptive Profile Diagram of Ouzo

224

All the samples of ouzo prepared without soaking of the seeds were scored using the above scheme, and the mean values are presented in Table 3. As shown, the score values for ouzo varied between 4.3-6.4. The results of ANOVA-2 showed that there was no significant difference between the samples. Table 3 Results of sensory evaluation of ouzo samples with different beginning and finishing distillation time of intermediate fraction using Quantitative Descriptive Profile (QDP) 10- point score scale and 5-point pleasure score scale. Beginning time (min)

Finishing time (min)

QDP score (10- point scale)

Pleasure score (5-point scale)

3 5 7 9 3 5 7 9 3 5 7 9 3 5 7 9

75 75 75 75 70 70 70 70 65 65 65 65 60 60 60 60

5.55 5.06 5.75 5.83 5.38 5.67 5.54 5.93 5.95 4.85 4.88 4.33 6.36 5.63 5.50 6.24

4.11 3.33 3.56 3.11 3.43 3.43 3.57 3.43 3.71 4.00 4.00 3.29 4.29 3.29 3.43 3.57

Samples of four were examined by sensory evaluation using differential tests with the same beginning but different finishing times of distillation and inversely; no significant difference was found between the samples. Since the examined values that determine the time of distillation of the heart fraction during ouzo production had no significant effect on the sensory characteristics of the product, the selection of the desired fraction for economic purposes may be done between 3-60 min. All the samples prepared without soaking of the seeds were also scored by a pleasure test (Table 3). The results showed that the most pleasant samples were with the finishing time of the intermediate fraction of distillation of 60 or 65 min (pleasure score around 4.0). A Quantitative Descriptive Profile analysis of ouzo samples with different soaking treatment and different contents of distillate (100%, 50%, 20%) was conducted. Two samples of high score were selected for tasting in each case. The mean values are shown in Table 4. No significant difference (ANOVA-2) was

225 found between the samples with different soaking processing or between samples prepared with different percents of distillate. Table 4 Results of sensory evaluation of ouzo samples with different soaking treatment and with different percent of distillate using Quantitative Descriptive Profile (QDP) 10- point score scale and 5-point pleasure score scale. Finishing time (min) 100% distillate

(5DP score Pleasure score (5-point scale) (10 - point scale) soaking medium none

65 60

4.89 4.54

no soaking

65 60

water ethanol 5.38 4.45

5.00 4.80

none

water

ethanol

3.86 3.43

3.29 3.00

2.57 2.57

percent of distillate to the product 100

50

20

100

50

20

5.16 5.58

5.48 5.48

6.06 5.48

3.57 3.29

4.00 3.00

3.90 3.43

The results of the pleasure test showed that significant differences (ANOVA-2) were found among the samples prepared with different soaking treatments and different percents of distillate in the product (Table 4). The tasters preferred samples produced from seeds without soaking and with added anethole when lower percents of distillate were used in the product. The tasting of samples with added anethole showed that the differences in turbidity and odor were perceptible by the tasters, while differences in flavor and aftertaste of the product were not easily perceived.

4. CONCLUSIONS Ouzo is produced by distillation with ethanol of the seeds: Illicum verum, Pimpinella anissum, Foeniculum vulgare, Coriandrum sativum and Pistacia Chia lentiscus (P.latifolia) selecting the distillation fractions between 3 min and 60-65 min. Addition of anethole to the distillate may be done so that the produced ouzo has the best possible aroma and flavor. The results of the sensory evaluation were in good correlation with the analytical data of alcohol titer and anethole concentration. The trained assessors perceived differences in the various sensory characteristics of ouzo, and they preferred the samples produced from distillates with high alcohol titer and high concentrations of anethole.

226 5. R E F E R E N C E S 1. 2. 3. 4.

5. 6. 7. 8. 9. 10.

Regulation 1576/89 of European Commission NSSG, Agricultural Statistics, Athens, Greece, 1995. A. Tsakiris, Alcoholic Drinks, Athens, Greece, 1994. E. Soufleros and A. Bertrand, Etude sur le Tsipouro, Eau-de-Vie de Marc Traditionelle de Grece, Precurseur de F Ouzo, Connaissance de la Vigne et du Vin 21(1987)93. M.B. Embong, D. Hadjiyev, S. Molnar, Can. J. Plant Sci., 57 (1977) 681. M. G. Kontominas, J. Agric. Food Chem. 34 (1986) 847. A. Geronti, Study of ouzo production, Dipl. Thesis, NTUA, Athens, Greece, 1996. G. V. Civille, B. T. Carr, Sensory Evaluation Techniques, 2nd Edition, CRC Press, Inc., 1987. J. R. Piggott, Sensory Analysis of Foods, Elsevier Applied Science, 1988. D.B. Duncan, Biometrics, 11 (1955) 1.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

227

Formation of inosinic acid as the taste compound in the fermentation of Japanese sake K. Fujisawa* and M. Yoshino^ ^Department of Nutrition, Chukyo Women's University, Ohbu, Aichi 474, Japan ^Department of Biochemistry, Aichi Medical University, Nagakute, Aichi 480-11, Japan

Abstract Japanese sake contained markedly higher level of inosinic acid in comparison with white wine. Glutamate was also detected in a considerable amount in Japanese sake but negligible in wine. The effect of fermentation process on the production of inosinic acid and glutamate was analyzed in Japanese sake. Inosinic acid was produced 3 days after fermentation, and its content was increased with sake fermentation time. Increase in glutamate occurred 5 days after fermentation. Baker's yeast with high AMP (adenosine monophosphate) deaminase activity can contribute to the accumulation of inosinic acid which is the taste compound in Japanese sake.

1. INTRODUCTION Fermented beverages and foods contain inosinic acid (IMP) and glutamate as taste compounds, which are produced through the fermentation process by yeast. Inosinic acid is formed from adenine nucleotides by AMP (adenosine monophosphate) deaminase, which acts as a control system of glycolysis in yeast [1, 2]. Stimulation of fermentation is, thus, closely related to the formation of inosinic acid in yeast. In the present study we determined contents of inosinic acid and glutamate in Japanese sake, a traditional fermented alcoholic beverage made from rice. Increased production of inosinic acid with the fermentation process of Japanese sake showed that a high AMP deaminase activity in yeast can contribute to the accumulation of inosinic acid, the taste compound, in Japanese sake.

228 2. MATERIALS AND METHODS Japanese sake and white wine were obtained locally. Japanese sake is classified into Ginjo and Honjozo according to the degree of polishing rice as fermentation materials: Ginjo sake is made from highly polished rice. Rice as a source material for Honjozo sake is not polished below 60%. Inosinic acid was determined after conversion to uric acid. Japanese sake or white wine (0.1 mL) was incubated with 10 jiL of 5'-nucleotidase (2.5 units/ml) at 37°C for 15 min to hyrolyze inosinic acid to inosine. Inosine formed was further converted to uric acid by addition of 10 |j,L of xanthine oxidase and purine nucleoside phosphorylase (1 and 2 units/mL, respectively). Uric acid was determined according to modified Hantsch reaction [3]. Glutamate was enzymatically determined with glutamate dehydrogenase [4].

3. RESULTS Japanese sake fermented alcoholic beverage contained a high level of inosinic acid: however, Ginjo sake made from highly polished rice showed a lower content of inosinic acid than Honjozo sake leavened with less polished rice (Fig. 1). Glutamate, another taste compound, was also determined in Japanese sake; its content was considerably lower than that of inosinic acid (Fig. 2). The concentration of inosinic acid was about 2 times higher than that of glutamate in Japanese sake, suggesting that inosinic acid acts as a principal taste compound in Japanese sake. On the other hand, white wine made from grapevine contained lower and negligible amount of inosinic acid and glutamate, respectively (Fig. 3).

HR

TU

HO

Kl

HA

WA

Brewerys Figure 1. The content of inosinic acid in the Japanese sake

ON

KA

229

H

M GINJO

•

H3NJQ2D

^ _

o E

CJ CO

E CO

1 ^^m ^^H—

^^|-^fl

^Hn

I—I

o HR

TU

HO

KI

HA

ON

WA

KA

KU

Brewerys Figure 2. The content of glutamate in the Japanese sake

Glutamic acid (

Brewerys

Inosinic acid (

fjLmol/mi)

0.1

0.2

/imo\/n£)

0.2

0.1

0.3

Japanese S French D German L M Italian T Figure 3. The content of inosinic acid and glutamate in white wine.

Effect of fermentation time on the production of inosinic acid and glutamate in Japanese sake was analyzed. Inosinic acid was produced 3 days after the initiation of fermentation, and the content was increased with the time of fermentation (Fig. 4A). However, glutamate increased 5 days after fermentation, and glutamate content was only half of the inosinic acid (Fig. 4B). These results suggest that glutamate was synthesized followed by adenylate degradation through the AMP deaminase reaction. Mean values of inosinic acid and glutamate concentrations in Japanese sake and wine are summarized in Table 1.

230

4

5

6

7

8

FermentBtion

9

10

n

12

13

14

15

(days)

Figure 4. Effect of fermentation time on the concentration of inosinic acid (A) and glutamate (B) in the Japanese sake

Table 1 The content of inosinic acid and glutamate in Japanese sake and wine (fimol/ml) Inosinic acid Japanese sake

Honjozo Ginjo

White wine

Glutamate

0.594±0.097 0.396±0.108*

0.299±0.072 0.259±0.074#

0.119±0.054

<0.01

* p<0.001 : significant difference in the inosinic acid concentration between Honjozo and Ginzo sake (Student's t-test) # p, NS : no signifincant difference inthe glutamate concentration between Honjozo and Ginjo sake (Student's t-test)

4. DISCUSSION Baker's yeast has a high AMP deaminase activity [5], and is responsible for the formation of a large amount of inosinic acid, a principal taste compound [1, 2, 5]. Thus, fermented beverages and foodstuffs may be expected to contain a high level of inosinic acid as the taste

231 compound. AMP deaminase can act as a control system of yeast fermentation: ammonium ion produced by AMP deaminase contributes to the activation of phosphofructokinase and pyruvate kinase, resulting in glycolytic stimulation in yeast [1, 2]. Increased accumulation of inosinic acid is, thus, a result of the enhanced fermentation. Japanese sake also contains glutamate as another taste compound, but its content is half of the inosinic acid level. Glutamate is formed by glutamate dehydrogenase catalyzing the reductive amination of 2-oxoglutarate in yeast [6]. AMP deaminase reaction can supply the ammonium ion for the substrate of glutamate dehydrogenase [6, 7]. Because of large Km value of glutamate dehydrogenase for the substrate ammonium ion [6], concentration of ammonium ion produced by AMP deaminase reaction is a rate-limiting step in the glutamate formation. Thus, AMP deaminase reaction is closely related to the formation of glutamate [6, 7]. Glutamate formation is consistent with the decrease in citrate in yeast cells [7]. Citrate is a potent and physiological inhibitor of phosphofructokinase, the regulatory enzyme in glycolysis [8]. Thus, increased ammonium ion by enhanced AMP deaminase reaction causes an increase in glutamate with a decrease in citrate, resulting in glycolytic stimulation [6]. AMP deaminase with a higher activity in yeast contributes to the stimulation of fermentation of Japanese sake, and further to accumulation of inosinic acid and glutamate, which may act as principal taste compounds.

5. REFERENCES 1. M. Yoshino and K. Murakami, J. Biol. Chem. 257 (1982) 2822. 2. M. Yoshino and K. Murakami, J. Biol. Chem. 260 (1985) 4729. 3. N. Kageyama, Clin. Chim. Acta 31 (1971) 421. 4. E. Bernt and H. U. Bergmeyer, Methods Enzymatic Anal. (Bergmeyer, H. U., ed), 2nd English Edn, 1704, Academic Press, New York, 1974. 5. M. Yoshino, K. Murakami and K. Tsushima, Biochim. Biophys. Acta 570 (1979), 157. 6. M. Yoshino and K. Murakami, Biochim. Biophys. Acta 706 (1982) 111. 7. M. Yoshino and K. Murakami, Int. J. Biochem. 25 (1993) 1723. 8. M. Yoshino and K. Murakami, J. Biol. Chem. 257 (1982) 10644.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

233

Volatile composition of southern European dry-cured hams p. Dirinck and F. Van Opstaele

Chemical and Biochemical Research Centre, Cathohc Technical University St.-Lieven, Gent, Belgium

Abstract Flavor is an essential parameter for consumer appreciation of meat products. Southern European countries (France, Italy, Spain) produce high quahty hams by a dry-curing process, followed by long ripening and drying periods (9 months or more). These hams impart an "aged" flavor and can be differentiated from the short-ripened (3 months) brine cured products, which are produced in northern Europe. In this study the aroma conq)ounds of several ItaHan and Spanish dry-cured hams were isolated by simultaneous steam distillation-extraction (Likens-Nickerson, SDE). About 60 volatile compounds were identified and quantified by gas chromatography-mass spectrometry: aldehydes, alcohols, ketones, lactones, sulfiir compounds and esters. It was shown that flavor formation in dry-cured hams is related to hpolysis-oxidation of fat and to proteolysis and amino acid degradation. Principal component analysis was used for visuaHsation of the complex data matrix and for classification of hams in relation to their volatile conq)osition.

1. INTRODUCTION The flavor of meat products is an irq)ortant quahty parameter and a major contributor to consumer preference. In the case of dry-cured ham it is the result of both enzymatic and nonenzymatic reactions and degradations of macromolecules (triacylglycerols, fatty acids, proteins) in the tissues of the hams during the drying and ripening periods. In addition to those pathways, secondary metaboHsms of microorganisms are involved in the development of volatile aroma corrq)onents (1). During the last few years, several research centers studied flavor formation in dry-cured hams. In France, the group of Berdague isolated the volatile fraction of French dry-cured hams by means of vacuum distillation (2) and investigated the influence of pig crossbreed on the composition, the volatile con^oimd content and the sensory quahty of dry-cured ham (3).

234

They found that the volatile fraction consisted of con^onents derived from sugars, lipids and proteins of the meat, from the pig feed and from technological processes. Buscailhon et al. (4, 5) studied the changes in intramuscular Hpids during the processing of French dry-cured hams and concluded that the rancid flavor character was mainly detenrdned by lipid oxidation products. Italian dry-cured hams were investigated by ItaHan and Danish workers. Barbieri et al (6) isolated the flavor confounds of dry-cured Parma hams by means of a dynamic headspace technique. According to this group a whole series of esters was a typical feature of Parma ham in comparison with Iberian and French hams, which had higher amounts of aldehydes and alcohols in their volatile patterns. Also Careri et al. (7) noted the importance of esters for the overall aroma of Parma hams. Danish workers (1) studied the role of microorganisms in Parma hams and pointed to the influence of these organisms on the development of flavor. Their conclusion was that many in^ortant volatile confounds (methyl-branched aldehydes, secondary alcohols, methyl ketones, ethyl esters, dimethyl trisulfide) were the result of secondary metaboUsm (amino acid cataboHsm) of microorganisms. Hinrichsen et al. (8) also studied the formation of peptides in Itahan dry-cured ham during processing. Interesting work on Iberian hams was performed by Spanish researchers. Lopez et al. (9) identified the volatile compounds of dry hams from Iberian pigs, which were produced using an extensive feeding regime based on acorns and grass, while Garcia-Regueiro and Diaz (10) made a comparison between the volatile fraction of hams produced from heavy and light Large White pigs. Aged dry-cured hams were characterised by a higher amount of ketones, alcohols and sulfiir conq)onents (11) and Enterobactehaceae, Micrococcaceae and lactic bacteria were the microorganisms involved in the spoilage of hams (12). Further, Hpid oxidative changes in the processing of Iberian pig hams were followed by Antequera et al. (13). These workers also made differentiation between Iberian hams from Iberian and Iberian X Duroc pigs by analysis of volatile aldehydes (14). Dirinck et al. (15) studied the influence of northern (brine curing, 3-4 months of ripening) and southern European (dry-curing, 9-24 months of ripening) production techniques on the volatile composition of hams. By means of a dynamic headspace technique and a simultaneous steam distillation-extraction procedure, it was shown that the southern European dry-cured hams (Serrano, Parma) had a more intense pattern of a whole series of volatiles related to fat oxidation and amino acid degradation in comparison with the northern European brine-cured hams. The objective of this study was to determine the differences between volatile fractions of several ItaHan (Parma) and Spanish (Serrano, Iberico) dry-cured hams. Both Italian Parma and Spanish Serrano hams irrq)arted an "aged" flavor, but the Serrano hams were characterised by a higher rancid note, while Parma hams were described as more fiiiity. The Spanish Iberico ham was a longripenedhigh quahty product produced from heavy Iberian pigs, fed on acorns and pasture. The isolation procedure used in this study was a simultaneous steam distillation-extraction (Likens-Nickerson extraction, SDE). As shown in an earher work (15), in comparison with dynamic headspace, SDE resulted in a more con^lete picture of different volatiles contributing to ham flavor and reUable and reproducible semi-quantitative data could be obtained with this technique.

235 2. MATERIALS AND METHODS

2.1. Materials and sample preparation Four commercial Serrano hams were obtained from two different Spanish cortq)anies. The two hams from the first corr^any were manufactured by conventional Spanish technology (Serrano 3 and 4), as described by Toldra (16), and ripened for nine months. We were informed that in the company which produced Serrano 1 and 2, higher ten^eratures than conventional were used during the final stage of ripening. The fifth Spanish dry-cured ham in this study was a long-ripened (18-24 months) Iberico ham, produced from heavy Iberian pigs with a feeding system based on acorns and grass. Five Itahan dry-cured hams were used in this study. Four of them were produced according the salting, drying and aging parameters for the production of Parma hams (12 months of ripening). The fifth Itahan sartq)le was a commercial Parma labelled ham which was purchased locally. On arrival at the lab the surrounding fat of the hams was removed and each ham was cut into cubes of about 1 cm^. The cubes were mixed thoroughly in order to obtain representative samples for analyses. Portions of 100 g were weighted and placed in plastic bags in a freezer at -20°C, where they were kept until the day of extraction.

2.2. Isolation of volatUes by Likens-Nickerson extraction A 'total volatile' isolation technique based on a simultaneous steam distillation-extraction (Likens-Nickerson, SDE) was used to isolate the volatile fraction of the hams. Frozen ham cubes (100 g) were ground in a domestic blender and suspended in 600 mL of distilled water in a 1 L round bottom flask. Dichloromethane (50 mL) and 15 |Lig of dodecane as internal standard were placed in a 100 mL flask. The solvent return loop was filled with 10 mL of dichloromethane and the flask with the suspended ham and the flask containing the solvent were attached to the Likens-Nickerson apparatus and heated to boiling. Extraction was performed for 4 hours. After this reflux time the dichloromethane fractions of the solvent flask and the return loop were collected and the extract was concentrated to a final volume of 200 |iL by means of Kudema Danish evaporation. The concentrated extracts were stored in the freezer at -20°C.

2.3. Gas chromatography - mass spectrometry The extract (1 ^L) was injected into a HP 5890 gas chromatograph coupled to a HP 5971 A MSD mass spectrometer (Hewlett-Packard, USA). The volatiles were separated by means of a capillary fiised sihca column (HP PONA cross-hnked methyl sihcone, 50 m x 0.2 mm x 0.5 fxm). Hehum was used as a carrier gas with a flow rate of ImL/min. The oven temperature was initially held at 40°C for 5 nun and subsequently programmed at a rate of 5°C min'l to 250°C. This final temperature was maintained for 13 min. The temperatures of the injection port and the detector interface were 250 and 280°C, respectively. A spht injection with a ratio of 1:5 was used. The chromatograms were obtained by monitoring the total ion current in the 40-260 mass range with a solvent delay of 6.8 min. Kovats indices and comparison of the obtained spectra with spectra of the NBS49K Hbrary and of a personal library allowed the identification of volatiles. Semi-quantitative determinations of volatiles

236 were obtained by relating the peak areas of volatiles to the peak area of dodecane as internal standard and e?q)ressed as ng g'^of ham. Mean values were calculated from tripHcate analyses.

2.6. Statistical analyses To visualize the complex data matrix and the relationship between different dry-cured hams and their volatile composition, a principal con^onent analysis was performed using Unscrambler 6.1 (Camo, Norway) statistical software.

3. RESULTS AND DISCUSSION

3.1. Volatile composition of dry-cured hams As an illustration in Figure 1 a typical GC-MS profile of the Likens-Nickerson extracts of an Iberico ham is compared to the profile of a Parma ham. Peak numbering is in accordance with the peak numbering in Table 1, which presents the semi-quantitative data (mean of triplicate analyses, dodecane as internal standard) of all hams in this study. About 60 conq)onents were identified in the Likens-Nickerson extracts of different southern European dry-cured hams. These volatile aroma compounds could be classified into several chemical groups : saturated, unsaturated and branched aldehydes, ketones, aromatic compounds, alcohols, esters, acids, pyrazines, lactones, sulfiu* compounds, fiirans, and hydrocarbons. Because of their relatively high threshold values, hydrocarbons probably made no significant contribution to ham flavor. In Figure 2 mean values of the sums of different chemical classes occuring in Serrano, Parma and Iberico hams are presented. The volatiles with the highest concentration in the extracts were aldehydes, which from a biochemical point of view could be classified as originating from hpid oxidation or from amino acid degradation (6). Saturated (pentanal, hexanal, heptanal, octanal, nonanal and decanal), unsaturated (2-pentenal, 2-hexenal, 2-heptenal, 2-octenal, 2-nonenal, 2-decenal, 2undecenal) and polyunsaturated aldehydes (2,4-nonadienal and the isomeric decadienals) could be related to autoxidation of unsaturated fatty acids. The highest levels of saturated aldehydes were found in the Serrano hams, while the Parma ham had comparable levels as the studied Iberico ham. However an obvious difference between the 3 types of ham was observed in the group of the unsaturated aldehydes, which had a higher level in the Spanish hams compared to the Itahan Parma hams. Furthermore the level of 2-nonenal, 2,4nonadienal, 2-undecenal and the isomeric decadienals m the Iberico ham, was twice the level found in the Serrano hams. Because these unsaturated aldehydes are very potent aroma compounds they could play a major role in the important rancid/oxidized note reminiscent of Iberico hams. The identified branched aldehydes (2- and 3-methylbutanal) and the aromatic phenylacetaldehyde were the result of oxidative deamination-decarboxylation of ammo acids or were formed by microorganisms and could be related to proteolysis (1, 6). Examination of the semi-quantitative data showed that proteolysis was more intense for the Serrano hams. This was especially due to the higher level of phenylacetaldehyde in this ham in comparison with the Parma hams. However, the level of the branched aldehydes was comparable for the Serrano and the Parma and was lower for the Iberico ham.

237 Figure 1. GC-MS analysis of the Likens-Nickerson extracts of a Spanish Iberico and an Italian Parma ham (peak numbering in accordance with table 1)

IBERICO

Abundance

29

17

31 32

34 36

bo

5000000

24

25

4000000

-1000000 -2000000 -3000000 H -4000000

PARMA

-5000000 8.00

10.00 12.OO 14.OO 16.OO 18.OO 2 0 . 0 0 22.OO 24.OO 26.OO 28.OO

^

Abundeince

43 44

47IBERICO

m

5000000 4000000

46

40 3000000

1

1 VU ^ 41

2000000 1000000

45

39 1 1

m

^nffi -1000000

49

51

^53

48

uWiMwu

m

r>r^||f

-2000000 -3000000 -4000000 A

PARMA 30.00

32.00

34.00

36.00

38.00

40.00

42.00

44.00

46.00

238

CD O


> c (0 0) (0

E (0

x: o

•D

< 0^

o o

<^ Ul m

CN CD

CO ^

o

<^ ^ T - CM

'*" T^ ^

O)

CN O )

?^^°i

•

CM CM

CO ^. -rT-

' « ^ C M h - l ^ ' ^ ^ ( 3 > ( O r - C N ^ .

"^

r^cNTfr-:f3JOcDr-:^^J^2

5 ^ § § § 5 r^-^ S 5^ j ; ;

<^ i n T t CO T-^

( Q CJ) CD CD ' r -

' «3 ^ ^ S S

^ TT o

° CN 2 : : S ?

oi ^

^ ^

"^ "^

r^

T-

^

C N CO

1^ S -* m

^

in T - h>CO C O " ' . C3) ""^ " ' . ^" ^ OCJ ' ^ CM CD O CN 0 0 CO CN 'TCO d 5 ! CNJ § g CN CD ^ «J CJ) r^ ; ; ! CM ^ ! ^ O) CO CO CN ^

^ P CD c r CO 0 0 CN CO L: ^ c: i n CM 5

r^ o CD ' ^ - ^ r^ ^ CN

^

CO

^ h CN r^ CO CO CD V h - CO CN _ P 3 >r rO i ft-; h - C N h - > ^ ± ; c : > l ^ , o O O i n ' * o ^ ^ C O i O C N i J i r ^ C O Or-J i ^ b •l O) i C O l O C N ' r - 2 j ; j T - T - ^ T - « ' ( O g ' « ^ T - T - T - ' < t ; ^ J ; : ' * ' * C D ' " ^ C J )

O ^ C N C O C J ) * ^ .

CN i n

O CJ) ^ . C O C DCO ^. ^. -t CO CO CO d CD ai ZZ r^ o6 (6 _ - . c s i T - ^ c j > i n T - ^ c N h ^ i n ' r - ^ i 2 CN CN ^ h - T - h - T - O O C N ' ^ - C O O O CO CN CM ! ^ § -

-;

. n ° ' ^ ^ J : ; f i : S!?.^ ro g 3en^TT S «5

>-co

CD C

o c

'T ^

c

CO CO C C CO CO0 - 3 "3 T rC JD X i o

- j^ - B c s^

«^

CO CL O Q. I CD CN C ^

-

^

0) ^c 2b p>. "o S ^ "S « ^ 0

.2 3 -9 -^ ¥ ^ 9in

CO h -

CM T - CO CM CN CM CO - ^

TJ ^ •«-

**l

CO

•

(D C

*

-

•

CO

o c

u JQ I VN »CN C

I

CD

o c I

I

CM CO ^ r

I

O c __ iS CO ^ 0) CO Q. in

>%

I

T-

CD

-fi >% i = "S CD c 5= -C CD g - !r E o

Ci. CO CN O)

Q . CO CN ^

o

-^-r^^ricDS^^

CM O CD ° ° ° ° r J c-> f r i f ^ ^

^

—

2

CM

CO

E

c o

^

C

I

o in

^. ^ ^ ^ CO P ^ ' ^ fo ' ^ d ^ '-'

-_

I

Q

C

CD TJ — >^ O

CD

CO -»^ C CD 2 X Q . CO
CM

CN

CM

S^

9^ ro I (D CN CN CM x : CN X i T in CO h-

-c x: Sr I

+ ou i^

CO

^

Its

S2?:l?:^::°SE;i;S2?^^?oSgj^2SScN5?S!;=^?S5

CO i n ""^f " ^ CO ^ r - <». ^ . ° P CN P ^ . ^ CO ^o "^ , ^ r p ; f r ; r J T - : O ^ ^ i n O ' ^ 0 0 ^ - 0 Q ' ^ 0 0 ' ^ l ^ i r C L n c o C D C N C D C N t n 0 0 ^ C D

o ^

2cN°r2::!:::j:52^!2Sg32

^ ' ^ " ^ ^ C N ' T ^ . ^ r ^ c j ) c o c j ) ^ ^ ^ !;:: h^ 0 0 S S f e o i c N g g o ) " ^ C D o 6 c D § - ; r ^ I I T - CN

CJ) O - ^ O i n T- o TT o CJ) CO h-' T-^ C ^ « ^ O ^ S O « O C N " ' ^ C J ) C D C O C N C O ^ C D S C O O ^ S ' O S C M ^ ^ O O SCD U ^d ^ c N O ) g 5 g T - ^ 2 2 i n c o c N i n c N ^ o ^ c N c o ^ 2 2 c N ^ ' ^ S ^ c o ! 2

^

^ CO h CD CO CJ) CO CN O CD ^ CJ) ^ i n CJ) i n c o T - ^ - T T - T - ^ ' i ^ ) CD \.^ cy^ vij CO CO h"- fY-i v^' r n ^ i - ^ * \jf \^ ffs csi T- ^ c o ^ ^ c ^ O i i n ^ T - c o ^ C N C O T - ^ T - ^ C N C N

CJ) CJ) i n o ^ in CJ) CN CN CO ^ r^ i n CO cxj T - " ^ op CO T-^ CJ) CO CD CN CD c D ^ i n o o ^ c ^ - ^ c N O ^ ^ i ^ T j - ^ J ^ ^ S i ^ ^ S c O o o ^ CD CJ) ' r - C J ) ' « ^ ' * 2 ^ ^ ^ 0 0 < O ^ ^ ' r - ^ S - T ^ ' ^ 2 ^ C 0 C g

T - h - CM CO -r- i n ^ C O O C J ^ C N l ^ O i O T f ^ f t ^ T - c O C O r - T - C D ^ C D i O C N f 5 c O ^ ^

T^ 2 CD h-- 4_

CN CJ) i n i^. P

(0

C (0

c 00

S ^ °°" 5 S ?

(Q CL CO

c g Q.

o E

8 o

>

(D

o E O)

iS ? T3

2-

> ^ 2 (0

.J.

• <^ - Q CO CO C \ - CO

(n

E (0 ^ a>

•D

3 O

i_

i

CD

-o c

5 "O


c en x: "c (0 Q.

O

CO c o o

E 0)

o o

E (D x:

OT

o

*•—

en

0)

•o

c

^ ^

"cD

>

o <0 (0

(U

0 JI

"D (D


i5

0)

(/)% >

lO

< ^ Q. < 3 Q. <

Q^

s

< <

S Q^ 0.

< Q^

S

•^

'^. T -

T

r^ • Q C D I O P ^ ^ .

OO CO -«-

^

T - CJ) CM CO O

CMCOP

cr o ^. _. ^ ^ ^ ^ ^ _jv3 c: o CM CM JJ5 5 S OO i n

r^ CO o

OO C O T-^ CO " ^ CM

T- If)

#NS

239

00 CM o in CO CO lO CM

rt-i ^sr '^a^ y j

r2 ^ i;^ 12

r*^ « ^ w-* ^ •*

c^fvj^ininco

^^^

5 2 (S J^ !5 j;^ 5

. o 00 2 ^ ^ P °9 od d K '^ '^ ^ '^

f F j i O ^ ^ l O ^ l O T - C M C M ' ^ ' ^ C O

CM y ^ CM O ) ^

r ^ O O O O T j - C M - r - C M C O C M f ^ C D O O ^ ^

'r-cocDc:)iomcoif)'^oo^'^°9 TrcOT^r^'«i^dcoo)55'^oS2

CM

If) O 00 T- lO rri 1^ '^ in CM '^^ CM i?. lO -^ CD CO

iq 00 CO -^ o) Q5 o> 00 r^ -^ u:) CO CO h-; CO jQ Q

. in OC) CM

O CM

^ ^ -

i n t o- * 00 CJ) o r^ i n (3) 'CD ^ in • ^-l q Q D - q - q - - - ^ c D g - o c 7 ) CM T— r^. 3 ^ 0 O ^ ^ U ^ T - - * - ' ' ^ i f ) c M O ) c c : c S S 2 CM g 5 ; i .° 5 2 ° o ^ ^

ooiJ^SJin^cMS^cMO)^^

O) O ) r ^ / v i 00 d cj) \jf «-»' ^^' ^ __ i *n^ ^ - _ CO vv* d ^-^ *—» V-" ^

T-r^

d

CO OO T- CJ)CO O)

IJ:!^cMlJ:coinT-T-T-^cooocMr^ooo)r^cMCM

00 If) Tf r^

00

cr o

c J

T—•

O CM

S J i d c D S ^ i ^ i ^ o o c j ) o i « r ; c o J ^ T r ^do c M C M o i o d ( d i r ) ^

O " ^ T - CM 00 G) CO -^ ^ CX> ' ^ 0 0 CM T- CO ^ r^ " ^ 0 0 CM CM CM CM CM ^ CM

^ ^ . 1 0 ^ 0 ) ^ 0 0 ) 0 )

^

J^JjcogJcO^CMCMCO

T -

-: P o CJ)

O CJ) [ ^ C D CO CO ^ ^ 1 -

Q^

0.

i< oocor^cMcocM

CO TT

(N CO

lO

^

CD CM

CO

''J-

'^ CM" o c

CO CO

CO

: C ^ D CM

CO

SECM

CO

Cfo

CO

• (O uo OO z ^ fQ- ix^ CO —.

C3)

O

2 o^ ^ '^•

^ ^ C O C M ^ ' ^ T - C O

d o I^SCOCM^S-^CDO

T5

(U

c 0 CQ C

o

- t

o

CM" O ) CM

">> c ^ 4 CO

0

c o

c

Jii CD "G 9> 0 i5 CO

si ^1

o

CM " 0

1

0 O CO O 0 •C D CO

ii

CO

T-:

0

T-:

T3

"O)

1

0 Q.

CM

5 "O)

_

O CD

P -r^

0

O) O

-c S ^ :£ 9 -G

0 -o o

CO o E X 0 CO

'2 ^ ?

CO

"co o c

Tl- 0 0 O CO CO

•D

O CO O O

x:

CO c o CO o 0 T3 0 CO T3 CO X X 0 0

^

CM T -

H . rv. o) P o D- o X-: r--^ O CM °° d O O CJ)

CD O

O" OO T- O CM

CD CO T f m

- ^

00 T - CO CM '^

0 T3 O

•D

O C c 0 •D o CO CO O CO c O o CO CO 0 ! 5 CO o CO O c O CO o c T3 c o c CO O c 0 CO o c CO T3 o 2 0 CO o 0 iS 0 " 0 o T3 •D

T-^ d c oO ^ ^ C M O gCM l ^r^T ^- O ^)

CO

CD 0 o C c (Q CD (Q CD O x: O +-• C C CD (0 o CU c c cn c ' C CO >s
CJ) CO T f CD

CD •D >s JC

CQ

8

,CO

c O 0) o 1 Q . CM

T—

S [- «P C3) CM t o

CO i n m in

d d

m CD . CD • 0 ) ^ c o i n^pT-r^ 00 CO ^ d d cr lo CO T q" cr . c o c D d c o J J ^ c o d d c f ^ S j o g S ^ c o - J '^ i ^iridS- a- j -^ ^ S"^g^^ --• S- •

m CO • OO CO -^t CO CO Tj- CO C CO

CO - ^ T - CO T - CM

coinT-cj)0'^ c c c

l O h - i u O C O C D ' r ^ ^ P ' ^

o S9

O 0^ Ul

UI (0

CO Ul (0

(£.

^ Ul

UJ

o >

3

Ul -J 1-

s o

Q.

D O

z

Q

(0

•I> "e^ n"- d '-H3

C CO (Q O" C (U

E -2

(D

E,

(U 0)

c

>

0) D (0

o

M -

' l _

Q.

o CO 15

^^ •D 0) 3 _C

c o o^ 0) J3

CO 0 O ) TD

240

High molecular weight aldehydes were also detected but their contribution to the flavor may be minor because of their low volatihty. These aldehydes were also found in raw meat (17) and they could be important as precursor in the formation of volatile alkanals and alkenals. Methylketones (2-butanone, 2-pentanone, 2-heptanone, 2-octanone, 2-decanone, 2-pentadecanone), li-oxidation products of fatty acids, and polyfunctional ketones (diacetyl, 3hydroxy-2-butanone, 2,3-pentanedione, 2,3-octanedione) were identified in the di&erent drycured hams. The highest amounts of ketones were detected in the Serrano hams, while the concentration of these confounds was rather low in the Iberico. The methylketones have a moderate aroma strength and give fiiiity, spicy, fatty tones to the flavor. Diacetyl and 3hydroxy-2-butanone are components with a strong buttery smell. The group of esters (ethyl octanoate, ethyl decanoate, ethyl tetradecanoate and ethyl hexadecanoate) was in^ortant for the classification of the hams. Ethyl octanoate was only identified in the Parma hams, while ethyl decanoate, ethyl tetradecanoate and ethyl hexadecanoate were not found in Serrano 1 and 2. Because Likens-Nickerson extraction was used for isolation of the volatiles no low molecular weight ethyl esters were detected in this study (15). According to Hinrichsen et al.(\) ethyl esters are formed enzymatically in the final stage of ripening by combining ethanol and acids. Therefore, microorganisms appear to play an important role in their formation. The y-lactones, products of dehydration and cycHsation of the y-hydroxyacids, are potent aroma compounds and their level was twice as high in the Iberico ham con]5)ared to the Serrano and Parma hams. These compounds contribute to fatty, creamy and coconut-like odors. Only 2 sulfixr containing con:^ounds, methional and 1,2,4-trithiolane, were isolated and identified among which 1,2,4-trithiolane was probably a conq)onent formed during the extraction procedure. In our previous study we demonstrated that, besides low molecular weight esters, dynamic headspace isolation also allowed to detect higher amounts of sulfur compounds (15). Rehable quantitative data of methional, which is known to be a very potent aroma component, could not be obtained as it was not separated from 2-heptanone. So it was not possible to conclude whether sulfixr-containing compounds played a role ia the diflferentiation of Serrano, Iberico and Parma hams. Acids of high molecular weight (decanoic acid, dodecanoic acid, tetradecanoic acid and hexadecanoic acid) were detected but because of lower volatihty then contribution to the ham flavor might be of less in^ortance.

3.2. Principal component analysis In order to visualize the con^lex data matrix in Table 1, a principal component analysis was performed on the semi-quantitative data, with the 10 hams as objects and all 59 volatile compounds as variables. However, instead of the absolute values in Table 1, procentage values were calculated and used for statistical analyses. Figure 3 shows the results in a 2dimensional scatter plot with objects and variables presented in the same plane. A plot of PCI vs. PC2 showed that hams were clustered in different quadrants. So the used analysis procedures based on Likens-Nickerson and gas chromatography for determination of semiquantitative data allowed to obtain a clear classification of the studied hams from different southern European origin.

241

Figure 2. Comparison of the volatile composition of Serrano,Parma and Iberico hams. Mean values for the sum of : 1. Alkanals C5-C9 ; 2. Alkenals C5-C11 3. 2,4-nonadienal, isomeric 2,4-decadienals ; 4. Methylketones C6-C7, 3-hydroxy-2-butanone, 2,3-pentanedione ; 5. gamma-lactones C8-C9 ; 6. 2-pentylfuran, 1-octen-3-ol; 7. 2-methylbutanal, 3-methylbutanal, phenylacetaldehyde

3500 3000 2500 D SERRANO •

PARMA

•

IBERICO

Figure 3. Principal component analysis of the volatile composition of dry-cured Southern European hams 1.0H

Bi-plot

PC2

2-phenylethanol gamma-nonalactone 1-hexanol ethyl decanoate 3-methy(l-butanol ethyl hexadecanoate gamma-octalactone PARMA nonanal 2-decenal ethyl octanoate benzaldehyde octanal , ethyl tetradecanopte

0.5

IBERICO

2-heptanone+methional

~ \RMA PARMA 3-methylbutanai

1-octen-3-ol 2-heptenal Pi|\RMA 1.2,4-tnthiolane 2-nonenal

ciecanoic acid hexadecanal heptanal

2-pentylfuran

2-undecenal heptane ^^^^^ 2,4-nonadienal c.t-2.4-decadienal

3-hydroxy-2-butanone diacetyl

°?hexenaf' 2-octenal "nvr^.inP tetradecanoic acid pyrazine furfl[iryl alcohol 1-pentanpl dodecanoic acid tetradecanal ph( nylacetaldehyde t,t-2,4-decadienal SERRANO pentanal hexadecanoic acid 2-pentinone 2-methylbutanal 1-hydroxy-2-propanone 2,3-pentanedi(| 2-pentadecanone hexanal 2-ethylfuran S E R R A N O

-0.5

$ERRANO SERRANO

-1.0-

PC1 -0.5

0.5

1.0

242

By presenting the objects and variables in the same plane it was possible to show which volatiles occured in a relatively greater concentration in different types of ham. The higher amount of oxidation products observed in the Spanish hams compared to Parma hams could be attributed to the use of higher ten^eratures enq)loyed during ripemng of Serrano hams and thus explain the higher rancidity note in the former products. A whole series of unsaturated aldehydes were dominant in the volatile pattern of the Iberico ham This was probably due to the feeding regime, based on acorns and pasture, which may result in a high degree of unsaturation of the fat. Because unsaturated aldehydes are known to be responsible for rancid odors, the volatile con^osition of the Iberico ham could explain the even highei' rancidity note of the Iberico ham compared to the Spanish Serrano hams. Although ethyl esters were detected in both the Iberico and 2 Serrano hams, these compounds had the highest relative importance in the Itahan Parma hams. Because of the lower amount of oxidation products in the Parma hams, esters could play a major role in the overall aroma and may be responsible for a morefinity-floweryodor character.

3.3. Conclusions Determination of the volatile compositions of hams, according to the described analysis technology (SDE-extraction, GC-MS identification and quantification of volatiles followed by principal component analysis) provides a better understanding of a) the biochemical pathways influencing flavor formation in dry-cured hams; b) determination of the volatiles responsible for the differences in flavor character of southern European hams from different origin and c) the influence of feeding systems and processing technology on ham flavor. These techniques could also be used for assessing the authenticity of products from different origin and for studying all kinds of parameters influencing flavor formation m hams, such as basic materials, curing technology andripeningconditions.

4. ACKNOWLEDGEMENTS The 'Vlaams instituut voor de bevordering van het wetenschappeHjk-technologisch onderzoek in de industrie (IWT)' is thanked forfinancialsupporting this investigation.

5. REFERENCES 1 L. Hinrichsen and S.B. Pedersen. Relationship amongflavor,volatile compounds, chemical changes and microflora in Itahan-type dry-cured ham processing. J. Agric. Food Chem, 43 (1995) 2932-2940. 2 J.L. Berdague, C. Denoyer, J. Le Quere and E. Semon. Volatile compounds of dry-cured ham J. Agric. Food Chem, 39 (1991) 1257-1261. 3 J.L. Berdague, N. Bonnaud, S. Rousset and C. Touraille. Influence of pig crossbreed on the composition, volatile confound content and the flavour of dry-cured ham. Meat Sci., 34(1993)119-129.

243

4

S. Buscailhon, J.L. Berdague and G. Monin. Time related changes in volatile composition of lean tissue during processing of French dry-cured ham. J. Sci. Food Agric, 63(1993)69-75. 5 S. Buscailhon, J.L. Berdague, J. Bousset, M. Comet, G. Gandemer, C. Touraille and G Monin. Relations between conq)ositional traits and sensory quahties of French dry-cured ham. Meat Sci., 37 (1994) 229-243. 6 G. Barbieri, L. Bolzoni, G Parolari, R.Virgih, R. Buttini, R, M. Careri and A. Mangia. Flavour compounds of dry-cured ham J. Agric. Food Chem, 40 (1992) 2389-2394. 7 M. Careri, A. Mangia, G Babieri, L. Bolzoni, R Virgih, R and G Parolari. Sensory property relationships to chemical data of Itahan dry-cured ham. J. Food Sci., 58 (1993) 968-972G. . 8 L. Hinrichsen, J.H. Miller and T. Jacobsen. Formation of peptides in Itahan dry-cured ham during processmg. 42th ICoMST, Meat for the consumer, L-14, 1996. 9 M. Lopez, L. De La Hoz, M. Cambero, E. Gallardo, G Reglero and J. Ordonez. Volatile compounds of dry hams from Iberian pigs. Meat Sci., 31 (1992) 267-277. 10 J. A. Garcia-Regueiro and I. Diaz. Volatile confounds in dry-cured ham produced from heavy and hght Large White pigs. 40the ICoMST, The Hague, The Netherlands, SVIA.09, 1994. 11 M.C. Vidal-Aragon, E. Sabio, C. Sanabria, A. Fallola and M. Elhas. Volatile confounds identified in altered dry-cured ham. 40th ICoMST, The Hague, The Netherlands, 1994. 12 C. Sanabria, A. Fallola, E. Sabio, M.C. Vidal-Aragon, A. Carrascosa and J.L. Ferrera. Microbial population in altered dry-cured ham. 40th ICoMST, The Hague, The Netherlands, S-VLV.01/1, 1994. 13 T. Antequera, C.J. Lopez-Bote, J.J. Cordoba, M.A. Garcia, M.A Asencio, J. Ventanas, J. A. Garcia-Regueiro and I. Diaz. Lipid oxidative changes in the processing of Iberian pig hams. Food Chem, 45 (1992) 105-110. 14 T. Antequera, L. Martin, L., J. Ruiz, R Cava, L. Timon and J. Ventanas. Differentiation of Iberian hams from Iberian and Iberian x Duroc pigs by analysis of volatile aldehydes. 42th ICoMST, Meat for the consumer, L-16, 1996. 15 P. Dhinck, A. De Winne, M. Casteels and M. Frigg. (1996). Studies on vitamin E and meat quahty. 1. Effect of feeding high vitamin E levels on time-related pork quahty. J. Agric. Food Chem, 44 (1996) 65-68. 16 F. Toldra. The enzymology of dry-curing of meat products. In New technologies for meat and meat products, eds. F.J. M. Smulders, F. Toldra &M. Prieto, ECCEAMST, Audet Tijdschriften B.V., pp. 209-231, 1992. 17 P. Dirinck, F. Van Opstaele and F. Vandendriessche. Flavor differences between northern and southern European cured hams. Food Chemistry, 59 (1997) 511-521.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

245

Role of sodium nitrite on phospholipid composition of cooked cured ham. Relation to its flavor A. S. Guillard', I. Goubet^ C. Salles^ J. L. Le Quere*" and J. L. Vendeuvre' * Centre Technique de la Salaison, de la Charcuterie et des Conserves de Viandes, 7 av. du general de Gaulle, 94700 Maisons-Alfort, France. ^ Laboratoire de Recherches sur les Aromes, INRA, 17 rue Sully 21034 Dijon Cedex, France.

Abstract The role of sodium nitrite on phospholipid composition was studied during the processing of cooked cured ham. Evolution of the different classes of phosphoUpids in raw meat, cured meat with brine injected at 0, 50 and 100 mg of sodium nitrite/kg meat, and cooked meat, was determined as well as fatty acid content of phosphatidyl choline and phosphatidyl ethanolamine in raw and cooked meat. The major effect of sodium nitrite was observed on phosphatidyl ethanolamine whose content was significantly lowered in the presence of this salt. This effect was observed at the end of the curing process and was not modified by the cooking. The fatty acid content between raw meat and cooked cured meat showed the strongest effect of sodium nitrite on arachidonic acid, for both phosphatidyl choline and phosphatidyl ethanolamine. This fatty acid was degraded preferentially in the presence of this salt, probably due to its high level of unsaturation. Known volatile odorant compounds typical from polyunsaturated fatty acid oxidation (hexanal, oct-l-en-3-ol, ...) were observed in lower amounts in cooked meat cured with sodium nitrite. The content of these volatile compounds is usually measured in order to evaluate the lipid oxidation level in meat. In our study, nitrite treated meat contained less of these compounds concomitant with a lowered phosphatidyl ethanolamine content, especially for one of its major fatty acid, arachidonic acid. Further investigations are needed to understand the oxidation route of this polyunsaturated fatty acid in the presence of sodium nitrite.

1. INTRODUCTION Curing of meat before cooking imparts a characteristic flavor to cooked cured ham. Among the ingredients added with brine, sodium nitrite is thought to be a major contributor to this flavor. For instance, it has been shown that addition of sodium nitrite changes the profile of volatile compounds of cooked cured meat qualitatively (i.e. formation of nitrogen

246 compounds) as well as quantitatively (i.e. decrease of volatile lipid oxidation products) [1,2]. Several compounds has been identified [3, 4] but, in spite of numerous studies, no single compound or class of compounds has been found to be responsible for the characteristic flavor of cooked cured meat products, nor have the involved mechanisms been elucidated [5]. In order to investigate the role of sodium nitrite in fatty acid oxidation during the process, the effect of this salt was studied by comparison of products processed with or without adding sodium nitrite to the brine. PhosphoUpid (PL) composition was studied as well as fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), the two major PLs of pork meat. The content of volatile odorant compounds generated by fatty acid oxidation was also determined in order to study the role of sodium nitrite on the generation of flavor compounds.

2. MATERIALS AND METHODS 2.1. Materials 15 kg of pork semi membranosus muscle from conmiercial sources were sorted for similar visual appearance and mean pH value 5.7, standard deviation 0.1, and divided randomly into three sets of 5 kg each. 10 % (weight/weight) intramuscular brine injection was performed with a pumping needle. For each set, the level of sodium nitrite was adjusted to inject 0, 50 or 100 mg/kg muscle. No spices were added to the brine in order to avoid their interference. The three hams were put in 5 kg sealed cook-in-bag pouches and cooked to a core temperature of 65°C. After cooling to a core temperature of 3°C and a further 24 hours stabiUzation, lOOg slices were made, wrapped in an aluminium sheet, placed individually in polyethylene bags sealed under vacuum, and frozen to -20°C, to avoid oxidation. The aluminium foil was used to protect the sample from oligomer migration of the polyethylene bag. The procedure descibed above was repeated three times. The nine hams obtained (3 hams with no sodium nitrite, 3 hams with 50 mg/kg and 3 with 100 mg/kg sodium nitrite) were then analysed according to the procedure described in Figure 1. 2.2. Phospholipid analysis Total Upids from 10 g of ground sample were extracted according to the Folch method [6]. PLs were then separated from neutral lipids by Uquid-solid extraction on silica cartridges according to Juaneda and Rocquelin [7]. PL class separation was performed by high performance liquid chromatography (HPLC) with a 250*7.5 nmi Lichrosorb Si60 column and detected using a Cunow LSD Ught scattering detector as described by Juaneda et al [8]. Each class of PL was quantified as percent of total phosphoUpids. PLs were analysed in raw meat, in meat after curing with brine containing 0, 50 or 100 mg sodium nitrite per kg meat, and after cooking.

247

10 g ground sample (raw meat, cured, cooked) at 0, 50 or 100 mg/kg sodium nitrite/raw meat Total lipids extraction (Folch 1957)

Z

Separation of polar lipids by solid-liquid extraction (silica cartridges. Juaneda and Roquelin 1985) Analysis of phospholipid composition (HPLC. Si60 column, light scattering detector) Semi-preparative separation of polar lipids (HPLC, 8160 column. PE and PC peaks collection) PC

PE

Z

Fatty acids analysis trans esterification (Morrisson and Smith 1963). CI7:0 used as internal standard. GC analysis (DBWax column).

Figure 1. Description of the analysis of phospholipids and fatty acids from phosphatidyl choline and phosphatidyl ethanolamine. 2.3. Fatty acid analysis Fatty acid composition was determined in PC and PE, the major classes of PL in pork meat. Required quantities (1 mg each) of PC and PE were obtained by semi-preparative HPLC separation with a 250*20 nmi Lichrosorb Si60 column. PC and PE were collected at the end of the column. Fatty acid composition was determined by gas-chromatography, after trans-esterification of 400|ig of PL according to Morrisson and Smith [9]. 40 jig of heptadecanoic acid was added as an internal standard. Fatty acid methyl esters were separated using a Hewlett Packard HP5890 series n equipped with a DB-Wax J&W Scientific fused siUca capillary column (30 m x 0.32 nun i.d., film thickness 0.5 jum), a split-spUtless injector and a flame ionization detector. A temperature gradient was programmed from 40 to 90°C at 10°C/min., from 90 to 240°C at 5°C/min. and maintained at 240°C for 30 min. 2.4. Volatile compound analysis 250 g cooked cured (50 mg/kg sodium nitrite) and uncured (0 mg/kg sodium nitrite) cooked meat were ground frozen. The volatile constituents of each sample were extracted by hydrodistillation under vacuum and collected in glass traps cooled with liquid nitrogen and further extracted with bidistilled dichloromethane as described elsewhere [10]. Volatile compounds were analysed by means of gas-chromatography using a Hewlett Packard HP5890 series n gas chromatograph equipped with a DB-FFAP J&W Scientific fused siHca capillary column (30 m x 0.32 mm i.d., film thickness 0.25 jum), a split-splitless injector and a flame ionization detector. A temperature gradient was progranmied from 40 to 220°C at 3°C/min., and maintained at 220°C for 10 min.

248

2.5. Statistics Differences between means from the replicate processes for PL, fatty acid methyl esters and volatile compound analysis were tested with the Student t-test.

3. RESULTS AND DICUSSION 3.1. Phospholipid content of raw, cured and cooked meat The major PL observed in raw meat were PC, PE and phosphatidyl inositol (PI) representing respectively 57, 25 and 12% of total PL (table 1). These results were consistent with the usual composition of pork meat [11]. Comparison of PL composition between raw, cured and cooked meat at different sodium nitrite levels showed significant differences for PC and PE (table 2). For these two major classes of PL, the effect of processing depended on the addition or not of sodium nitrite with brine. The effect observed was not altered by the cooking step, as there was no significant difference in the PC and PE levels before and after cooking. For PC, addition of sodium nitrite to brine, for the two levels studied, led to a preservation of this class of phosphoHpids as the level observed was not different from raw meat. In meat treated without this salt, there were significantly less PC observed in comparison to meat treated with this salt. The effect of sodium nitrite on PC could be interpreted as a protective action against oxidation for this class of PL. A stronger and opposite effect was observed for PE, as there were significantly less PE in meat treated with sodium nitrite. The degradation of PE observed during the curing step in the presence of this salt could be due to the composition of this class of PL, rich in polyunsaturated fatty acids such as linoleic and arachidonic acids. Interaction of sodium nitrite, or most probably its reduced form nitric oxide (NO), with fatty acids has already been demonstrated by Goutefongea [12]. And this binding was related to the fatty acids degree of unsaturation [13]. Thus, the fatty acid composition of these two classes of phospholipids was studied in the presence and absence of sodium nitrite.

Table 1 Mean phospholipids composition of raw meat in percent of total phospholipids. Mean and standard deviation (SD) were calculated from three replicates. Phospholipids Phosphatidyl choline (PC) Phosphatidyl ethanolamine (PE) Phosphatidyl inositol (PI) Phosphatidyl serine (PS) SphingomyeUn (SM) Lysophosphatidylcholine (LPC) Cardiolipids (CL)

Mean (%) (SD) 57.5 (1.5) 24.8 (1.5) 11.7(1.4) 3.6 (0.4) 1.6 (0.1) 1.1 (0.1) 1.3(1)

249 Table 2 Mean phospholipids composition of meat (raw, after curing and after cooking, in presence of 0, 50 and 100 mg/kg sodium nitrite) in percent of total phospholipids. Means were calculated from three repUcates. ab ' Means in a raw with different superscript letters are different (p<0.10). PL

Raw meat 0

Cured meat 100 50 sodium nitrite (mj?/kg)

0

Cooked meat 100 50 sodium nitrite (mg/kg)

PC

57.5^"

56.8^

60.1*

59.4*

55.6"

59.4*

59.6*

PE PI PS SM LPC CL

24.8^ 11.7 3.6 1.6 1.1 1.3

26.5* 11.0 2.3 1.7 1.2 0.7

21.0*' 12.6 3.0 2.3 1.7 1.0

21.6" 10.6 3.7 2.4 1.0 1.2

24.0* 12.8 2.2 1.5 1.3 1.5

19.4" 12.3 3.7 2.1 1.9 1.0

19.6" 11.9 3.9 2.5 1.0 1.5

Table 3 Mean composition (standard deviation) of fatty acid methyl esters of PC and PE in raw meat, in ng/|Lig. Means were calculated from three replicates. Fatty acids ng/|Lig phosphatidyl choUne ng/|ig phosphatidyl ethanolamine 16:0 18:0 16:1 18:1 18:2 18:3 20:2 20:3 20:4 Total saturated Total monounsaturated Total polyunsaturated

73.1 (6.8) 9.9(1.3) 4.1 (0.5) 30.7 (4.0) 60.2(10.5) 3.9 (0.9) 0.8 (0.3) 1.1(0.3) 8.1 (1.2) 83 34.8 74.1

2.3 (0.5) 5.9 (0.8) 0.6 (0.4) 4.2 (0.4) 7.3 (0.9) 0.2(0.1) 0.4(0.1) 0.4(0.1) 5.9 (0.5) 8.2 4.8 14.2

3.2. Fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine in raw and cooked meat Fatty acid methyl ester composition of PC and PE in raw meat (table 3) were consistent with the usual composition observed in pork meat [11]. Major fatty acids of PC are palmitic acid (C16:0, 38%), linoleic acid (C18:2n-6, 31%) and oleic acid (C18:ln-9, 16%). Major

250 fatty acids of PE are linoleic acid (C18:2n-6, 27%), arachidonic acid (C20:4n.6, 22%), stearic acid (C18:0, 22%) and oleic acid (C18:ln-9, 15%). 52% of the fatty acids from PE are polyunsaturated. The effect of sodium nitrite was studied by comparison of the fatty acid content in raw meat and cooked meat treated with 0 and 50 mg/kg sodium nitrite. For the major fatty acids of PC (figure 2), palmitic acid (C16:0) and linoleic acid (C18:2n-6)» a significantly lower amount was observed after processing in the presence and in the absence of this salt. In the case of palmitic acid, a stronger decrease was observed in its absence. Concerning oleic acid (C18:ln-9) and arachidonic acid (C20:4n-6)» a significant effect of sodium nitrite was observed as a lower amount of these two fatty acids was quantified only after processing in the presence of this salt.

C18:1

C16:0

cookwct 0 iTi9^kg

COOIMC^ 50 mgflm

cooked, SOmgAcg

C20:4

C18:2

lisl

• J l li i i cooked, 0 mgflcg

cook*d,Om^g

cooked, SO tvg/kg

cooked, 50 mgAcg

Figure 2. Fatty acids composition in ng/|ig of phosphatidyl choline (PC) in raw meat (raw) and cooked cured meat without (0 mg/kg) or with sodium nitrite (50 mg/kg). Means with different letters are different (p<0.10). For the major fatty acids of PE (figure 3), only Unoleic acid (C18:2n.6)» arachidonic acid (C20:4n.6) and stearic acid (CI8:0, 22%) were significandy affected by the processing. As

251 observed in PC, the content of linoleic acid was significantly lowered by the processing, in presence and in absence of sodium nitrite. A significant effect of sodium nitrite was observed for arachidonic acid as the content of this fatty acid was lowered in the presence of this salt. Referring to the decrease observed for PE after curing in the presence of sodium nitrite, the effect was mainly observed for arachidonic acid. This effect was also observed for arachidonic and oleic acid in PC. 3.3. Comparison of volatile odorant compounds from fatty acid oxidation in cooked meat cured with 0 or 50 mg sodium nitrite per kg meat Volatile odorant compounds resulting from unsaturated fatty acid oxidation were quantified in cooked meat treated with 0 and 50 mg/kg sodium nitrite (table 4). A study of the volatile compounds showed significantly lower amounts of hexanal, heptanal, octanal, decanal, oct-2-enal, non-2-enal and oct-l-en-3-ol in the sample treated with sodium nitrite.

C18:1

C18:0

111 L L 11 11 I i raw

cooked, 0 m^kg

cooked, SO

raw

C18:2

cooked, 0 mg^g

cooked,

C20:4

Is S a

I Q.

I" "5 JZ

o.

1^

I 4 a

raw

cooked, 0 mg/kg

coolwd, 50 tvg/kq

raw

cooked, 0 mg/kg

cooked, 50 mg/kg

Figure 3. Fatty acids composition in ng/|ig of phosphatidyl ethanolamine (PE) in raw meat (raw) and cooked cured meat without (0 mg/kg) or with sodium nitrite (50 mg/kg). Means with different letters are different (p<0.10). The strongest effect was observed for hexanal and oct-l-en-3-ol. This effect of sodium nitrite is well known and explained by an antioxidant effect of this salt on unsaturated fatty

252

acid oxidation [2, 5, 10]. Saturated aldehydes with 7 to 10 carbon atoms are typical from the oxidation of n-9 unsaturated fatty acids such as oleic acid. Nonanal is the major oxidation product of oleic acid [1]. The other compounds are typical from oxidation of n-6 unsaturated fatty acids such as linoleic or arachidonic acid. The olfactive impact of these oxidation products has been well studied in the case of Unoleic acid [14]. These compounds were all odor active, as detected by gas-chromatography-olfactometry. The fatty acid content of PC and PE (figures 2 and 3) showed no specific effect of sodium nitrite on Unoleic acid, a lower amount of arachidonic acid in the presence of this salt, and also for oleic acid from PC. Thus, sodium nitrite's effect on unsaturated fatty acids is, on one hand antioxidant as volatile oxidation products were detected in lower amounts and, on the other hand, leads to a lower amount of some fatty acids in sodium nitrite treated products. The action of this salt has to be further studied in order to understand the deviation of unsaturated fatty acids from their usual oxidation route.

Table 4 Quantification of volatile compounds extracted from cooked meat treated with 0 and 50 mg/kg sodium nitrite. Means were calculated from three replicates. * : significant differences between means (p<0.10). Compound Retention Content in the volatile extract in |ig/kg, relative to internal index standard pentyl pentanoate, of cooked meat with (DB-FFAP) 0 mg/kg sodium nitrite 50 mg/kg sodium nitrite Hexanal

1070

651.4

54.3*

Heptanal

1173

24.5

9.6*

Octanal

1272

13.4

4.3*

Nonanal Decanal

1374 1472

32.6 5.9

25.2

oct-2-enal

1400

7.8

0*

non-2-enal

1508

8.0

2.9*

2,4-decadienal oct-l-en-3-ol

1758 1430

1.6 103.9

0*

0 7.2*

4. CONCLUSION The effect of sodium nitrite on the phospholipid composition of cooked cured ham during its processing was observed for the two major phospholipids of pork meat, phosphatidyl choline and phosphatidyl ethanolamine. The content of these two classes of phospholipids was clearly more modified during the curing than during the cooking. The major effect of sodium nitrite was observed on phosphatidyl ethanolamine whose content was significantly lowered in the presence of this salt. The evolution of the fatty acid content between raw meat

253 and cooked cured meat showed the strongest effect of sodium nitrite on arachidonic acid, for both phosphatidyl choHne and phosphatidyl ethanolamine. This fatty acid was degraded preferentially in presence of this salt, probably due to its high level of unsaturation. The content of known volatile odorant compounds typical from oxidation of this polyunsaturated fatty acid (hexanal, oct-l-en-3-ol, ...) were observed in lower amounts in cooked cured meat treated with sodium nitrite. The content of these volatile compounds is usually measured in order to evaluate the lipid oxidation level in meat. In our study, nitrite treated meat contained a lowered content of these compounds concomitant with a lowered content of some unsaturated fatty acids. The action of this salt has to be further studied in order to understand the deviation of these fatty acids from their usual oxidation route. The specific flavor of cooked cured ham seems to be due to the action of sodium nitrite, not only by its effect on lowering some typical fatty acid oxidation compounds, but also by a deviation of some fatty acids from their typical oxidation route. If other odorant compounds resulting from this action of sodium nitrite are generated, they have not yet been identified. Acknowledgements The authors would Uke to thank P. Juaneda from the Unite de Nutrition Lipidique, INRA Dijon for his contribution to the phopholipids analysis.

5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14

S. Erduran and J. H. Hotchkiss, J. Food Sci., 60 (5) (1995) 946. D. S. Mottram, S. E. Croft, R. L. S. Patterson, J. Sci. Food Agric, 35 (1984) 233. D. S. Mottram, J. Agric. Food Chem., 32 (1984) 343. F. Shahidi, L. J. Rubin, L. A. D'Souza, CRC Crit. Rev. Food Sci. Nutr., 24 (1986) 141. N. Ramarathnam and L. J. Rubin, 'Flavour of Meat and Meat Products', ed. F. Shahidi, Blackie Academic & Professional (1994) 174. J. Folch, M. Lees, G. H. Sloane-Stanley, J. Biol. Chem. 226 (1957) 497. P. Juaneda and G. Rocquelin, Lipids, 20 (1) (1985) 40. P. Juaneda, G. Rocquelin, P. O. Astorg, Lipids, 25 (11) (1990) 756. W. R. Morrisson and L. M. Smith, J. Lip. Res. 5 (1964) 600. A. S. Guillard, J. L. Le Quere, J. L. Vendeuvre, 'Flavour Science. Recent developments', ed. A. J. Taylor and D. S. Mottram, The Royal Society of Chemistry (1996) 231. E. Ngah, C. Alasnier and G. Gandemer, 40th ICoMST, The Hague (1994) s-v.l5. R. Goutefongea, R. G. Cassens, G. Woolford, J. Food Sci., 42 (6) (1977) 1637. M. Mouloud, phD thesis, Nantes (fr) (1990) 60p. F. Ulrich and W. Grosch, Z. Lebens. Unters. Forsch. 184 (1987), 277.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

255

Influence of fat on the flavour of an emulsified meat product F.F.V. Chevancea and L.J. Farmera^

^Department of Food Science, The Queen's University of Belfast and the ^Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK

Abstract The effect of fat (triglyceride) content on the flavour of an emulsified meat product was investigated using frankfurters containing 5%, 12% or 30% fat. Profiling studies indicated that decreasing the fat content significantly increased the intensity of spicy, peppery and smoky flavours. Terpenes and phenols, some of which contributed to these flavours, were collected in greater quantities from the low-fat frankfurters by static headspace and 'nose-space' analysis but not by Likens-Nickerson extraction. Thus, the differences in flavour may be explained by the more rapid release of lypophilic terpenes, phenols and other compounds when the proportion of fat is reduced.

1. INTRODUCTION

The growing demand by consumers for 'healthier' foods has stimulated the development of processed meat products with a reduced content of triglyceride lipid. Unfortunately, the eating quality of these low-fat' products often does not compare favourably with their traditional, 'full-fat', counterparts. Thus, a certain amount of fat is necessary to ensure desirable texture, mouthfeel, tenderness, juiciness, flavour, appearance and overall acceptability [1, 2]. Most of the research on low-fat processed meats has concentrated on rheological and structural aspects or the textural aspects of sensory quality. Where fat reduction was accomplished with 10% or less added water, the resulting products were firmer [3], more rubbery, and less juicy [4-6]. However, increasing amounts of added water along with decreasing fat content can reduce or eliminate some of the detrimental effects on texture [7-9]. Fewer studies have addressed the effect of fat content on flavour and these have yielded conflicting results. Some studies

256 on beefburgers [10-12], frankfurters [6] and sausages [13] have found a tendency for higher fat contents to give higher scores for overall flavour intensity, whereas other studies on beefburgers [14] and frankfurters [5] have found the opposite effect; a reduction in fat content increased the overall intensity of the flavour. Other work showed no effect of fat level [15-18]. Some of the disagreement between the reported effects of fat on flavour may be due to the various fat contents examined. Matulis et al [19] found that at fat levels above 5%, the influence of fat on flavour intensity of frankfurters appeared to be minimal. Reitmeier et al [20], also reported that pork flavour intensity was not different amongst pork patties with 9, 18 and 23% fat, but patties with 4% fat were scored lower in pork flavour intensity. The different sensory methods and descriptors used for the measurement of flavour may also explain some of the discrepancies in the reported results; many of the studies mentioned measured only the overall flavour intensity or the acceptability of the flavour. Recent studies [21, 22] have examined the effect of fat content on the intensity of individual flavours of frankfurters and sausages. Low-fat sausages [21] had a significantly higher intensity of "taste of smoke" and meat flavour than the full-fat sausages while a recent hedonic scaling study [22] found that low-fat frankfurters had increased intensity of smokiness, spiciness, and saltiness and reduced overall acceptability of the flavour compared with the full-fat counterparts. The human perception of flavour is closely related to the nature and quantities of volatile aroma compounds released from the food. So far, very little information is available concerning the influence of fat content on flavour volatiles in meat products [23, 24]. The aim of the present study was to evaluate the effect of fat content on the flavour quality of an emulsified meat product of the frankfurter tjrpe using detailed sensory methods and to investigate the reason for any differences by instrumental analysis of the aroma volatiles. Frankfurters containing 5%, 12% and 30% fat were subjected to quantitative descriptive analysis (profiling) and the volatile compounds were collected by Likens-Nickerson extraction, by headspace analysis and from the human nose during eating.

2. MATERIALS AND METHODS

2.1. Emulsified meat products Fresh beef was obtained from the National Food Centre abattoir. Pork and pork backfat were obtained from the Dairygold (Michelstown, Ireland) meat processing plant. The products were formulated with 1.5% nitrite salt [99.4% sodium chloride with 0.6% sodium nitrite (E250), Colorozozout, Degens, Holland], 0.5% spice mix (Vienna Gold, Rasp & Co, Germany), 0.05% sodium ascorbate (E301, Dalgety Food Ingredients) and 0.25% sodium tripolj^hosphate (E450b, Albright & Wilson Ltd, England).

257

'Low-fat', 'medium-fat' and *full-fat' frankfurters were prepared in conjunction with the National Food Centre (Dublin, Ireland), by adjusting the amount of pork adipose tissue (back fat) included in the composition to give nominal fat contents of 5%, 12% and 30%. In the reduced-fat products, water was added to replace the fat to ensure the same protein level in all formulations. Two types of smoking were used; 'frankfurters with liquid smoke' were prepared using 0.05% hickory smoke (D402V, Dalgety Food Ingredients) added to the batter [22], while 'traditionally smoked frankfurters' were prepared by a similar method but using a natural smoking process with solid aerosol smoke. Analyses of the triglyceride content indicated that the exact fat levels in the cooked frankfurters with liquid smoke were 5.3%, 13.8% and 30.2% fat and in the traditionally smoked frankfurters were 5.3%, 12.3% and 29.9% fat. 2.2. Profiling studies Six experienced and trained panellists took part in these studies. During training, the panellists discussed and agreed upon those attributes that best described frankfurters. Descriptors were chosen for the aroma and the flavour at three stages of the eating process: at initial bite, during the chewing process, and after swallowing. Frankfurters with liquid smoke (5%, 12% and 30% fat) were steam cooked for 8 min to an internal temperature of 92°C and were presented to the panellists in pieces of 2 cm length x 2 cm diameter in a warmed covered pot (50°C). During one complete session, each of the products, coded with a 3 digit random number, was served to the panellists with an interval of 8 min between two consecutive samples. To cleanse the palate, panellists used natural yoghurt (Spelga, Dromona Quality Foods Ltd, Belfast, UK), rinses with 'Kali water' (carbonated water and potassium bicarbonate; Cantrell and Cochrane Ltd, Belfast, UK) and water biscuits (Jacob's biscuits, Dublin, Ireland) followed by rinses with tap water passed through a water filter (The Boots Company pic, Nottingham, UK); this regime was agreed during training sessions to give the most effective removal of the mouthfeel and strong spicy after-taste from the previous sample. Each attribute was scored on a line scale which was anchored with appropriate terms at the 5% and 95% points, such as 'very weak' and 'very intense', respectively, using a computerized data system (PSA 1.64, Oliemans Punter and Partners bv, Utrecht, The Netherlands). All tests were conducted in individual, partitioned booths under red illumination to mask visual differences between samples. Two sessions were conducted during the same morning; a break of 15 mins was imposed between the two sessions. A total of ten replicate analyses were completed. The data were analysed by analysis of variance using Genstat statistical software (Genstat V, Release 3.1, Lawes Agricultural Trust, Rothamsted Experimental Station). Where the effect of treatment was significant, a Fisher's least significant difference test was applied to the mean scores.

258

2.3. Simultaneous distillation extraction The volatile compounds from cooked frankfurters prepared with liquid smoke were simultaneously steam distilled and extracted with pentane using a Likens-Nickerson apparatus [25]. Approximately 480 g of frankfurters were chopped for 1 min using a food processor (Robot Chef, Robocoupe, Bagnolet, France) and mixed with 100 mL distilled water. The resulting slurry (500 g) was placed in a round bottom flask (1000 mL, Quickfit, J. Bibby Science Products Ltd, Stone,UK) which was fitted with a Likens-Nickerson apparatus. A pearshape flask (50 mL, Quickfit), containing 20 mL of pentane (A.R., Rhone-Poulenc Ltd, Manchester, UK), was attached to the solvent arm of the apparatus. Bromobenzene (Aldrich Chemical Co Ltd, Dorset, UK) was added to the pentane prior to collection as an internal standard (10 |iL, 2980 ng jaL^- The water/meat mixture and the pentane were maintained at boiling point by use of a heating mantle and water bath, respectively. Water, maintained at 0-2°C with ice, was circulated through the condenser. After 30 min to 'prime' the system, the volatiles from the frankfurters were extracted for 2 hrs. The dilute extract, which possessed a very strong aroma ('plastic, eucalyptus, smoky') was concentrated to about 1 mL by evaporation using a gentle stream of nitrogen (1000 mL min-^). Extractions were performed on the frankfurters with liquid smoke containing 5% and 30% fat in triplicate. Volatile compounds from 1 |iL of each extract were separated and analysed using a CP WAX52CB capillary column (50 m x 0.32 mm i.d., Chrompack Ltd, London, UK) in a Hewlett Packard 5890 Series II gas chromatograph fitted with a 'split/splitless' injector. The carrier gas was helium, with a column head pressure of 80 kPa. The oven temperature was maintained at 40°C for 5 mins, increased to 220°C at 4°C min-l, and maintained at 220°C for 30 mins. The volatiles were detected using a flame ionisation detector. The areas of integrated peaks were divided by the peak area of the internal standard (bromobenzene) to give relative peak areas. 2.4. Static headspace analysis and gas chromatography-odour assessments Frankfurters (20 g) were chopped into 1 cm slices and then into 4 sectors and placed in a 100 mL bottle (Duran, Davidson and Hardy Ltd, Belfast, UK) sealed with a screw cap with PTFE protective seal and fitted with two bulkhead unions (1/16 x 1/16 inch, S.G.E. Ltd, Milton Keynes, UK) into which a trap (95 x 0.8 mm i.d.) containing 2.6 mg Tenax GC (S.G.E. Ltd) and a 10 mL gas-tight syringe (Series II, S.G.E. Ltd) could be fitted. A short length of stainless steel tubing (1 cm length x 1/16 inch external diameter) joined the union fitted with the syringe with a 6.5 cm long PTFE tubing (1/16 inch i.d.) so that the flow of air from the syringe would be directed to just above the food sample. The bottle was placed in a water bath at 70°C and the syringe fitted. The other union remained open avoiding a pressure build-up in the system. After 20 min, a conditioned trap (pre-conditioned at 250°C with a flow of helium at ca. 1 mL min-i for 20 mins) was fitted to the second union and 10 mL of air was injected through the syringe

259 into the bottle, displacing headspace on to the trap. After 5 min, the trap was removed and purged for 5 min with nitrogen (50 mL min-i) to remove moisture from the trap. Four collections were made for each of the frankfurter formulations (5%, 12% and 30% fat, both with liquid smoke and traditionally smoked). Gas chromatography (GC) was performed using a Hewlett Packard 5890 Series II gas chromatograph, fitted with a 'Unijector' (S.G.E. Ltd). The collected volatiles were thermally desorbed at 250°C for 5 min and re-focused on to a 10 cm region at the front of a fused silica capillary column (CP WAX52CB, 50 m x 0.32 mm i.d., Chrompack Ltd), which had been pre-cooled for 5 min with solid CO2. After desorption, the oven was heated rapidly to 60°C, maintained at this temperature for 5 min before increasing at 4°C min-i to 220°C (30 mins). The effluent from the column was split between a flame ionisation detector and an odour port, which comprised a heated fused silica transfer line (220°C) connected to a PTFE cup, flushed with an auxilliary flow of moist air {ca. 14 mL min-i). GCodour assessments were conducted by 4 assessors on each of the frankfurters. Mean peak areas of integrated peaks were calculated. 2.5. Nose-space a n a l y s i s Pieces of traditionally smoked frankfurters {ca. 5g) were steam cooked for 5 min to an internal temperature of 80°C, and allowed to cool for 1 min to 65°C prior to collection. The food sample was placed in the mouth of a subject who chewed normally. Volatiles from the nose were collected on to a trap (140 x 3 mm) containing Tenax TA (S.G.E. Ltd). This was effected without disturbing the eating pattern using a 5 mL pipette tip (Alpha Laboratories Ltd, Eastleigh, UK) from which the end had been removed to leave a 10 cm tube, connected to the trap with a short length of flexible tubing. The subject held the broad end of the pipette tip against (without sealing) one nostril and the exhaled air was drawn through the trap using a Teflon vacuum pump (Model 1, Whatman International Ltd, UK) at a flow rate of 250 mL min'^ for the first minute of eating. The time of swallowing was not dictated but occurred naturally at around 35-40 s for the two subjects involved. Four replicates were performed for each of the samples (5%, 12% and 30% fat frankfurters) and for each of the subjects. Blank nose-space samples (collected without food) were also collected from the subjects for 1 min. Traps were thermally desorbed, as described previously, using a Hewlett Packard 5890 Series II gas chromatograph, in this case fitted with a CHIS injector (S.G.E. Ltd), and chromatography was performed using a CP SIL8CB capillary column (25 m x 0.32 mm i.d, Chrompack Ltd). The oven temperature was programmed to start at 40°C, rise at 5°C min-i to 100°C, then at 14°C min-^ to 200°C and maintained at this temperature for 2 min. Volatiles were detected using a flame ionisation detector and the mean peak areas of integrated peaks calculated.

260 2.6. Gas Chromatography - Mass Spectrometry (GC-MS) The headspace volatiles from frankfurters were collected at 70°C on to traps (95 x 0.8 mm i.d.) containing Tenax GC (S.G.E. Ltd) by dynamic headspace concentration using a stream of nitrogen (50 mL min-i) for 30 min. The volatiles were analysed using a HP 5890A gas chromatograph connected to a HP 5970 mass selective detector operated at 70 eV in the E.I. mode over the range 35-450 a.m.u. The capillary column, injection technique and oven program were as described for static headspace analysis.

3. RESULTS AND DISCUSSION

3.1. Profiling studies A total of 25 different attributes for flavour and texture, detected at different stages of the eating process, were defined for frankfurters during the profiling studies (Table 1). Panellists were asked to concentrate on attributes relevant to the perception of flavour. However, these were judged to include some aspects of texture. The results of the studies comparing the three types of frankfurters are presented in Table 2. No significant differences were found between the three frankfurters for any of the aroma attributes, except for 'turf smokiness' which was more intense for the full-fat product. Many significant differences were observed for texture and flavour especially between the 5% and 30% fat frankfurters. Very highly significant differences were obtained for the textural characteristics, with the low-fat frankfurters being the most rubbery, tough, solid, gristly, gritty, lumpy and mealy in texture. The full-fat frankfurters scored most highly for 'melts in mouth. For many of these attributes, significant differences were observed between the low-fat and medium-fat, and between the medium fat and full-fat frankfurters. Such textural changes have been well documented [1-9] and could influence the release and perception of flavour during eating. In contrast with those reports which have suggested that, at fat contents exeeding 5%, there was little effect on flavour [19, 20], these data indicate that there were almost as many significant differences in flavour between the 12% and 30% fat products as between the 5% and 12% fat frankfurters. The panellists were asked to assess aspects of flavour at several stages during the eating process which provided some temporal information on the flavour perceived. Figure 1 illustrates the evolution of some of the flavour attributes during the eating process. The overall flavour was perceived to be more intense for the frankfurter with 5% fat, significantly so after swallowing. While this difference was not significantly different between the three fat levels at the time the frankfurter was placed in the mouth (initial bite), or during the chewing process it was significantly different for the residual flavour just after swallowing and for

261 Table 1 "Definitions" of the attributes used during sensory profiling Attributes

Definition

]Flavour/ T e x t u r e ^

Odour 1

2

3

4

5

Odour/Flavour: Mealy Meaty Porky Fatty/greasy Spicy Peppery Smoky b Sweet flavour Synthetic flavour Salty flavour

V V V V V V V

Cereal, animal feed

V V V V V V V V

Herby burst Overall flavour

V

V V V

V

Meaty odour or flavour more Uke beef t h a n other meats

V V V V V

Pork meat odour or flavour Fatty, greasy flavour Spicy, aromatic Black pepper Hke Smoky odour or flavour Sweet, sugary

V V V V

Medicinal, artificial-Uke

V V V

Perception of saltiness Sudden burst of herby flavour

V

V

Intensity of the overall flavour

Texture: Greasy texture Rubbery texture Tough/Firm Solid texture Juicy burst Mealy texture Crispy skin Sticky skin Separation Gristly texture Gritty texture Lumpy texture "Melts in mouth"

V V V V V

Greasiness around t h e mouth

V V

Springiness when chewed Degree of resistance to chewing Brakes into small particles Sudden burst of Hquid in mouth

V V V V V V V V

Texture hke animal feed Crackly skin Adheres to teeth Separation of outer a n d inner layers Refers to bits which do not chew up Small brittle particles Refers to size of bits of fat a n d meat Refers to sample t h a t disappears to nothing very rapidly when chewed

^ Flavour and texture attributes were defined at different stages of the eating process: 1 = initial bite; 2 = chewing process; 3 = after swallowing; 4 = 45 s after swallowing; 5 = 90 s after swallowing. " Three types of smokiness were defined for aroma attributes: "Maple cure smokiness" refers to the sweet smoke occurring with cured products; "Wood smokiness" corresponds to the smoke odour from wood fires; whereas 'Turf smokiness" refers to the odour arising from practice widespread in Ireland, of burning dried peat blocks, known as turf, as fuel on open fires.

262

Table 2 Effect of fat content on the mean sensory profiling scores for frankfurters Attributes

5% fat

12% fat

30% fat

Sig.^

SEM^

Mealy

20.8

19.3

Meaty

18.0

17.7

18.8

NS

0.93

19.1

NS

0.97

Porky

28.9

29.5

30.7

NS

1.39

Aroma

Fatty/greasy

17.8

17.1

17.0

NS

0.48

Spicy

23.4

24.3

25.6

NS

0.89

Peppery

25.2

24.9

26.3

NS

0.91

Maple cure smokiness

33.1

29.9

31.8

NS

1.57

Wood smokiness

20.5

21.0

22.6

NS

0.79

Turf smokiness

16.9^

16.2a

19.4b

*

0.87

Rubbery texture

60.2C

55.4b

45.6a

***

1.43

Tough texture

51.3c

44.6b

26.0a

***

1.81

Solid texture

37.3c

31.4b

24.7a

***

1.40

Greasy texture

22.6

22.4

19.0

NS

1.47

Greasy flavour

27.5

26.8

23.8

NS

1.10

Juicy burst

33.4

34.2

37.3

NS

1.50

Initial flavour intensity

36.5

35.4

35.7

NS

1.28

Porky flavour

29.0

28.0

30.7

NS

1.44

Sweet flavour

17.5a

19.2a

25.0b

***

1.03

Synthetic flavour

36.7c

28.2b

22.4a

***

1.08

Smoky flavour

32.5b

27.5a

28.8a

*

1.12

Salty flavour

26.9

26.0

25.2

NS

0.88

*

1.02

***

0.87

Initial bite

Spicy flavour

30.8^

29.2ab

26.3a

Peppery flavour

36.3c

32.7b

29.3a

30.7

31.9

26.5

NS

1.74

21.7a

***

1.50

**

2.06

*

1.48

***

1.74

Chewing process Crispy skin Sticky skin

34.8^

31.6b

Separation

36.9^

36.8b

26.0a

Firm texture

50.5b

50.7b

45.1a

Rubbery texture

51.ic

43.6b

32.6a

263 Table 2 continued Effect of fat content on the mean sensory profiling scores for frankfurters Attributes

5% fat

12% fat

30% fat

Gristly texture

24.9b

21.7a

20.9a

Gritty texture

24.0^

21.3ab

Lumpy texture

36.9^

Mealy texture "Melts in mouth" Herby burst

SigJ

SEM2 0.76

18.9a

** *

32.iab

29.4a

*

1.97

27.5c

23.lb

17.8a

***

1.36

20.9a

25.7a

31.lb

*•

1.71

**

1.41

•*•

1.38

33.33b

30.3b

26.1 a

1.16

Synthetic flavour

34.8^

26.50a

23.5a

Porky flavour

24.9

25.8

26.6

NS

0.80

Overall flavour intensity

38.2

33.9

35.0

NS

1.32

Salty flavour

31.6C

27.5b

24.7a

***

0.78

••*

0.97

Peppery flavour

43.6C

33.9b

30.6a

Spicy flavour

35.0c

29.4b

25.6a

***

1.05

39.7c

34.6b

31.3a

***

1.07

After s w a l l o w i n g Residual flavour intensity Meaty aftertaste

17.4

17.1

16.9

NS

0.36

Porky aftertaste

26.9

26.3

25.4

NS

0.97

Green aftertaste

15.8^

13.5ab

12.6a

*

0.87

Greasy aftertaste

22.0

20.5

21.3

NS

1.20

Smoky aftertaste

38.2^

34.2ab

30.3a

*

1.90

Salty aftertaste

31.5b

26.6a

25.4a

***

0.83

Spicy flavour in mouth

34.4c

30.4b

26.2a

***

1.14

Peppery flavour in mouth

37.3c

32.9b

29.2a

***

0.99 0.68

Spicy flavour in throat

30.5c

26.2b

24.2a

***

Peppery flavour in throat

36.7c

30.9b

26.3a

***

1.10

Flavour intensity after 45 s

41.6b

34.9a

32.2a

***

1.27

Flavour intensity after 90 s

37.5c

32.4b

28.5a

***

1.18

^ Degree of significance between the three fi*ankfurters (analysis of variance) : NS = no significant difference; * = P<0.05; ** = P<0.01; *** = P<0.001 2 SEM = Standard Error of Means a,b,c Values are the means of ten replicate analyses by 6 panellists. For each attribute, values which do not share a common superscript are significantly different (P<0.05) according to Fisher's LSD test

264

Overall flavour

Porky flavour • 5 % fat

-12% fat -30% fat

^40 +

2

3 4 Stage of eating (a)

Stage of eating

Pepperiness

Spiciness

0)

§40.'A

O

^ o ^0 1 Ifm "^^ 11 —

• •• A

'

?,0-

'

"

"•"

"

A

—

~

-

1 Stage of eating

Smokiness

Stage of eating

•

'

-

1 Stage of eating

Saltiness

Stage of eating

Figure 1. Temporal changes in sensory scores during the eating process for selected profiling attributes of frankfurters, (a) 1 = initial bite; 2 = chewing process; 3 = taste after swallowing; 4 = 45 s after swallowing; 5 = 90 s after swallowing

265 the after-taste intensity measured 45 and 90 s after swallowing. Some workers [5] have shown similar increases in overall flavour in low-fat frankfurters or beefburgers, but other studies on frankfurters, beefburgers or sausages [6, 10-13] found that the full-fat products gave highest scores for the overall intensity of flavour. Assessment of individual flavours may be more reliable than assessment of overall flavour, since the descriptor is better defined. The present study showed significant differences between the frankfurters with extreme fat levels for peppery and spicy flavours at all stages of eating, and for smoky flavour, which was assessed for the initial bite and after swallowing only. This is in good agreement with the results obtained by Hughes et al [22], who conducted hedonic scaling tests, using six flavour parameters, on the same frankfurters as in the present study; they found an increase in spiciness and smokiness with the decrease of fat. These results also agree with the increase in spice flavour reported in low-fat frankfurters by Yang et al [26], and in low-fat sausages by Solheim [21] compared with their full-fat counterparts. Saltiness was not significantly different between the three fat levels at the initial bite, but was found more intense for the low fat frankfurter for the other stages of eating. This is perhaps surprising as the salt concentration was the same in the frankfurters at all fat contents, and one might expect that the greater proportion of water in the low-fat frankfurters would dilute the salt and decrease its impact. However, these results agree with those of Hughes et al [22] conducted on the same frankfurters but using a different panel and sensory methods. In contrast, porky flavour, one of the few flavour attributes clearly derived from the basic meat ingredients of the frankfurters, was not significantly altered by fat content at any stage of eating. Thus, the main difference in flavour between the three frankfurters was due to changes in the spicy, peppery and smoky flavours rather than any changes in the basic meaty or porky flavour of the meat. 3.2. Analyses of volatile compounds In order to determine the identities of the compounds responsible for the flavour attributes described by the panellists, the volatiles released from the frankfurters were analysed by GC-odour assessment and GC-MS. Odours included 'spices, green, pepper' (LRI 1022), 'medicinal, cough syrup' (LRI 1205), 'flower, carnation' (LRI 1540), 'plastic, medicinal' (LRI 1733), due to terpenes (tentatively identified as a-pinene or P-thujene, cineole, linalool, and an unknown terpene, respectively); 'smoky, frankfurter, plastic' (LRI 1852), 'smoky, frankfurter' (LRI 1934) probably due to 2-methoxyphenol (guaicol) and 2methoxy-4-methyl phenol (creosol); and 'caramel, sweet' (LRI 982), 'mushrooms' (LRI 1300), 'meaty, metallic, geranium' (LRI 1373), 'cooked potatoes' (LRI 1451), 'potatoes' (LRI 1505), 'meaty, roasted' (LRI 1663), 'biscuity, pop corn' (LRI 1751) tentatively identified as 2,3-butanedione, l-octen-3-one, dimethyl trisulfide + unknown, methional, unidentified, 2-methyl-3-(methyldithio)-furan, and unidentified. Of these, the terpenes and phenols would seem to be responsible for the flavour differences between the different frankfurter formulations observed by sensory profiling. These compounds originate from the spices and smoke

266 incorporated into the product (Chevance and Farmer, unpublished data) while the remaining compounds identified are known odour compounds in cooked meats [27, 28]. The total quantities of volatiles extracted fi:om the 5% and 30% fat frankfurter using a Likens-Nickerson procedure were similar (Figure 2a). This would be expected as the same amount of spices and liquid smoke were added to both formulations. However, as may be observed in Figure 2b, considerable differences occurred in the quantities of volatiles released into the headspace, as measured by static headspace analysis. The compounds identified were mostly terpenes and a few phenols. These compounds were not necessarily the key odour compounds responsible for the odours described in the previous paragraph, but were from the same compound classes and may be affected similarly by fat content. While Figure 2 shows the effect of fat content for only a few selected compounds, most of the compounds monitored were released in greater quantity as the fat decreased. The greater release of terpenes and phenols from the low-fat product coincides with the harsher perception of spicy, peppery and smoky flavours detected by profiling studies compared with the full-fat frankfurter. In order to assess the importance of mastication and the mouth/nose environment on the release of flavour compounds, the volatile aroma compounds were also collected from the nose during eating. These studies were conducted on traditionally smoked frankfurters and the results are compared with those using static headspace collection for the same product (Figures 3a and 3b). Similar results were obtained for static headspace collections from frankfurters prepared with liquid smoke (Figure 2b) or traditionally smoked (Figure 3a), except that the peak for guaicol was considerably smaller in the frankfurters prepared with liquid smoke. The greater quantities of volatiles collected from the nose-space compared with the headspace method is due to the differences in method. Table 3 reports the ratios between quantities of volatiles released from the 5% and 30% fat traditionally smoked frankfurters analysed by static headspace and nosespace collection. Slightly higher ratios were obtained for the volatiles collected from the nose, but these differences were not large. The effect of fat on the release of these volatile components is largely unaffected by whether the food is chewed in the mouth or is placed, chopped, in a bottle. This may suggest that the release of these compounds is primarily dictated by the availability of lipid to act as a solvent for these lipophilic flavour compounds, rather than by mastication or salivation effects.

0)

nc a V

^ .2 CO

0 en

Ci

__^___

H

Kd

n*S T I

^^ 00 rH

>

>

1—*

t—(

(N

j

1—1

rH rH 1—1

-t^ /-^

+J .CU .^ ^ 1

-M .CU

Tt^

^

CO


rH

1

1

1

>

1-1

o

1

HH

1

>

I >

HS L

!

^^^H 1

Tt*

gOT ^ SB9JB 5[B8d UB8p\[

1

Tt^

CD

"^^^H

1

00

I-|H

h

(N

01 X seaxB 5[e8d ue8j/\[

co

IC

eg CO

TO

CO

Xr

0

T^ .5 CD

a

^

O

0

O

-^

03

0

0

'^

^

•rH

0

^ s^

0

> o

PH

2

0

C»

0

0

o

0)

a

O

o

CO

c« o CD

^

-fH

(D

CO

0

^ CO

pel

0.? > ^

C«

^a

CO A ^ 0 0

X

a o > S

•fH

(B :=!

^^

0)

- 0 CO ^

>

sa

0

2 I '^ 0)

¥

H i fi

1-1 iC

<3i

^

t 0) 0

O

a

'-•3

1—1—1—1—1— o o o o o c5

D ES •

CO

\ ^ "^ ^ O (N ^

^^^^^B 1

h


1—1 Tt^

C5

1H 1 1- H

"^^^^H 1 1—1 CO

CO

1

>

H[J

,

c5

1^ i o

^ (M B8JB 5[B8d u e a p

o

_ _ ,

gOT ^ SB8JB 5[B8d u e a j ^

X

1 o CO

SI o^ F-i

Q ^ ^ Q

^

f^

2

K ^

CO


CO

-i

e

267

b

^" t CO

^ o

S=

<^

CO

5*. 13

O

O >

-^ p -2

3 "OH

ir

CJ (?<|

\^.

§

^

II s^

CJ

e

s ^ CO

268 Table 3 Ratios of the mean peak areas for volatiles released by the frankfurter collected by the static headspace and nose-space Compounds Static Nose-space headspace Assessor 1 3-carene 1.78 3.48 2.98 a-terpinene 2.51 limonene 2.51 3.18 2.91 cineole 3.07 2.52 p-cymene 3.58 guaicol 1.10 3.81 Values represent the ratios of means of 4 replicates.

5% and the 30% fat methods Nose-space Assessor 2 3.74 3.66 2.84 4.52 4.07 3.28

The structure of frankfurters has been described as a complex emulsion in which globules of fat are stabilized in an aqueous/protein matrix [29, 30]. A previous study [24] has attempted to interpret the flavour release from these frankfurters in terms of the model proposed by McNulty [31] to explain the release of flavour from emulsions. This model assumes that the frankfurters are oil in water emulsions which are diluted by the addition of saliva on eating. Calculations according to this model suggest that the equilibrium aqueous terpene concentration should be increased by ca. 6 fold when the fat content is reduced from 30% to 5% [24]. The results described in this paper show an increase of ca. 2-4 fold in the quantities of terpenes and phenols released in the headspace or nose-space between the 30% and the 5% fat frankfurters (Table 3). Thus, the model partially explains the observed increase in the release of terpenes and phenols from the low-fat frankfurter compared with the full-fat frankfurter. However, other factors, such as the presence of additional components (eg. proteins) and the fact that cooked frankfurters are not true emulsions may explain the differences observed between calculated and observed differences in flavour release.

4. CONCLUSION

Decreasing the fat content from 30% to 5% in an emulsified meat product such as frankfurters alters the flavour quality, giving a low-fat product with strong peppery, spicy and smoky flavours. This flavour imbalance appears to be related to a higher release from the low-fat product of the terpenes and phenols responsible for these flavours, rather than to the overall quantities of volatiles present. The quantities of volatiles released in the nose closely reflects that measured in the headspace, indicating that mastication and dilution with saliva have only a small effect under these experimental conditions. A model based on

269 an oil-in-water emulsion can partially explain the observed differences between frankfurters with different fat contents, suggesting that the additional fat in the full-fat frankfurters plays an important role as a solvent for the lipophilic flavour compounds, reducing the intensity of some flavours. Thus, the perceived flavour differences between frankfurters formulated with differing the proportions of fat may be explained by the more rapid release of lypophilic terpenes, phenols and other compounds in the low-fat meat product.

5. ACKNOWLEDGEMENTS

We gratefully acknowledge the funding and collaborative support received from the National Food Centre, Dublin as part of E.U. programme, AIR2-CT93-1691.

6. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

E.S. Troutt, M.C. Hunt, D.E. Johnson, J.R. Claus, C.L. Kastner, D.H. Kropf, and S. Stroda, J. Food ScL, 1992, 57, 25. J.T. Keeton, Meat ScL, 1994, 36, 261. S. Barbut and G. Mittal, J. Food ScL Technol, 1989, 22, 124. L.W. Hand, C.A. HoUigsworth, C.R. Calkins, and R.W. Mandigo, J. Food ScL, 1987, 52, 1149. E.J. Marquez, E.M. Ahmed, R.L. West, and D.D. Johnson, J. Food ScL, 1989, 54, 867. J. Park, K.S. Rhee, J.T. Keeton, and K.C. Rhee, J. Food ScL, 1989, 54, 500. P.O. Ahmed, M.F. Miller, C.E. Lyon, H.M. Vaughers, and J.O. Reagan, J. Food ScL, 1990,55,625. J.R. Claus, M.C. Hunt, and C.L. Kastner, J. Muscle Foods, 1989, 1, 1. J. Park, K.S. Rhee, and Y.A. Ziprin, J. Food ScL, 1990, 55, 871. B.W. Berry, J. Food ScL, 1992, 57, 537. B.W. Berry, J. Food ScL, 1993, 58, 34. E.S. Troutt, M.C. Hunt, D.E. Johnson, J.R. Claus, C.L. Kastner, and D.H. Kropf, J. Food ScL, 1992, 57, 19. CM. Schultz, R.W. Mandigo, and C.R. Calkins, in Annual Reciprocal Meat Conference of the American Meat Science Association', 1991,44, 206. J.J. Mize, Southern Cooperative Series Bulletin, 1972, 173. S.R. Drake, L.C. Hinnergardt, R.A. Kluter, and P.A. Prell, J. Food ScL, 1975, 40, 1065. H.R. Cross, B.W. Berry, and L.H. Wells, J. Food ScL, 1980, 45, 791. B.W. Berry and K.F. Leddy, J. Food ScL, 1984, 49, 870.

270

18. 19. 20. 21. 22. 23. 24.

25. 26. 27. 28.

29. 30. 31.

K.K. Kregel, K.J. Prusa, and K.V. Hughes, J. Food ScL, 1986, 51, 1162. R.J. Matulis, F.K. McKeith, J.W. Sutherland, and M.S. Brewer, J. Food ScL, 1995, 60, 42. C.A. Reitmeier and K.J. Prusa, J. Food Sci„ 1987, 52, 916. R. Solheim, Appetite, 1992, 19, 285. E. Hughes, S. Cofrades, and D.J. Troy, Meat ScL, 1997, 45, 273. S.B. El-Magoli, S. Laroia, and P.M.T. Hansen, Meat ScL, 1996, 42, 179. K.E. Ingham, A.J. Taylor, F.F.V. Chevance, and L.J. Farmer, in 'Flavour Science: Recent Developments', eds. A.J. Taylor and D.S. Mottram. The Royal Society of Chemistry, Cambridge, 1996, p. 386. G.B. Nickerson and S.T. Likens, J, Chromatog., 1966, 21, 1. A. Yang, G.R. Trout, and B.J. Shay, in '41st International Congress of Meat Science and Technology', 1995, San Antonio, Texas, USA, 435. D.S. Mottram, in 'Volatile Compounds in Foods and Beverages', eds. H. Maarse. Marcel Dekker, New York, 1991, p. 107. P. Werkhoff, J. Bruning, R. Emberger, M. Guntert, and R. Hopp, in 'Recent Developments in Flavor and Fragance Chemistry', eds. R. Hopp and K. Mori. VCH, Weinheim, 1993, p. 183. L.L. Borchert, M.L. Greaser, J.C. Bard, R.G. Cassens, and E.J. Briskey, J. Food ScL, 1967, 32, 419. K.W. Jones and R.W. Mandigo, J. Food ScL, 1982, 49, 1930. P.B. Mc Nulty, in 'Food Structure and Behaviour', eds. J.M.V. Blanshard and P. Lillford. Academic Press, London, 1987, p. 245.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

271

Aroma-impact compounds in cooked tail meat of freshwater crayfish {Procambarus clarkii) K. R. Cadwallader' and H. H. Baek^ ^Department of Food Science and Technology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Box 9805, Mississippi State, MS 39762 ^Department of Food Engineering, Dankook University, Chunan 330-714, South Korea

ABSTRACT Volatile components from cooked crayfish tail meat were isolated by vacuum steam distillation-solvent extraction (VSDE) and by headspace techniques (static and dynamic modes). Predominant odorants in VSDE extracts were evaluated by aroma extract dilution analysis (AEDA), while the intense odorants in the sample headspace were detected by gas chromatography-olfactometry (GC-0) of decreasing headspace volumes for static headspace sampling (GCO-H) and of decreasing purge gas volumes for dynamic headspace sampling (GCO-DHS). A total of 28 odorants (with average logsflavor dilution (FD) factors > 2) were detected by AEDA. The most intense odorants (average logsFD factors > 4) were 2-acetyl-lpyrroline (popcorn), 3-(methylthio)propanal (cooked potato), (E,E)-2,4-decadienal (fatty, fried) and several unknowns with roasted potato/nutty, crayfish shell/stale/hay, and crabby/fried fish aroma notes. Results of GCO-H and GCO-DHS revealed a total of 16 and 12 intense odorants, respectively, in the headspace above cooked crayfish tail meat. Odorants requiring the lowest static headspace (<2.5 mL) or purge gas volume (<100 mL) for detection were hydrogen sulfide (cooked egg), trimethylamine (cooked fish), methanethiol (rotten/sulfurous/putrid), acetaldehyde (sweet/ethanolic), l-octen-3-one (mushroom), dimethyltrisulfide (cooked cabbage), 3-(methylthio)propanal, and an unknown (wild onion/garlic).

1. INTRODUCTION The crayfish industry is the largest commercial crustacean aquaculture industry in the United States with an annual harvest exceeding 45 million kg (1). The predominant commercial species is the red swamp crayfish {Procambarus clarkii). Crayfish have the same general appearance as lobsters, but adults are much smaller in size. During processing, the smallest grade of live crayfish are boiled (or steam-cooked) and then "peeled" to recover the tail meat. Crayfish tail meat is an essential part of Louisiana Cajun-cuisine, which has attracted considerable attention in the last decade. Studies have been conducted on the volatile constituents of cooked crayfish tail meat (2-3), crayfish hepatopancreas (4), and crayfish processing by-product (5-7). However, little is known about the compounds responsible for the characteristic aroma of cooked crayfish tail meat.

272

Gas chromatography-olfactometry (GC-0) can be used to determine the predominant odorants in a volatile flavor isolate (8). One such procedure is aroma extract dilution analysis (AEDA), in which serial dilutions of a volatile extract are evaluated by GC-0 to obtain a flavor dilution factor (FD-factor) for each odor-active compound (9). The FD-factor for an odorant is equal to the ratio of its concentration in the initial extract to its concentration in the highest dilution at which it is detected by GC-0 (9). FD-factors are useful for understanding the role each compound plays in the overall aroma of a food. AEDA has been previously employed in the study of the aromas of crustaceans (10-12). A major limitation of AEDA, however, is that it does not allow for the evaluation of odorants having boiling points lower than the solvent used for extraction. These odorants may be fully or partially lost during concentration of the extract or may co-elute with the solvent during GC-0. Alternatives to AEDA have been developed in order to determine the relative impact of the highly volatile compounds on the aroma of a food. One such technique involves GC-0 of static headspace samples (GCO-H)(13). In GCO-H, a decreasing series of sample headspace volumes is evaluated by GC-0. Odorants detected in the lowest headspace volume have the highest odor-impact in the headspace of the sample. An alternative to GCOH is the GC-0 evaluation of dynamic headspace samples (GCO-DHS), in which a decreasing series of purge gas volumes is evaluated by GC-0. Like GCO-H, odorants detected at the lowest purge gas volume during GCO-DHS are considered to have the highest odor activity. The aim of the present study was to identify the predominant odorants in cooked crayfish by AEDA, GCO-H, and GCO-DHS analysis. 2. MATERIALS AND METHODS 2.1. Materials. Live aquacultured crayfish (Procambarus clarkii) harvested from earthen ponds during March-May of 1996 and 1997 were obtained from the Aquaculture Research Unit of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. After being washed in tap water, crayfish were boiled for 7 min at 100° C (14) and then immediately cooled in an ice-water bath. Boiled crayfish were "peeled" by hand to recover the tail meat. Hepatopancreas tissue and any digestive tract adhering to the tail meat were removed using a knife. Tail meat was coarsely chopped prior to analysis. 2.2. Chemicals. Reference aroma compounds listed in Tables 1-3 were obtained from the following commercial sources: nos. 1-6, 8, 9, 12-14, 16, 19, 20, 22, 27, 33, 34, 38, 39 (Aldrich Chemical Co., St. Louis, MO); 31 (Bedoukian Research Inc., Danbury, CT); 13 (Lancaster Synthesis, Inc., Windham, NH). Standard no. 15 was obtained from Dr. R. Buttery (USDA, ARS, WRRC, Albany, CA) and 29 from USDA, SRRC (New Orleans, LA). Compound 17 was synthesized according to Ullrich and Grosch (15). 2.3. Vacuum simultaneous steam distillation-solvent extraction (VSDE). VSDE was performed on 1-kg of crayfish tail meat as previously described (7). The extract was concentrated to 100 |iL under a stream of nitrogen and stored at -20°C until analysis. 2.4. Aroma extract dilution analysis (AEDA). The gas chromatography-olfactometry (GCO) system consisted of a Varian 3300 (or 3400) GC (Varian Instrument Group, Walnut Creek, CA) equipped with a flame ionization detector (FID) and a sniffing port. Serial dilutions (1:3) of VSDE extracts were prepared in dichloromethane. Each dilution (1 ^iL) was injected into a

273

capillary column (DB-WAX or DB-5ms, 30 m length x 0.25 mm i.d. x 0.25 fam film thickness (df); J&W Scientific, Folson, CA.). Column eluent was split 1:1 between FID and sniffing port using deactivated fiised silica capillaries (1 m length x 0.25 mm i.d.). GC conditions were the same as those of GC-MS except that the oven temperature was programmed from 40°C to 200°C at a rate of 8°C/min with initial and final hold times of 5 and 60 min, respectively. FID and sniffing port were maintained at a temperature of 250°C. Sniffing port was supplied with humidified air at 30 mL/min. Further details of AEDA have been previously reported (16). 2.5. Gas chromatography-olfactometry of headspace samples (GCO-H). A 10 g sample of crayfish tail meat was placed in a 250-mL round bottom flask which was sealed with a septum. The flask was incubated in a 60°C water bath for 20 min and a headspace sample was drawn from the flask using a preheated (60°C) gastight syringe. The headspace sample was immediately injected at a flow rate of 5 mL/min into an HP5890A GC (Hewlett-Packard, Co., Palo Alto, CA) equipped with a packed column inlet modified for capillary GC injection (Uniliner Sleeve Adaptor; Restek Corp., Bellefonte, PA). Separations were performed on DB-WAX and DB-5ms fused silica capillary columns (30 m length x 0.53 mm i.d. x 1 |im df for DB-WAX (or 1.5 |Lim df for DB-5ms); J&W Scientific). Prior to injection, a 15 cm section of column was cooled in liquid nitrogen to cryofocus the volatiles. The GC was rapidly heated and the run started when the oven temperature reached 40°C. Other GC conditions were the same as those described for AEDA. A fresh crayfish tail meat sample was used for each analysis. 2.6. Gas chromatography-olfactometry of dynamic headspace samples (GCO-DHS). A Tekmar 3000 Purge and Trap Concentrator/Cryofocusing Module (Tekmar Co., Cincinnati, OH) coupled with an HP5890 series II GC was used for GCO-DHS. A crayfish tail meat sample (3 g) in a 25-mL purge tube was preheated to 60°C for 5 min. The volatiles were purged at 60°C with helium (40 mL/min) for 1.25, 2.5, 5, 10, or 20 min onto a Tenax TA trap (part no. 12-0083-303, Tekmar Co.) maintained at 0°C. After sampling, the trap was dry purged for 5 min and then the volatiles were desorbed (180°C for 1 min) and subsequently cryofocused (-120°C) onto a 15 cm section of 0.53 mm i.d. deactivated fiised silica capillary column. Transfer lines and valves were maintained at a temperature of 175°C. Trap pressure control was set at 4 psi. Helium flow during thermal desorption of the Tenax (20 mL/min) and cryofocusing (1.4 mL/min) traps was controlled by the split/splitless electronic pressure control pneumatics of the GC. Cryofocused volatiles were thermally desorbed (180°C for 1 min) directly into the analytical GC column. All other GC and GC-0 conditions were the same as described for AEDA. A fresh crayfish tail meat sample was used for each analysis. Between each analysis, the system was purged after installation of clean glassware followed by baking of the Tenax TA trap (225°C for 15 min). 2.7. Gas chromatography-mass spectrometry (GC-MS). The GC-MS system consisted of an HP5890 Series II GC/5972 mass selective detector (MSD, Hewlett-Packard, Co.). Separations were performed on fiised silica capillary columns (DB-WAX or DB-5ms, 60 m length X 0.25 mm i.d. x 0.25 |im df; J&W Scientific). The carrier gas was helium at a constant flow of 0.96 mL/min. Oven temperature was programmed from 40°C to 200°C at a rate of 3°C/min with initial and final hold times of 5 and 60 min, respectively. MSD conditions were

274

as follows: capillary direct interface temperature, 280°C; ionization energy, 70 eV; mass range, 33-350 a.m.u.; EM voltage (Atune + 200 V); scan rate, 2.2 scans/s. VSDE extract (3 jiL) was injected in the splitless mode (30 s valve delay; 200°C injector temperature). For DHS, the purge and trap system was connected to the MSD and the previously mentioned 0.25 mm i.d. capillary columns were used for analysis. 3. RESULTS AND DISCUSSION Freshly cooked crayfish tail meat has a weak and delicate aroma that can be described as sulfurous, potatoey, earthy, fishy, and fatty. In our initial experiments, we used simultaneous steam distillation-solvent extraction under reduced pressure (VSDE) to extract exhaustively the volatile constituents from the tail meat. The VSDE isolated volatiles were subsequently evaluated by aroma extract dilution analysis, which revealed the presence of 28 intense odorants (average logsflavor dilution (FD) factors > 2; Table 1). These odorants were grouped into nine general categories according to their aroma characteristics (listed in descending order of intensity, which was based on the sum of the logsFD factors of the group constituents); fatty/fishy/stale (nos. 12, 22, 27, 31, 32, 34, 36, 37), popcorn/meaty (nos. 14, 15,18, 25, 26, 33), potato/nutty (nos. 19-21, 24), earthy/musty (nos. 13, 29, 35), burnt, plastic (nos. 10, 30), phenolic (nos. 38, 39), sulfurous (no. 16), dark chocolate/malty (no. 8), and buttery (no. 9). Two unknown odorants (nos. 32, 37) within the fatty/fishy/stale group had intense and distinctive cooked crayfish shell- and crayfish hepatopancreas fat-like aroma notes. A third unknown (no. 36) was described as green/fishy. The identified odorants in this group were lipid-derived unsaturated aldehydes and contributed fatty/fried (no. 34), cucumber/melon (no. 27), fresh fish/melon (no. 31), stale/bitter/nutty (no. 22), rancid/crabby (no. 12) aroma notes. Compounds nos. 12, 22, 27, 31 and 34 were previously reported as volatile constituents of crayfish processing by-products (5-7), while only no. 22 was previously found in the tail meat (2). These alkenals probably originated from polyunsaturated fatty acids through enzymemediated oxidation and/or retro-aldol condensation reactions (17-19). All the odorants in the popcorn/meaty group were described as popcorn-like except for 2methyl-3-furanthiol (MF, no. 14) which had a vitamin/meaty note. MF has been previously reported as a potent odorant in many foods, including tuna (20) and beef (21). The most intense odorant in this group was 2-acetyl-l-pyrroline (AP, no 15). This Maillard reaction product (22) has been identified in crayfish processing by-products (7) and is an important component of the aromas of cooked crustaceans (10-12). Another important odorant in this group was identified as 2-acetyl-2-thiazoline (AT, no. 33). Like AP, this Maillard reaction product contributes to the aromas of a number of foods (23). 3-(Methylthio)propanal (MP, no. 20), a Strecker aldehyde (24), was the most intense odorant in the potato/nutty group, followed by nos. 24,19, and 21 in decreasing order of odor intensity. MP was previously shown to contribute a strong cooked potato aroma note to cooked crustaceans such as blue crab (10, 11) and spiny lobster (12). Compound no. 19, a lipid-derived odorant, was identified as a volatile constituent of the crayfish processing byproduct (6, 7). Compounds contributing earthy/musty aroma notes included l-octen-3-one (no. 13), 2methylisobomeol (no. 29) and an unknown (no. 35). Compound no. 13 (mushroom-like) may have been formed via enzyme-mediated lipid oxidation (17-19) and is a common volatile

275 Table 1. Predominant odorants in volatile flavor extracts prepared by vacuum steam distillation-solvent extraction. No.'

RIb

Compound

Aroma description'^

DB-WAX DB-5ms

8 9 10 12 13 14 15 16 18 19 20 21 22 24 25 26 27 29 30 31 32 33 34 35 36 37 38 39

3-methylbutanal^ 2,3-butanedione^ unknown (Z)-4-heptenaf l-octen-3-one^ 2-methy 1-3 -furanthiol 2-acety 1-1 -pyrroline*^ dimethyltrisulfide^ unknown (E)-2-octenal^ 3 -(methy lthio)propanaf unknown (E)-2-nonenal^ unknown unknown unknown (E,Z)-2,6-nonadienal^ 2-methylisobomeol^ unknown (E,E)-2,4-nonadienal^ unknown 2-acetyl-2-thiazoline^ (E,E)-2,4-decadienaf unknown unknown unknown p-cresol^ 3-methylindole^

932 969 1100 1238 1298 1308 1333 1375 1425 1428 1455 1496 1531 1558 1576 1585 1587 1600 1622 1712 1732 1769 1816 1827 1897 1985 2096 2501

658 606 898 978 864 912 968 909 1161

1155 1196 1219 1162 1323

1080 1405

Av Logs FD-Factor'^

dark chocolate, malty buttery sour, plastic rancid, crabby mushroom, earthy vitamin, meaty popcorn cooked cabbage popcorn raw nut, earthy, potato cooked potato roasted potato skin stale, bitter, nutty roasted potato, nutty popcorn, roasted popcorn, roasted cucumber, melon earthy, soil burnt agar fresh fish, green, melon crayfish shell, stale, hay popcorn, roasted fatty, fried leather, moldy, earthy green, fishy crabby, fried fish fecal, stable chlorine bleach, pungent

3 2 3.5 3 3 3 4.5 3 2.5 3 4.5 2 3 4 2.5 3.5 3.5 3 3.5 3.5 5 3.5 4.5 3 2 4.5 3 3

'Numbers correspond to those in Tables 2 and 3. ^ Retention indices calculated from GC-0 results. ^ Aroma description as perceived by panelists during GC-0. Average logsflavor dilution factor (n=2). ^ Compound identified based on comparing its mass spectrum, RI values on two capillary columns and aroma properties with those of the reference compound. ^MS signal too weak to interpret-compound identified using remaining criteria in footnote e. constituent of crustaceans (7, 10-13). The occurrence of compound no. 29, a well known environmental off-flavor in aquaculture products (25), was described as earthy/soil-like. Burnt/plastic notes were contributed by two unknown odorants (nos. 10 and 30). The presence of the phenolic group of odorants was considered undesirable because of their malodorous fecal/stable (no. 38) and chlorine bleach/pungent (no. 39) notes. The sulflirous note (no. 16) also was considered to be undesirable because of its cooked cabbage note. This

276 compound was previously identified as negatively impacting the aroma of enzymehydrolyzed crayfish processing by-product (7). Results of AEDA can indicate the potent odorants among the intermediate and low volatility aroma fractions. However, this technique cannot assess the importance of components possessing higher volatilities than the solvent. For example, it is not possible to accurately assess the importance of 3-methylbutanal (no. 8) because it co-elutes with the solvent peak. Furthermore, these odorants are more likely to be important when the aroma is perceived orthonasally. Therefore, GCO-H and GCO-DHS were performed to evaluate the contribution of the highly volatile components to the aroma of cooked crayfish tail meat. Results of GCO-H and GCO-DHS revealed a total of 16 and 12 intense odorants, respectively, in the headspace above cooked crayfish tail meat (Tables 2 and 3). The headspace volatile profile of cooked crayfish tail meat was similar to that reported for canned salmon (26), boiled salmon (27), and boiled trout (13). Odorants requiring the lowest static headspace (<2.5 mL) during GCO-H were hydrogen sulfide (no. 1), trimethylamine (no. 2), methanethiol (no. 3), l-octen-3-one (no. 13), and an unknown (no. 7). Compound no. 3

Table 2. Lowest static headspace volumes required to detect odorants by gas chromatography-olfactometry of cooked crayfish tail meat. No."

RIb

Compound

Aroma description^

DB-WAX DB-5ms

1 2 3 4 6 7 8 11 12 13 15 16 17 20 23 28

hydrogen sulfide^ trimethylamine^ methanethiol^ acetaldehyde^ 2-methylpropanaf unknown 3-methylbutanal^ unknown (Z)-4-heptenaf l-octen-3-one^ 2-acety 1-1 -pyrroline^ dimethyltrisulfide^ (Z)-l ,5-octadien-3-one^ 3 -(methy lthio)propanaf unknown dimethyltetrasulfide^

<600 620 640 672 812 870 908 1125 1234 1305 1355 1392 1413 1471 1548 1604

<500 <500 <500 <500 537 653 897 975 919 979 996 911 1246

cooked egg cooked fish rotten, sulfurous, putrid sweet, ethanolic dark chocolate, malty wild onion, garlic dark chocolate, malty skunky, rubbery rancid, crabby mushroom, earthy popcorn cooked cabbage mushroom, planty cooked potato stale, bitter cooked cabbage

Volume (mL) 2.5 2.5 1 20 10 2.5 20 10 20 2.5 10 5 5 10 5 20

^Numbers correspond to those in Tables 1 and 3. ^ Retention indices calculated from GC-0 results. ^ Aroma description as perceived by panelists during GC-0. ^ Lowest static headspace volume required for detection by GC/0. ^ MS signal too weak to interpretcompound identified by comparing its RI values on two capillary columns and its aroma Properties with those of a reference compound. ^Reference unavailable—compound identified by comparison of its RI on DB-WAX and its aroma properties with published data (13).

277

Table 3. Lowest dynamic headspace purge gas volumes required to detect odorants by gas chromatography-olfactometry of cooked crayfish tail meat. No."

RIb

Compound

Aroma description^

DB-WAX DB-5ms

2 3 4 5 6 7 8 13 15 16 20 28

trimethylamine^ methanethiof acetaldehyde^ dimethylsulfide^ 2-methylpropanal^ unknown 3-methylbutanal^ l-octen-3-one 2-acetyl-1 -pyrroline^ dimethyltrisulfide^ 3 -(methy lthio)propanal^ dimethyltetrasulfide^

624 643 679 721 816 894 915 1300 1355 1381 1456 1590

<600 <600 <600 <600 <600 617 654 991 925 982 907 1217

Volume'* (mL)

cooked fish rotten, sulfurous, putrid sweet, ethanolic canned com dark chocolate, malty wild onion, garlic dark chocolate, malty mushroom, earthy popcorn cooked cabbage cooked potato cooked cabbage

100 50 100 400 200 100 400 50 800 50 100 400

^Numbers correspond to those in Tables 1 and 2. ^ Retention indices calculated from GC-0 results. ^ Aroma description as perceived by panelists during GC-0. ^ Lowest dynamic headspace purge volume required for detection by GC-0. ^ Compound identified by comparing its mass spectrum, RI values on two capillary columns and its aroma properties with those of a reference compound. MS signal too weak to interpret—compound identified using remaining criteria in footnote e. ^Reference unavailable—compound identified by comparison of its RI on DB-WAX and aroma properties with published data (13). imparted a rotten/sulfurous/putrid note and was the most intense odorant in this group. Odorants requiring the lowest purge volume (<100 mL) for detection by GCO-DHS were similar to the those indicated by GCO-H and included trimethylamine (no. 2), methanethiol (no. 3), acetaldehyde (no. 4), l-octen-3-one (no. 13), dimethyltrisulfide (no. 16), MP (no. 20), and an unknown (no. 7). The most intense odorants in this group were nos. 3, 13, and 16. Compound no. 1 was not detected during GCO-DHS. This might be due poor adsorption of this highly volatile and polar compound onto the Tenax trap. In general, a group of compounds with sulfurous/sulfide notes (nos. 1, 3, 5, 16, 28) were predominant in the headspace of crayfish tail meat, especially nos. 1, 3, and 16. The origin of these sulfur-containing compounds in fish has been recently reviewed (19). Other odorants included the Strecker aldehydes (nos. 6, 8, 20), (Z)-l,5-octadien-3-one (no. 17), (Z)-4heptenal (no. 12) and two unknowns (no. 11, 23).

4. CONCLUSIONS The combined results of AEDA, GCO-H, and GCO-DHS reveal that aroma-impact compounds of low, intermediate, and high volatility are found in cooked crayfish tail meat.

278

Use of these complementary techniques led to the identification of 2-acetyl-l-pyrroline, MP, (E,E)-2,4-decadienal and several unknowns with roasted potato/nutty, crayfish shell/stale/hay, and crabby/fried fish aroma notes as predominant odorants within the intermediate and low volatility aroma group; odorants with mostly sulfurous/sulfide aromas have the greatest impact on the headspace aroma. Journal article J9237 of the Mississippi Agricultural and Forestry Experiment Station.

7. REFERENCES 1. S.P. Meyers, INFOFISHMarketing Digest, 3 (1987) 31. 2. W. Vejaphan, T.C.-Y. Hsieh, S.S. Williams. J. FoodSci., 53 (1988) 1666-1670. 3. T.C.-Y. Hsieh, W. Vejaphan, S.S. Williams, J.E. Matiella, in: T.H. Parliament, C.T. Ho and R.J. McGorrin, (Eds), Thermal Generation ofAromas, ACS Symposium Series no. 409, American Chemical Society, Washington, DC, 1989, pp. 386-395. 4. T.E. Kinlin, J.P. Walradt, and W. Denton, in: J.W. Avault Jr. (Ed.), Fresh Water Crayfish, International Symposium on Freshwater Crayfish, Louisiana State University, Baton Rouge, LA, USA, 1974, pp. 175-184. 5. U. Tanchotikul, T.C.-Y. Hsieh, J. FoodSci., 54 (1989) 1515-1520. 6. Y.-J. Cha, H.-H. Back and T.C.-Y. Hsieh, J. Sci. FoodAgric, 58 (1992) 239-248. 7. H.H. Back and K.R. Cadwallader, J. Agric. Food Chem., 44 (1996) 3262-3267. 8. T. Acree, in: C.T. Ho and C.H. Manley (Eds.), Flavor Measurement, Dekker, New York, 1993, Chapter 4. 9. W. Grosch, Trends FoodSci. TechnoL, 4 (1993) 68-73. 10. H.Y.Chung and K.R. Cadwallader, J. Agric. Food Chem., 42 (1994) 2867-2870. 11. H.Y. Chung, F. Chen, and K.R. Cadwallader, J. FoodSci., 60 (1995) 289-291,299. 12. K.R. Cadwallader, Q. Tan, F. Chen, and S.P. Meyers, J. Agric. Food Chem., 43 (1995) 2432-2437. 13. C. Milo and W. Grosch, J. Agric. Food Chem., 43 (1996) 459-462. 14. G.A. Marshall, M.W. Moody, Hackney, C.R. and J.S. Godber, J. FoodSci., 52 (1987) 1504-1505. 15. F. Ullrich and W. Grosch. J. Am. Oil Chem. Soc, 65 (1988) 1313-1317. 16. H.H. Back, K.R. Cadwallader, Marroquin, E. and J.L. Silva, J. FoodSci., 62 (1997) 249252. 17. R.C. Lindsay, Food Rev. Intern. 6 (1980) 437-455. 18. J.B. Josephson, in: H. Maarse (Ed.), Volatile Compounds in Foods and Beverages, Dekker, New York, 1991, pp. 179-202. 19. T. Kawai, Crit. Rev. FoodSci. Nutr., 36 (1996) 257-298. 20. D.A. Withcombe and C.J. Mussinan, J. FoodSci., 2 (1988) 658, 660. 21. R. Kerscher and W. Grosch, Z. Lebensm Unters.Forsch A, 204 (1997) 3-6. 22. P. Schieberle, Z Lebensm. Unters. Forsch., 185 (1990) 206-209. 23. T. Hoffmann and P. Schieberle, J. Agric. Food Chem., 43 (1995) 2946-2950. 24. D.A. Forss, J. Dairy Res. 46 (1979) 691-706. 25. J.F. Martin, C.P. McCoy, C.S. Tucker and L.W. Bennett, Aquacult. Fish. Manage., 19 (1988) 151-157. 26. B. Girard and S. Nakai, J. FoodSci. 59 (1994) 507-512. 27. C. Milo and W. Grosch, J. Agric. Food Chem., 44 (1996) 2366-2371.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

279

Comparison of flavor characteristic of domestic chicken and broiler as affected by different processing methods A. Apriyantono and Indrawaty Department of Food Technology and Human Nutrition, Bogor Agricultural University, Kampus IPB Darmaga, PO Box 220, Bogor 16002, Indonesia Abstract Two types of chickens from the Indonesian market, namely domestic chicken and broiler, were used in this study. Difference in the sensory properties and volatile composition with respect to the lipid content and composition of each variety was examined. The two types of chicken were boiled, fried or roasted. Boiled, fried or roasted domestic chicken was generally more accepted by 30 semi-trained panelists which characterized these as having a higher intensity of all taste and aroma descriptions than those of broilers. However, the fatty note and bitter taste of the broiler variety exceeded those of domestic type. Broilers used contained 6.4% total lipid as compared to 2.6% for domestic chickens. Both chicken lipids contained palmitic, oleic and linoleic acids as their major components. Quantitatively, total volatiles of boiled and fried broilers were higher than those of domestic chickens. Boiled broiler was characterised by the presence of higher amounts of long chain aldehydes and fatty acids than those of the boiled domestic chicken. This was in line with a higher intensity of fatty aroma of boiled broilers. Surprisingly, no sulfur volatiles were detected in boiled domestic chicken, but a large number of sulfur compounds were present in the boiled broilers.

1.

INTRODUCTION

In general, Indonesians consume more chicken meat than meat from other species, mainly because of cost factors. Two types of chicken, namely domestic and broiler, are generally consumed in Indonesia. People prefer domestic chicken as compared with broiler since they believe it has a better flavor. However, very little information is available on the differences in their flavor, either sensory characteristics, volatile composition, or correlation between their sensory characteristics and their volatile composition. In addition, very few studies have reported the dependence of chicken flavor on different processing methods [1]. This study was focused to investigate the differences of sensory characteristics and volatile composition of domestic chicken and broiler as affected by different processing methods and their relation with the lipid content and composition of each variety.

280

2.

MATERIALS AND METHOD

2.1.

Materials Domestic chickens were obtained from a local restaurant supplier, whereas broilers were obtained from a local chicken farm. The chickens used were in the condition of post rigor (2.5 - 4h after slaughtering, without chilling treatment), and only the breast part (without skin) was used in this study. The age of domestic chickens was 3.5 months, whereas that of broilers was 40 days (this age difference was intentionally chosen, since in both ages the chickens were sold and normally available in the market). Frying oil used was refined, bleach, deodorized palm oil and was a gift from PT. Sayang Heulang (Jakarta). Some aroma standards for sensory description analysis, i.e. Firanova boiled A, Firanove roasted B, Firanova boiled C, and 2,4decadienal were gifts from PT. Firmenich, Indonesia. Other materials, namely salt, sugar, monosodium glutamate (MSG) were purchased from a local market, whereas caffeine was from Cica Company (Japan). All chemicals used analytical grade (Merck, Germany). Standards of fatty acid methyl esters were obtained from Nucheck (Denmark). 2.2.

Method

2.2.1. Cooking procedure Chicken breasts were boiled for 20 min, where at the end of boiling the internal temperature of the chickens was 750C. Frying of the breast chickens was done using refined, bleached, deodorized palm oil for 20 min and the final internal temperature of the chickens was 80'C. Roasting of the breast chickens was done using an oven at a temperature of 2500C for 30 min and the final internal temperature of the chickens was 900C. 2.2.2. Sample preparation Chicken breasts were skinned and deboned, the meat was then homogenised using a blender. The meat used for analysis of volatile composition and sensory was fresh, whereas that used for analysis of total lipid, fatty acid composition and moisture content was stored in a freezer for 7 days. 2.2.3. Analysis of moisture content, lipid content and fatty acid composition Moisture content was determined using an oven drying method, whereas lipid content was determined using a Soxhiet method with petroleum ether (b.p. 40 -600C) as the solvent. Analysis of fatty acid composition was done in two steps, first methylation of the fatty acids of chicken oil using BF3methanol [2]-l second analysis of the fatty acid methyl esters obtained using a gas chromatograph (Shimadzu GC 9AM) and margaric acid methyl ester as the internal standard. The capillary column used for fatty acid analysis was DB-23 (30 m, i.d. 0.25 mm, and film thickness 0.25 [im, J&W, USA).

281 2.2.4. Sensory analysis Consumer preference (hedonic) test for aroma, taste and general acceptance of processed chickens was done using 30 semi-trained panelists with a hedonic scale ranging from 1 to 7 (dislike very much to like extremely). Quantitative flavor description analysis of processed chickens was done using a modified QDA (Quantitative Descriptive Analysis) procedure [3,4]. This procedure involved several steps, i.e. selection of panelists, test of panelists, training of panelists and measurement of the samples. In order to quantify the intensity of the flavour descriptions, a series of standard intensities of each description was used (Table 1). Table 1 Standard of Intensity of taste and aroma description for quantitative sensory description analysis Description Aroma Boiled chicken

Definition Aroma of boiled chicken

Fried chicken Aroma of fhed chicken Aroma of fat Fatty Roasted

Savoury

Aroma of roasted chicken

Aroma of savoury (tasty or delicious)

Taste Sweet

Sweet taste

Salty

Salty taste

Bitter

Bitter taste

Umami

Characteristic of MSG taste (savory)

Score^

Standard Firanova boiled A^' NaCI salt Firanova boiled A^' NaCI salt Fried chicken leg 2,4-Decadienal 2,4-Decadlenal Firanova roasted B^ NaCI salt Firanova roasted B^ NaCI salt Firanova boiled C'' Firanova boiled C'' Sucrose Sucrose NaCI salt NaCI salt Caffeine Caffeine MSG MSG

0.3% 0.5% 0.7% 1.0% 0.2% 1.0% 0.3% 0.5% 0.7% 1.0% 0.4% 0.7%

1.5% 5.0% 0.2% 1.0% (D.05% (D.08% 0.2% 0.5%

30 130 100 20 130 20 125 35 140 25 140 25 135 30 135 20 125

^Tradename of the flavoring ^The score was selected arbritarily based on preliminary examination of the relative intensity of each description and the logarithmic relatitionship between concentration and sensory Intensity

2.2.5. Volatile composition Volatiles of processed chickens were extracted using a Likens-Nickerson apparatus with diethyl ether as the extraction solvent and 2h heating time. The

282

extracts were then dried with anhydrous sodium sulfate, concentrated using a rotary evaporator followed by flushing using nitrogen until the volume was about 0.5 mL. The extracts were analysed using Shimadzu QP5000 GCMS with DBS capillary column (30 m long, i.d. 0.25 mm, film thickness 0.25 Vtm) and identification was done by matching the mass spectra obtained with those present in the NIST Library or published literature. Identification was confirmed by matching their LRI values with those reported in the literature. Quantification of volatiles was carried out by GC-MS using 1,4dichlorobenzene as the internal standard where the internal standard was put in the Likens-Nickerson apparatus before distillation. 2.2.6. Data analysis Some data were analysed using ANOVA (analysis of variance) followed by Duncan multiple range test analysis. Multivariate analysis of some data was done using The Unscrambler software version 6.1 (Camo AS, NonA/ay, 1996). 3.

RESULTS AND DISCUSSION

3.1. Lipid content and fatty acid composition Based on the analysis of lipid content using Soxhiet method and petroleum ether (b.p. 40 - 60°C) as the solvent, it was revealed that lipid content of broiler was 6.4% and was higher than that of domestic chicken which was only 2.6%. In addition, water content of broiler was 71 % and was higher than that of domestic chicken, i.e. 67%. Fatty acid composition of broiler and domestic chicken was similar, where palmitic, oleic and linoleic acids were dominant. Broiler contained higher amounts of most fatty acids, except for 20:0, 20:4 and 22:6. Special note should be taken of the palmitic and oleic acids content of broiler which were almost four times higher than that of the domestic chicken. Linoleic acid content of broiler were almost twice those of domestic chicken. Paimitoleic acid content of broiler was also much higher than that of domestic chicken, i.e. 256.0 mg/IOOg whereas domestic chicken contained paimitoleic acid only at an amount of 15.3 mg/IOOg (Table 2). The high content of unsaturated fatty acids of both chicken samples would result in formation of high amounts of volatiles derived from the fatty acids when both chicken are heated. It is also expected that higher content of unsaturated fatty acids would result in higher amounts of volatiles formed. 3.2. Consumer preference In general, domestic chicken, either boiled, fried, or roasted, was more preferred by 30 semi-trained panelists than broilers for its taste, aroma, and general acceptance (Table 3). The results confirm the preference of the public who believe that domestic chicken is more delicious than broilers. It is also interesting to note that boiled chicken is less preferred than fried or roasted chicken. The sensory scores for domestic chicken were ranged from 3.5 to 4.6 (between dislike slightly and like slightly to between like slightly to like moderately), where the scores for broilers were lower. This phenomenon relates with the sensory description and also the volatile composition of chicken (see below).

283

Table 2 Fatty acid composition of broiler and domestic chicken Domestic chicken Fatty acid 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:4 22:6 Unidentified

GC % area

Broiler

mg/IOOg meat

0.6 0.9 20.9 1.1 8.1 30.5 22.9 0.6 0.4 5.9 1.2 7.1

GC % area

mg/IOOg meat

0.3 1.0 27.1 6.2 5.6 39.3 13.8 0.6 0.1 0.7 0.3 5.2

12.2 36.5 913.3 256.0 183.0 1664.0 611.8 26.9 3.2 32.6 16.6 nd

7.5 10.6 231.1 15.3 87.1 425.9 335.1 9.4 3.9 96.5 23.7 nd

nd = not determined

Table 3 Average hedonic scale of consumer preference for taste, aroma and general acceptance of processed chicken as judged by 30 semi-trained panelists Chicken type Preference for

Taste Aroma General acceptance

Domestic

Broiler

Boiled

Fried

Roasted

Boiled

Fried

Roasted

4.4<= 4.4''

5.r

5.5^ 5.8'

3.5' 3.8''

5.1^ 4.8"=

4.9" 5.0"

s.r

3.6"

5.1"

4.9"

5.4^ 4.6'=

5.5^ Note: 1 = dislike very much; 2 = dislike moderately; 3 = dislike slightly; 4 = like slightly; 5 = like moderately; 6 = like very much; 7 = like extremely. The same superscript letter at the same row indicates that the value is not significantly different (p=0.05) based on Duncan statistical test.

3.3. Sensory description The quantitative flavor description of taste and aroma of broiler and domestic chickens are presented in Tables 4 and 5, respectively. It can be seen that boiled, fried or roasted domestic chicken possessed a higher intensity for all taste and most aroma descriptions than those of broiler, except for the bitter taste and the fatty note.

284

Table 4 Average quantitative taste description^ of chicken as judged by 15 trained panelists in 3 replicates Chicken type

Taste description Sweet Salty Umami Bitter

1Domestic chicken

Broiler

Boiled

Fried

Roasted

Boiled

Fried

Roasted

77.9 58.4 74.1 8.8

54.8 113.0 98.9 12.9

63.7 72.5 80.9 25.9

69.5 50.4 61.9 11.7

54.2 102.8 93.3 15.2

54.4 62.9 72.9 33.0

The score is based on the intensity of each taste description standard presented in Table 1

Table 5 Average quantitative aroma description^ of chicken as judged by 15 trained panelists in 3 replicates Chicken type Aroma description Boiled Fried Roasted Fatty Savoury

Broiler

Domestic chicken Boiled 99.1 2.5 2.0 73.5 30.9

Fried 2.8 90.7 3.5 39.7 98.1

Roasted 2.6 3.3 108.8 27.1 66.6

Boiled 66.5 4.0 2.5 81.5 26.6

Fried 2.5 73.2 2.9 57.0 87.1

Roasted 2.0 2.9 96.1 30.2 54.1

The score is based on the intensity of each aroma description standard presented in Table 1

The higher intensity of fatty note of broilers may relate to the higher content of its lipid than that of domestic chickens. The higher intensity of fatty note of broilers may also reflect the lesser acceptability of broilers in comparison with domestic chickens, especially for the boiled or fried samples, and again this may correlate with the higher content of its lipids. Principal Component Analysis (PCA) was used in order to further characterize the flavor description of both chickens. Results of PCA for taste description suggested use of 3 Principal Components (PC), i.e. PCI, PC2 and PCS, where PCI explains 8 1 % , PC2 explains 15% and PCS explains only S% variation of the data. Therefore, plot of PC1-and PC2 (the two PC would explain 96% of variation of the data) would be appropriate for characterizing the taste description; the biplot of score and loading of PCI and PC2 is presented in Figure 1. Figure 1 shows that boiled broilers, or domestic chickens were characterized by sweet taste, fried chickens were characterised by umami and salty taste, whereas roasted chickens were characterised by a bitter taste. This means that sweet taste, for example, was a more dominant

285

Bi-plot Roasted chicken (B) o

o Bitter

0 Roasted chicken (D)

0 Umami

Boiled chicken (D) o

<- u o Fried chicken (B) 0 Fried chicken (D)

Boiled chicken (B) o

0 Sw

PC1 -1.0 X-expl: 8 1 % , 1 5 %

-0.5

B = broiler, D = domestic chicken Figure 1. Score and loading plot of principal component of chicken taste

1.0 -^

PC1

Bip lot 0 Savoury 0 Fried chicken (D)

0.5

0 Roasted chicken (D) 0 Fried chicken (B) 0 Roasted chicken (B)

-

0 -

0.5 -

0 Fatty 0 Boiled chicken (D) 0 Boiled chicken (B)

1.0 -

PC1

-1.0 x-expl: 63%,63%

B = broiler, D = domestic chicken Figure 2. Score and loading plot of principal component of chicken aroma

taste description in boiled chicken and significantly different from the sweet taste of fried or roasted chicken and this applies for either broilers, or domestic chickens. Results of PCA for only 2 aroma descriptions, i.e. fatty and savory note suggested the use of only one PC, i.e. PCI which explains only 63% variation of the data. The exclusion of the other aroma descriptions was due to their specific note for

286 one type of chicken, either boiled, fried or roasted. Loading and score plot of PCI and PCI of aroma description is presented in Figure 2. It can be seen from Figure 2 that boiled chickens were characterized by fatty note, whereas fried or roasted chickens were characterised by a savory note. 3.4. Volatile composition Aliphatic aldehydes, hydrocarbons, aliphatic alcohols and fatty acids dominated the volatiles of domestic chickens and broilers (Table 6). These compounds are very likely derived from their lipid degradation. Other classes, such as pyrazines were present only in roasted broilers and domestic chickens, where 2,3-diethylpyrazine was only present in roasted domestic chickens, whereas tetramethylpyrazine was only present in broilers. The presence of pyrazines only in both roasted chickens may be due to the higher internal temperatures of the chickens can be reached during roasting (i.e. 900C) and the longer heating time (i.e. 30 min) as compared to those applied for boiled or fried chickens. This means the Maillard reaction, the reaction which forms the pyrazines, took place in a greater extent in both roasted chickens. In addition, furans with long chain substituents dentified in both chickens, namely 2hexylfuran, dihydro-5-pentyl-2(3M-furanone, and 2octylfuran may be formed by the interaction of the Maillard reaction and lipid degradation products. This interaction apparently took place in a greater extent with increasing temperature since furans were present only in roasted broilers. It is surprising that a large number of sulfur compounds were present only in boiled broilers while no sulfur compounds could be detected in boiled or roasted domestic chickens. In addition, only one sulfur compound was detected in both fried chickens, i.e. 5,6-dihydro-2,4,6-trimethyl-4H-1,3,5-dithiazine. Isopentylthiosulfidewas the only sulfur compound detected in roasted broilers. The absence of sulfur compounds in the volatiles of boiled domestic chickens may be due to a lower amount of sulfur-containing amino acids present in domestic chickens compared to those present in broilers. Theoretically, the higher the temperature, the higher would be the amount of sulfur compounds formed. However, since the amount of aliphatic aldehydes and fatty acids were much higher in fried or roasted broilers as compared to their boiled counterpart, most sulfur compounds formed were not detected in fried or roasted broilers. The amount of aliphatic aldehydes present in boiled broilers was higher than that present in boiled domestic chickens. This was parallel with a higher amount of unsaturated fatty acids present in broiler. Special attention should be paid to the higher amount of long chain aldehydes, i.e. (E)-2-decenal, (E, E)-2,4-decadienal, (E)2-undecenal, tetradecanal, pentadecanal and hexadecanal, present in boiled broilers as compared to those present in boiled domestic chickens, in accordance with the higher intensity of fatty note in broilers as compared to domestic chickens. Moreover, the amount of fatty acids present in the volatile extract of broilers was higher than that of domestic chickens. The long chain aldehydes along with fatty acids may be responsible for the higher intensity of fatty note of broilers compared to that of domestic chickens. As can be seen from Table 2, total amount of fatty acids of broiler,particularly the unsaturated ones was higher than that of domestic chickens. These unsaturated fatty acids were believed as being the precursorsof aldehydes and were perhaps somewhat responsible for the higher intensity of fatty note of broilers.

287 Table 6 Volatile components (in ppm) present in boiled, fried or roasted domestic chicken and broiler No. Component

LRI

LRI Boiled Boiled (B) (D)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Aliphatic aldehyde Pentanal Hexanal (E)-2-Hexenal Heptanal (E)-2-Heptenal Octanal (E,E)-2,4Heptadienal (E)-2-0ctenal Nonanal (E,Z)-2,6Nonadienal (E)-2-Nonenal (Z)-4-Decenal (Z,Z)-2,4Nonadienal Decanal (E,E) 2,4Nonadienal (E)-2-Decenal (Z,Z)-2,4Decadienal Undecanal (E,E)-2,4Decadienal (E)-2Undecenal Dodecanal (E,E)-2,4Dodecadienal Tetradecanal An unsaturated aldehyde Pentadecanal Hexadecanal An unsaturated aldehyde Heptadecanal Heptadecenal (Z,Z) 9,17-Octadecadienal {E)-9Octadecenal

Roasted Roasted

Fried

Fried

(D)

(B)

(D)

(B)

1.5

0.1 1.9

0.2 3.7

0.2 3.5

<0.1

<0.1

<0.1

<0.1

0.2 0.2 0.1 0.1

0.2 0.2 0.1

0.1 1.5 nd 0.2 0.1 0.1 0.1

0.4 0.3 0.1 0.2

0.6 0.5 0.1 0.2

0.4 0.6 nd

0.3 1.1 nd

0.5 0.9 <0.1

0.5 1.9 nd

0.1 0.1

0.1 0.1

<0.1

<0.1

0.2 0.3 0

0.3 nd nd

Ref.

0.4 5.0 0.1 0.4 0.3 nd 0.1

<0.1

1010 1018

697' 800' 854' 899' 954' 1001' 1013^

1062 1108 1156

1062^ 0.7 1102' 0.8 1155' <0.1

0.3 2.0 <0.1

1162 1195 1199

1160' 1193' 1194^

0.2 0.2 0.1

0.3 0.1 nd

1208 1217

1204' 1218^

0.1 0.1

0.2

0.1

0.1

<0.1

<0.1

<0.1

0.2 nd

<0.1

1265 1297

1261' 1295^

0.2 0.2

0.5 0.2

0.2 0.2

0.2 0.1

0.4 0.2

0.5 0.2

1306 1321

1305' <0.1 1316^ 0.5

<0.1

1.1

0.5

0.3

0.1 0.6

0.2 0.5

1367

1376'

0.3

0.6

0.2

0.2

0.4

0.6

1411 1424

1407'

0.1

0.1 0.1

<0.1 <0.1

0.1 0.1

0.1

0.2

<0.1

<0.1

1616 1696

1611'

0.3 nd

<0.1

1.7 0.1

<0.1

804 854 904 958

1719 1833 1882

<0.1

1831'

<0.1

<0.1

<0.1

0.2

0.2

0.6

0.3

0.5

<0.1

<0.1

0.5 5.4

1.0

0.8

0.5

1.7

1.1

10,2

16.8

0.1

nd

23.5 <0.1

13.8

<0.1

11.4 <0.1

0.2 nd

0.5 0.5 0.1

0.1 nd 0.1

nd 1.4 0.2

nd 0.4 0.1

0.8

4.8

0.1

2.8

1923 1931 1994

<0.1

0.1 nd nd

2003

1.0

0.1

nd

288 Table 6. Continued No 32 33 34

1 2 3 4 5 6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Component (Z)-13Octadecenal (Z)-9Octadecenal Octadecanal Total amount Aliphatic ketone 2-Heptanone 3-Octanone 2,3-Octanedione 2-Nonanone 2-Dodecanone (E)6,10Dimethyl-5,9undecadien-2one 2-Heptadecanone Total amount (ppm) Hydrocarbon Decane 2-Methyldecane 3-Methyldecane Undecane 4-Decyne Dodecane Tridecane 3-Dodecyne 1-Tridecene 8-Methyl-1undecene 1-Tetradecene Tetradecane 3,3-Dimethyl1,6-heptadiene 1-Pentadecene Pentadecane 1-Hexadecane 1-Heptadecene

LRI

LRI Ref.

2005

Boiled Boiled (D) (B) ""01 nd

Fried (D) 01

Fried (B) 02

Roasted Roasted (D) (B) 1.8 2.7

2014

nd

nd

7.1

nd

0.1

0.5

2028

0.8 18.3

1.6 21.3

2.5 28.8

1.5 28.8

4.4 44.7

3.2 34.6

<0.1 nd nd

0.2 nd 0.1

0.2 nd <0.1

<0.1 <0.1 nd

<0.1 <0.1 nd

893 990 1065

889^ 986^

0.2 0.3 nd

<0.1 nd nd

<0.1 nd nd

1095 1399 1456

1091' 1396^ 1434'

<0.1 nd <0.1

<0.1 nd nd

<0.1 nd <0.1

nd

nd

nd

nd

0.5

0.1

01

0.1

0.3

0.4

0.2 0.1

0.5 nd

nd nd

nd nd

nd nd

nd nd

<0.1

nd

nd

nd

nd

nd

0.3 nd <0.1 0.1 nd nd <0.1

nd nd <0.1 0.1 nd nd nd

<0.1 nd 0.1 0.1 nd nd <0.1

<0.1 nd <0.1 <0.1 nd nd nd

nd nd 0.1 0.2 nd <0.1 <0.1

nd <0.1 0.1 0.1 <0.1 <0.1 nd

nd <0.1 <0.1

nd 0.01 nd

nd <0.1 <0.1

nd <0.1 <0.1

<0.1 0.2 <0.1

<0.1 <0.1 nd

nd 0.1 <0.1 <0.1

nd 0.2 0.1 <0.1

nd 0.2 nd <0.1

nd 0.3 nd 0.1

<0.1 0.4 0.1 0.1

<0.1 0.3 <0.1 0.1

1907

1005 1066

1000^

1072 1102 1126 1201 1301 1327 1339 1348 1393 1401 1451 1495 1502 1601 1679

1099' 1199' 1299'

1392' 1399^

1500' 1600'

<0.1 nd 0.1

<0.1

0.1

289 Table 6. Continued No 18 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3

1 2

Component Heptadecane Octadecane Total amount (ppm) Aliphatic alcohol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octen-3-ol {E)-2-0cten-1ol Octanol 2-Nonanol 1-Nonanol 1-Nonen-3-ol 1-Dodecanol 1-Tridecanol 1-Tetradecanol 1-Pentadecanol 1-Hexadecanol

LRI 1701 1797

LRI Boiled Boiled Ref. (D) (B) 1700" <0.1 0.1 nd 1800^ nd 1.1 1.0

Pyrazine 2,6-Dimethylpyrazine 2,3-Diethylpyrazine Tetramethylpyrazine Total amount (ppm) Sulfur compound Methylpropenyl disulphide Dihydro-3(2H)thiophenone

Fried (B) 0.1 nd 0.6

Roasted Roasted (D) (B) 0.4 0.2 0.2 nd 1.3 1.3

0.4 0.2 0.1 0.8 0.1

0.5 0.1 0.1 0.7 0.1

0.1 nd nd <0.1 0.1 nd <0.1 nd

0.3 0.1 nd <0.1 0.3 nd 0.1 0.2

0.5 <0.1 <0.1 0.1 0.1 0.2 0.1 nd

0.1

0.1

0.2

nd

0.1

0.1

0.2

0.3

0.2

nd

nd

0.2

nd

nd

nd

<0.1

0.1

nd

nd

0.3

0.3

3.6

3.1

1.3

1.3

3.3

3.1

0.4 0.1 0.1 1.0 0.5

0.2 <0.1 <0.1 0.5 0.1

0.2 nd nd nd <0.1 0.2 <0.1 nd

0.6 nd nd 0.1 0.1 0.1 <0.1 nd

0.1 nd nd <0.1 <0.1 Nd <0.1 nd

<0.1

nd

<0.1

877 978 989 1075

768^ 867' 969' 988' 1067'

0.7 0.6 0.1 1.4 0.2

1078 1100 1179 1286 1482 1583 1684 1784

1070' 1098' 1171' 1293^ 1473' 1575' 1676' 1778'

1888

1879'

(E)-2-Tridecen- 1893 l-ol A long chain 1899 alcohol 2-Pentadecyn- 1898 1-ol Total amount (ppm)

Fried (D) 0.1 nd 0.6

0.1 <0.1 <0.1 0.4 0.1

916

912'

nd

nd

nd

nd

0.1

<0.1

1083

1082'

nd

nd

nd

nd

0.1

nd

1091

1085'

nd

nd

nd

nd

nd

<0.1

nd

nd

nd

nd

0.2

<0.1

nd

0.1

nd

nd

nd

nd

nd

0.1

0

nd

nd

nd

917 944

953'

290 Table 6. Continued No

Component

"3~ 5,6-Dihydro2,4,6-trimethyl4H-1,3,5dithiazine 4 Methyl 2propenyltrisulfide 5 5-Hydroxymethyl-2methyl-3furanthiol 6 Isopentyl thiosulfide 7 2,4-Dimethyl3-thiophenethiol 8 2-Formyl-3thiophenethiol 9 A thiophene derivative 10 2-Methyl-3-(2methyl-3thienylthio) tetrahydrothiophene 11 A thiophene derivative (MW 216) Total amount (ppm)

1

2 3

4 5 6 7 8

Benzene derivative 4-Ethyl-1,2dimethylbenzene Benzaldehyde 1-Methyl-2(1methylethyl) benzene Benzeneacetal dehyde 4-Ethylbenzaldehyde 4-pentylbenzaldehyde A benzene derivative Naphthalene Total amount

LRI

LRI Ref.

1141

Boiled Boiled (D) (B) nd nd

Fried (D) 0.2

Fried (B) 0.4

Roasted Roasted (B) (D) nd nd

1149

nd

0.1

nd

nd

nd

nd

1188

nd

1.6

nd

nd

nd

nd

1196

nd

nd

nd

nd

nd

0.2

1214

nd

1.6

nd

nd

nd

nd

1219

nd

0.1

nd

nd

nd

nd

1796

nd

0.1

nd

nd

nd

nd

nd

0.1

nd

nd

nd

nd

Nd

0.2

nd

nd

nd

nd

0

3.9

0.2

0.4

0

0.2

1801

I8O5J

1961

1085

1094^

<0.1

nd

nd

nd

<0.1

nd

960 1026

961^

0.2 0.1

0.1 nd

0.1 nd

0.2 nd

nd nd

nd nd

nd

nd

nd

<0.1

<0.1

0.1

1046 1165

1171'

0.1

nd

0.1

nd

0.1

nd

1464

1476^

0.1

0.1

0.1

0.1

0.1

0.1

0.1

nd

0.1

nd

0.1

nd

<0.1 0.4

0.1 0.4

0.1 0.4

<0.1 0.2

1775 1184

1179

<0.1 0.6

<0.1 0.3

291

Table 6. Continued No.

1 2 3

4 5

1 2 3 4 5 6

7 8

1 2

Component Furans 2-Pentil furan 2-Hexylfuran Dihydro-5pentyl 2(3H)furanone 2-Octylfuran Methyl 8-(2furyl)octanoate Total amount Fatty acid Decanoic acid Dodecanoic acid Tetradecanoic acid 9-Hexadecenoic acid Hexadecanoic acid (Z,Z)-9,12Octadacadienoic acid Oleic acid Octadecanoic acid Total amount Miscellaneous D-Limonene Ethyl citrate Total amount (ppm)

LRI

LRI Ref.

Boiled Boiled (D) (B)

Fried (D)

Fried (B)

993 1093 1158

994^ 1092^^

0.5 nd nd

1296 1627

1300^

Roasted Roasted (D) (B)

0.2 nd nd

0.3 nd nd

0.3 nd nd

0.6 nd nd

0.4 <0.1 <0.1

nd nd

nd nd

nd nd

nd nd

nd <0.1

0.1 nd

0.5

0.2

0.3

0.3

0.6

0.5

1388 1586

1568'

nd 0.2

nd nd

nd 0.1

nd 0.4

<0.1 0.3

<0.1 0.1

1791

1770*

0.4

0.3

0.7

1.3

0.3

0.1

nd

nd

<0.1

0.2

nd

0.1

0.7

3.2

0.5

6.0

0.4

1.3

<0.1

0.1

nd

0.1

<0.1

0.1 0.1

0.7 0.2

0.5 0.2

6.5 1.0

0.1 <0.1

0.1 0.1

1.5

4.4

2.0

15.5

1.2

1.8

0.3 nd 0.3

<0.1 nd <0.1

<0.1 <0.1 <0.1

<0.1 0.2 0.2

<0.1 <0.1 <0.1

0.1 nd 0.1

1954 1983

1961'

2144

2150 2173

1030 1669

<0.1

2157'

1031'

1 2

Unknown unknown Unknown

820 857

nd nd

nd <0.1

nd nd

nd nd

0.1 nd

0.3 nd

3 4 5 6 7 8 9 10

Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown

931 953 1049 1137 1233 1236 1271 1282

nd nd 0.1 nd <0.1 nd 0.1 nd

nd nd 0.2 <0.1 nd nd nd nd

nd <0.1 <0.1 nd nd <0.1 nd <0.1

nd nd nd nd nd nd nd nd

<0.1 nd nd nd nd <0.1 0.1 nd

0.1 nd 0.1 nd nd nd nd nd

292 Table 6. Continued No. 11 12 13 14 15 16 17 18

Component Unknown Unknown unknown Unknown unknown unknown unknown unknown Total amount (ppm) Grand total amount (ppm)

LRI 1291 1313 1374 1476 1568 1686 1864 1874

LRI Ref.

Boiled Boiled (D) (B) nd nd nd nd nd <0.1 nd nd nd nd nd nd <0.1 nd nd <0.1 0.2 0.3

Fried (D) <0.1 nd nd nd <0.1 0.1 0.1 nd 0.3

Fried (B) nd nd nd nd 0.3 nd nd nd 0.3

2 6 ^ 6 " ""34:6

34

47.9

Roasted Roasted (D) (B) nd nd <0.1 nd nd nd <0.1 <0.1 nd nd nd <0.1 nd nd <0.1 0.1 0.3 0.6

52.2

427^

Notes: The amount of volatile presented in the table is the average of 3 replicates. LRI : Linear Retention Index on DB-5 column nd : not detected D : domestic chicken B broiler a. Ref. [5], DB-5 column b. Ref. [6], DB-5 column c. Ref. [7], DB-5 column d. Ref. [8], DB-5 column e. Ref. [9], DB-5 column f. Ref. [10], DB-5 column g Ref. [11], DB-5 column h. Ref. [12], DB-5 column 1. Ref. [13], DB-5 column Ref. [14], DB-1 column jRef. [15], DB-5 column

The higher intensity of roasted note in domestic chickens (Table 5) may relate to the higher amounts of pyrazines present in them when compared to those present in roasted broilers (Table 6). However, the higher amount of long chain aldehydes in roasted domestic chickens may not correlate with the intensity of fatty note in chickens, since the intensity of fatty note of roasted domestic chickens was a bit lower than that of roasted broilers, despite their higher content of long chain aldehydes. This may be due to the contribution of fatty note of the fatty acids present in roasted broilers, since total fatty acids of broilers was higher. Volatile composition of fried domestic chickens and broilers was similar, the only significant difference, in addition to the difference in fatty acid composition, was a higher amount of some long chain aldehydes such as heptadecanal, (Z)-9octadecenal and octadecanal in fried domestic chickens, but the content of hexadecanal and (E)-9-octadecenal were lower. This difference may not explain the higher intensity of fried note of domestic chickens compared to that of fried broilers, when fried. In order to correlate volatiles composition with aroma description of domestic chickens and broilers, multivariate analysis was carried out using PLS-2 (Partial Least Square) where aroma description was used as Y matrix and volatiles as X matrix.

293

Since the unit of aroma description differed with that of volatiles, all variables were weighted using their own standard variation (weights = 1/standard deviation). This allows all variables to contribute to the model, regardless of having a small or large standard variation from the outset [16 ]. Results from PLS-2 analysis showed that total variance of matrix X from PCI and PC2 was 18%, whereas total variance of matrix Y was 95%. The total variance of X from PCI and PC2 was very low; this means that volatiles of chickens may not be used to predict their aroma description.

4.

ACKNOWLEDGEMENT

The authors wish to thank Prof. F. ShahidI of Memorial University of Newfoundland, for a fruitful discussion and suggestion, Ir. Budi Nurtama, MAgr of Bogor Agricultural University for helping in statistical analysis, PT. Sayang Heulang for providing the refined, bleached,deodorized palm oil, and PT. Firmenich Indonesia for providing some standards for sensory description analysis.

5. REFERENCES 1

D. S. Mottram. Meat. In Volatile Compounds In Food and Beverages, H. Maarse (ed.). Marcel Dekker, Inc., New York, 1991. 2 lUPAC (international Union of Pure and Applied Chemistry). Standard Methods for the Analysis of Oils, Fats and Derivatives. Blackwell Scientific Publications, Oxford, 1988. 3 I. B. Hashim, A. V. A. Resureccion and K. H. McWafters, J. Food Sci., 60 (1995) 664. 4 K. L. Zook and J. H. Pearce. Quantitative Descriptive Analysis. In Applied Sensory Analysis of Foods, Volume 11, H. Moskowitz(ed.), CRC Press, Inc., FL, 1988. 5 R. Adam. Identification of Essential Oil Component by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, IL, 1995. 6 M. S. Madruga and D. S. Mottram. J. Sci. Food Agric, 68 (1995) 305. 7 E. Gomez, C. A. Ledbetter, and P. L. Hartsell. J. Agric. Food Chem., 41 (1993) 1669. 8 R. Triqui and G. Reineccius. J. Agric. Food Chem., 43 (1995) 1883. 9 M. S. Madruga. LRI values of authentic compounds on DB-5 column (GCMS). Department of Food Science and Technology, Reading University, Reading, 1993. 10 C. Macku and T. Shibamoto. J. Agric. Food Chem., 39 (1991) 1987. 11 N. Ramarathnam, L. J. Rubin and L. L. Diosady. J. Agric. Food Chem., 41 (1993) 939. 12 A. Leseigneur and P. Heinen. LRI values of authentic compounds on DB5 column (GC). Department of Food Science and Technology, Reading University, Reading, 1990.

294 13 S. Lee, C. Macku, and T. Shibamoto. J. Agric. Food Chem., 39 (1991) 1972. 14 M. Guntert, J. Bruning, R. Emberger, R. Hopp, M. Kopsel, H. Surburg and P. Werkhoff. Thermally degraded thiamine-. A potent source of interesting flavor compounds. In: Flavor Precursors. Thermal and Enzymatic Conversions, ACS Symposium Series 490, American Chemical Society, Washington, D.C, 1992. 15 M-F. King, M. A. Mafthews, D. C. Rule and R. A. Field. J. Agric. Food Chem., 41 (1993)1974. 16 Camo AS. The Unscrambler User's Guide. Camo, AS, Trondheim, 1996.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

295

Comparison of Flavor Components in Fresh and Cooked Tomatillo with Red Plum Tomato Robert J. McGorrin* and Ludmila Gimelfarb Kraft Foods, Technology Center, 801 Waukegan Road, Glenview, IL 60025

Abstract The tomatillo or husk tomato (Physalis ixocarpa Brot.) is a solanaceous fruit vegetable used to prepare green sauces and salsas in various Mexican dishes. It is increasingly being utilized in a variety of "fusion cooking" recipes, which blend and adapt Latin American flavor themes for contemporary North American tastes. While similar in appearance to a green tomato, the tomatillo exhibits a unique flavor profile. However, little information has been previously reported on the identity of key aroma and taste components which impact tomatillo flavor, and how they compare with those found in fresh tomato. This chapter describes the characterization of volatile and non-volatile components in fresh and cooked tomatillos in relative comparison to red plum tomato. In this study, over 50 volatile compounds were identified in tomatillo, of which aldehydes and alcohols including (Z)-3-hexenal, (E,E)-2,4decadienal, nonanal, hexanal, hexanol and (Z)-3-hexen-l-ol were the most significant in providing the "green flavor" impact. Compounds unique to tomatillo flavor included hydroxy esters, aromatic esters, 8- to 12-carbon aldehydes, decanoic acid and terpenes. The nonvolatile profile of fresh tomatillos was found to be dominated by citric acid, which contributes to its acidic taste. Odor Unit values were used to evaluate the relative significance of identified volatiles and assess their overall impact on the distinctive flavor of this novel food ingedient.

1. INTRODUCTION The tomatillo, also referred to as the Mexican husk tomato or Chinese lantern plant, {Physalis ixocarpa Brot. syn. Physalis philadelphica Lam.) is one of the important solanaceous fruit vegetables of Mexico, and a minor crop in California and other regions of the Americas [1]. The tomatillo is related to, but horticulturally distinct from, the ground cherry {Physalis pruinosa L.) which is grown in parts of Europe [2]. Tomatillos comprise the major ingredient of the fresh and cooked green chili sauces used in Mexican cooking. In appearance, tomatillos are surrounded with a tan/green paper-like husk. Removal of the husk exposes a sticky-waxy substance on the surface of the fruit skin. Typically, the fruit color ranges from light- to medium-green, however tomatillos which begin to yellow are over-ripe and are considered low in culinary quality [3].

296 An emerging trend is the increased use of new flavor ingredients to provide new culinary experiences and variety [4]. Tomatillos are readily available as fresh vegetables in the produce section of U. S. supermarkets, and therefore are increasingly being utilized in a variety of "fusion cooking" recipes. In this context, tomatillos are combined with ingredients from several Latin American dishes and heated to create unique flavor blends which are suitable for mainstream acceptance, yet have an "authentic" flair [5]. However, while the flavor chemistry of fresh and thermally processed tomatoes has been well documented in two reviews [6,7] and in significant research by Buttery et al. [8-10] and Karmas et al. [11], little data is available on the key components of tomatillo flavor. Previously published studies on tomatillo are limited to information on non-volatile flavor components derived from compositional data [1]. We undertook this study to isolate and identify the potent volatile and non-volatile flavor components from fresh and cooked tomatillos, and to compare similarities and differences to those previously reported in tomato.

2. MATERIALS Samples of tomatillos and red plum tomatoes were purchased from a local food supermarket in the Chicago metropolitan area of the United States. Both were grown in the Curical, Sinaloa region of Mexico and harvested in March, 1997. A sample of 4-heptanone (internal standard) was obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Tenax TA (20/35 mesh) was obtained from Chrompack (Raritan, NJ). Silanized glass wool was obtained from Supelco Inc. (Belefonte, PA).

3. EXPERIMENTAL METHODS 3.1. Sample Preparation Fresh tomatillos and plum tomatoes (1.5 lb. of each, approximately 15 count) were separately pooled, rinsed with water (after removing the husks from the tomatillos) and dried. After removing the stems, the samples were pureed in a Sunbeam food processor. For heattreated samples, the individual purees were heated with constant stirring in an open stainless steel pan to 100 °C, held for 2 minutes, and rapidly cooled in an ice bath. Samples requiring volatile flavor measurements were analyzed by headspace gas chromatography-mass spectrometry (GC-MS) within one day of preparation. 3.2. Sensory Evaluation An informal sensory panel comprised of four experienced members trained in the Sensory Spectrum method was used for evaluation of flavor profile differences among fi-esh and cooked samples. Descriptor terms were generated by the panelists, and intensity scores were anchored on a four-point scale from least to most intense. 3.3. Analysis of Non-Volatile Components Analyses of moisture, pH, and titratable acidity were performed at Silliker Laboratories (Chicago Heights, IL) using standard AOAC analytical methods. Sugars (glucose, fructose.

297 sucrose) were assayed using an AOAC reversed phase liquid chromatography procedure [12]. Titratable acid content was calculated as citric acid after titration with 0.1 N NaGH. Organic acids (citric, malic, lactic, oxalic, acetic, tartaric) were determined using an ion exchange chromatography method [13]. Normalized fatty acid distribution was determined on methyl ester derivatives of lipid extracts using a capillary GC method on a 30 m x 0.32 mm (0.25 jim film) DB-WAX column (J&W Scientific, Folsom, CA) [14]. 3.4. Dynamic Headspace Analyses of Volatile Components Individual tomatillo and tomato purees (3.0 g) were placed in a 100-mL Envirochem (Kemblesville, PA) sparging vessel and mixed with 5 g of distilled water. An internal standard (0.16 ppm based on the total weight of sample) was spiked into each matrix. Samples were purged for 45 min. at 50 °C with a nitrogen flow rate of 106 mL/min onto a glass desorption tube (3.0 mm i.d. x 16 cm length) packed with 100 mg 20/35 mesh of Tenax-TA adsorbent. Prior to thermal desorption, the tube was purged for 15 min at 50 °C with dry nitrogen gas (30 mL/min) to remove traces of moisture. 3.5. GC-MS Identifications of Volatile Components Tenax cartridges were thermally desorbed into the GC-MS using a Model 4010 Thermal Desorption System (Chrompack, Raritan, NJ). The Tenax trap was heated at 220 °C for 10 minutes and volatiles were cryofocused onto a fused silica cold-trap held at -140 °C. Volatiles were directly desorbed for 1 min at 220 °C with a helium carrier gas flow rate of 1.0 mL/min (37.5 cm/sec linear velocity) onto a cooled capillary column. The GC column was a 30 m x 0.25 mm DB-5MS fused silica open-tubular capillary column, 0.25 [xm film thickness (Hewlett Packard, Wilmington, DE). The GC oven temperature was ramped using a multistage temperature program starting at -10 °C for the first 3 min, increasing at 10 °C /min to 40 °C (0 min hold); then slowing to 3 °C/min to 140 °C (0 min hold); and finally 8 °C/min to 230 °C, with a 5 min hold at the upper limit. The GC column was directly interfaced to a Hewlett Packard 5972 mass selective detector via a heated transfer line maintained at 280 °C. The mass spectrometer was operated in the electron ionization mode (70 eV) scanning masses 33350 m/z at 2.2 scans/sec. The electron multiplier voltage was increased 10% to provide optimal sensitivity. All mass spectra were background-subtracted and library-searched against the National Institute of Standards and Technology mass spectral reference collection. The Wiley/NBS Registry of Mass Spectra and DB-5 Kovats retention time indices («-alkane standards) were used to facilitate compound identification.

4. RESULTS AND DISCUSSION 4.1 Sensory Comparisons A contrast of sensory panel flavor profile differences between fresh tomatillo and tomato is outlined in Table 1. Relative to tomato, the characteristic aroma of tomatillo manifests a green, weedy note, and lacks the typical tomato vine/Z^o-butyl thiazole aromatic. The tomatillo taste profile is dominated by an intense citric acid sourness, which has an oxalic acid-like impression accompanied by a moderately astringent mouthfeel. Reduced brothy/mono-sodium glutamate (MSG) flavor was observed relative to tomato, however a

298 Table 1 Flavor Profile Comparison of Fresh Tomatillo vs. Tomato Green Tomatillo Aroma

Green, weedy

Red Plum Tomato Tomato vine

Taste Sweet Sour Bitter Brothy (MSG) Capsicum (heat)

+ ++++ + + +

+ +

Flavor Aromatics Green (hexenal/hexenol) Viney (thiazole) Sulfur (Me sulfide) Geranial

++++ + + +

++

++

slight bitterness and capsicum-like heat sensation was evident. Tomatillo flavor aromatics are predominantly green/hexenol, lacking the viney, sulfur, and geranial flavors typical of tomato. Cooking of tomatillos tended to diminish the green notes and harsh acidic taste, producing a more rounded flavor profile. 4.2. Analyses of Non-Volatile Composition A summary of compositional analyses for tomatillos is presented in Table 2. Data from this study includes previously reported protein, total lipid, carbohydrate and mineral assays. Sensory evaluation of fresh tomatillo provides a dominant sour taste. The perceived increase in sourness of tomatillo compared to tomato flavor is supported by a lower pH (3.83 vs. 4.47) and increased titratable acidity (1.11% vs. 0.46%, as citric acid). Relative acidity values are unchanged with cooking, while moisture levels declined slightly as expected. Of interest is that tomatillos have 4-fold increases in total lipid and iron contents vs. tomato, which may have flavor precursor implications. Quantitative data on organic acids by ion exchange HPLC analysis are presented in Figure I. The predominant organic acid in tomatillo was found to be citric acid, accompanied by small amounts (< 0.06%)) of malic and lactic acids. Citric acid levels were 4-fold higher than tomato. Despite the sensory impression of oxalic acid, very low but comparable quantities of oxalic (0.026%)) were measured in fresh tomatillo and tomato. Acetic and tartaric acids were below the 0.01% detection limit for the HPLC method. Sugar composition results provided some intriguing comparisons (Figure 2). At first glance, the data show different distributions and overall higher levels of sugars, predominantly fructose, in cooked vs. fresh tomatillo and tomato. However, if the individual sugars are summed for each set, it was observed that total sugar content was comparable for fresh

299 Table 2 Composition Data for Tomatillo and Tomato Tomatillo Fresh Water (%)

pH Titratable acidity (as citric acid) Protein (N x 6.25) (%) Lipids Carbohydrates Carotenoids (mg %) Minerals (mg %) Calcium Iron

93.0 3.83 1.11 1.0^ 0.7^ 4.5^

4« 18« 2.3«

Tomato

Cooked

Fresh

Cooked

91.2 3.85 1.23

94.5 4.47 0.46 1.1^ 0.2^ 4.7^ 8.2<^

93.5 4.47 0.50

I3Z? 0.5^

^ M. Cantwell, et al. (1992), Reference 1. b W. A. Gould (1992), Reference 15. c W. Friedrich (1988), Reference 16. tomatillo and tomato (2.5% vs. 2.4%), and also cooked tomatillo and tomato (3.2% vs. 3.0%). The increase of simple sugars during cooking suggests the breakdown of complex polysaccharides during heating in the presence of acid. Of interest is that the relative amounts of sucrose were higher for tomatillo. This compositional difference in sugars should conceivably influence the generation of alternate flavor compounds for tomatillos through participation in Maillard pathways during the cooking process. The results of the fatty acid profile comparison are presented in Table 3. Of significance is that, of the approximately 3-fold higher lipid concentration present in tomatillo vs. tomato (Table 2), a significantly greater proportion of the lipid fraction in tomatillo is comprised of decanoic acid (CI0:0, 20%). The other predominant fatty acids are linoleic (CI8:2, 30%) and palmitic (CI6:0, 20%). The profile data for cooked tomatillo reflect a significant increase in the proportion of CI8:2 in the total lipid fraction after heating, with a simultaneous decrease in CI0:0. This suggests that fatty acid precursors are available for formation of flavorsignificant saturated and unsaturated aldehydes in the maturation stages of the fruit and during cooking. 4.3. Volatile Headspace Concentration Techniques such as dynamic headspace concentration and simultaneous distillationextraction (SDE) have been commonly used for the analysis of tomato and other natural flavors [6,10]. Volatile headspace trapping on Tenax adsorbent resins has become a mainstay for flavor analysis laboratories because it is moderately sensitive, can be performed rapidly, and is a mild sampling technique which is not prone to generate thermal artifacts, as frequently happens for SDE. Because we were interested in quantifying the effects of sample cooking, we selected this technique to compare flavor differences among fresh and heated

300

1.75

H Citric B Malic • Lactic I n Oxalic

Fresh Tomatillo

Cooked Tomatillo

Fresh Tomato

Cooked Tomato

Figure 1. Organic acid composition of fresh and cooked tomatillo and plum tomato.

n Fructose | B Glucose • Sucrose

Fresh Tomatillo

Cooked Tomatillo

Fresh Tomato

Cooked Tomato

Figure 2. Sugar composition of fresh and cooked tomatillo and plum tomato.

jj

o

a o H cd

a

o ^ ^ Q

•^

'o

<:

ON C^

VO ON

O I> oo ON

en

oo o O 3

o o

I-tH

;H

C/5

t^

O

o O

O

T^ (D

o

m

o

oo

oo r-

O vo ^ o

ON

en (N

iri

en en

1-H ON

0^ 00

^

3

in vd

ON m ON ir^

O

ON (N i n 00 00 ON

o

a

3

00 ON

(N ON

O 00 (N ^

l> 00

^^

en ^ O 00

o (N

C/5

^ ^ cd

'-j-J

o o U

o H

^ U

o 00

U

»-H

u ^

o o

o

o H o

I u

O

o o

U 5^

o OH

T3

c3

a

a o H

o o o

I I -§

E

o

301

302 tomatillos. The repeatability of the technique has been estabUshed with model mixtures, and we chose 4-heptanone as an internal standard for quantification using MS total ion peak area. 4.4. GC-MS Identifications The volatiles from 3g of tomatillo or tomato samples were concentrated in approximately 5 liters of headspace through a Tenax adsorption cartridge and desorbed directly onto a cooled DB-5 capillary column. The GC-MS total ion chromatogram (Figure 3) from the volatile fraction displays the identities of 20 key compounds with flavor significance. Table 4 provides a more detailed list of volatile compounds identified by GC-MS, along with Kovats GC retention indices and quantitative data, which was obtained relative to a 4-heptanone internal standard. The GC retention indices enabled comparison of flavor compound profiles to those reported in the literature on DB-5 columns. Figures 4 and 5 provide GC-MS profiles of fresh tomato and cooked tomatillo, respectively. The GC-MS volatile profile obtained for fresh tomatillo was highly complex, as is typical for natural products (Figure 3). A total of 52 volatile compounds were identified, of which 22 were unique to tomatillo in comparison with compounds identified from fresh tomato (Figure 4, Table 4). The most predominant volatiles were saturated and unsaturated 6-carbon aldehydes and alcohols. (Z)-3-hexenal and (^-2-hexenal were present in significant concentrations, and contribute a green, leafy herbaceous aroma. These compounds have been previously associated with the characteristic green, tomato fruit-like aroma released by tomato leaves [9]. The reduction products, (Z)-3-hexen-l-ol and (F)-2-hexen-l-ol, were observed at lower concentrations; (Z)-3-hexen-l-ol was previously reported by Buttery and Ling [9] to form in tomato via a reductase conversion pathway. Hexanal, one of the major aldehydes in tomatoes, is considered to be important for fresh tomato flavor [6,8], and is also a contributor to the green, fatty component of tomatillo aroma. Similar to the unsaturated aldehydes, it is derived from a linoleic acid breakdown pathway [6]. 1-Hexanol provides a winey, cider-like character and is produced via bioreduction of hexanal. Other significant aldehydes include 8to 10-carbon enals including 2-octenal, 2-nonenal, and 2,4-decadienal, which contribute fattygreen aromatics. After the aldehydes, the largest volatile class of compounds identified in tomatillos were esters. Among these were butyl, iso-hutyl, hexyl, (Z)-3- and (^-2-hexenyl acetates, methyl hexanoate, and ethyl octanoate. Aromatic esters include methyl and ethyl benzoate, and methyl salicylate. Two unique hydroxy esters were identified by matches with the mass spectral library: methyl-(2-hydroxy-3-methyl)-valerate, and methyl-2-hydroxy-/5'o-valerate, the latter which was only found in cooked tomatillo (Figure 5).

"0CH3 OH MethyI-(2-hydroxy-3-methyI)-valerate

^

y

"0CH3

OH Methyl-2-hydroxy-/so-valerate

in

(U

c

E

CO

O Ob o

2 CM

il

CO

11 E

^

(D

1

co^ C O E o O

uo do (do rdo flo

.

CD (0 C C 0) 0) 0)

0)

T3 "2 c c oto0 (00)o o o OJ (0 Q Q o Js o c t5 — — ^1- ^f CO _" J ro 0) O 2 *? " t—

S E rf? o 5 jjl Itf 9 ^ X Zi

o '

22 i "o c


T

0)

T-

>» 0) Q. CO

^ ^

CNJ CO ' ^ lO <0 1 ^ 00 O) O

B CO

8 ^ ^

fvi

S S SJ pi?

3 c -V -V -f cu

CD ^<0

^

1 fi?^ -§

y CQ 5 CM CO CO 0) CD 1 1 1 X 1 1 1 X N CM
o o o

x-CMC0TriOCD00C:>OT-

IxSSS

If)

cs

o o o o

o o

o o

o o

:^

ID

o o

o o

CM

> o

o -a c

3-

o )£

2 o

o o o

o o 00

o o

o o

303

o

o

s .2

I o

3

en

u o

I

304

• CN CNI

coh-ooooT-cNco^rmcD

rao

75

T-T-x-f-CNCNiCMCMCNCNJCNJ

5:$ X CO

X <0 I I — C I t i0^ N C c s' i a^ ) >X» or c -M '< 5t

0) <5 f. 0) <1> c c 0) 0) $ -7^ -C -R 0 •«

iCNJ CO I

2 XI _ g X

CN

o o o o o o

ao CN

in

0 ^

JO

o o o o o

i ro

o o o o o

in H

CM

o o o o o o

o o o o o

in

-0

in

o

o

1-0

3

a i -H JE±J

O O

o o

o o

o o CO

§ 2

> o

c

CM ^ 0 T3

2 o

o o o

o o

o o

o

o> 00


^

c

E O CO CM

Ob o O

If 39 ~E "T^

CO

•

O E 1

(0

•

b)

o Q_

'

CO

C4

0)

uo CO njo roo

fe§

r

(D -J. c o X 0)

— (0 c

o c (1)

X ^ E go g g 1 C N i £ : < i > o c \ i J 5 S 0 ro

o O

i

>

CD

0) "(0 Q)

CO T T i n (D T— T— T— T—

X

1

X

1

1 - 1 - "i;;

g 0) 0 — O

"

.

in T-

,

,

1

o o o o o o CN

.

T-

O)

^

0)

.

C

1"^ st X

sl>lsls^ r-

O) • -

O

T3

CD X

•^5C\IC0C0
.

cocNco'^mcoi^oo

1

CN

o o o o o in

(0

.

r-

o o o o o

1

H

ID

to

.

. o, o o o o o H

CO T—

1^

_

o' o o o o in

o>

-o

.

o

-o o in

-o o

in

< o ^ -co " "

-o o

o < \^ -=s^_ "^ ^ ^ -

•

J -3 o

-o

-o -^5 ^ o

^

^

- ^

-^

J

^

^

««" ~:^—-,

^

3

^g ^^^ ^ ^ ^

o

" -o o

-in

~o _o

-=^^ ^ -H = ^

,__J

-o o in -H

^ --—^ — o • ^ ^ -CN

,

1 1

A

9i B •H

o

oo

in

^

o T-

o o CO T-

o o <>l T-

o o TT-

o o o

T-

o o o>

o o 00

o o (O

^

i

^

)^ X
c

•D

42 > o :^

305

O

C/3

^>i^

C/D

fD.^ O

ti o

td T3 O o o o r;J O

o

s o o

-S

• 1—1

U O

op

306 Table 4 Volatile Compounds Identified by Headspace GC-MS in Tomatillo and Red Plum Tomato Estimated Concentration (ppb) ^

No.«

la 1 2 3 4 5 6 7

8 9

10 11

12 13 22 14

RI^ (DB-5) 723 736 753 775 800 815 839 849 875 895 897 913 932 951 952 957 984 986 997 998 1004 1011 1014 1016 1029 1033 1054 1058

Compound

Fresh Tomatillo

2-Methyl-2-pentenal 20 Dimethyldisulfide tr. Acetic acid iso-Butyl acetate 240 Hexanal 5,400 Butyl acetate 20 (^-2-Hexenal 2,000 (Z)-3-Hexenal 21,000 (Z)-3-Hexen-l-ol 2,700 1-Hexanol 26,000 Methyl-(2-OH)-/5'o-valerate 2,4-Hexadienal 110 Methyl hexanoate 25 Benzaldehyde 540 (( (E)-2-Heptenal tr. Dimethyltrisulfide 2-Pentylfuran tr. 240 6-Me-5-hepten-2-one 820 Methyl-(2-OH-3-Me)-valerate 110 Octanal 180 (Z)-3-Hexen-1 -yl acetate 250 Hexyl acetate tr. (£)-2-Hexen-l-yl acetate 270 Limonene wo-Butylthiazole tr. Benzyl alcohol 120 (£)-2-0ctenal 300 43,7^2,99,71,55,41,69

Cooked Tomatillo

Fresh Tomato

tr. 820 820

1,200

160 3,300 800 7300 320

160 1,600 4,300 22,000

2,200 ((

400 (( tr. 630 670

510 110 tr. 120 300

380 140

70 90

700

Continued on next page

307

Table 4 Continued Estimated Concentration (pph)c

No.^

23

15 24

16

17 25 18 19 20 21 26

RI^ (DB-5) 1079 1085 1088 1098 1102 1114 1153 1163 1167 1175 1183 1187 1191 1197 1231 1237 1252 1263 1285 1291 1309 1373 1374 1400 1443 1473 1510 1619

Compound a-Terpinolene Guaiacol Methyl benzoate Linalool Nonanal 2-Phenylethanol (^-2-Nonenal Ethyl benzoate Terpinen-4-ol Cumic alcohol Methyl salicylate Myrtenol Ethyl octanoate Decanal Neral 43,55,70,83,92,125 (£)-2-Decenal Geranial (E,Z)-2,4-Decadienal 1 -Nitro-2-phenylethane (E,F)-2,4-Decadienal Decanoic acid p-Damascenone Dodecanal Geranyl acetone p-Ionone Methoxy safrole Cadinene

Fresh Tomatillo

Cooked Tomatillo

Fresh Tomato

tr. 450 95 tr. 2,000 tr. 60 tr. 50 85 tr. tr. 320 25 430 tr. 60 22 160 tr. 1 85 90 49

tr. 710 40

tr. 180 390 90

40 50 75

300

70 45

200 150 740 110 190 3 120

30

^ Numbers refer to Figures 3-5. ^ Kovats retention index, relative to «-alkane hydrocarbon standards. ^ Concentrations calculated from 160 ppb 4-heptanone internal standard.

1 1,300 46 410

308 Carotenoid-derived terpene compounds comprise a third class of volatiles identified in tomatillo flavor. The oxidative decomposition of carotenoids, particularly lycopene and p-carotene, has previously been shown to lead to the formation of terpene and terpene-like compounds in tomato flavor [6]. Unique terpenes identified in tomatillo include aterpinolene, terpinen-4-ol, myrtenol, and cadinene. Identifications of other terpene-derived tomatillo volatiles previously reported in tomato include 6-methyl-5-hepten-2-one, geranylacetone, P-ionone, and P-damascenone. As a general observation, lower levels of these compounds were observed in tomatillo, correlating with a -50% reduction in the levels of carotenoid precursors relative to tomato (Table 2). p-Damascenone has previously been shown to arise from thermal pH 4 hydrolysis of tomato glycosides [9], and this finding is supported by its increased level in cooked vs. fresh tomatillo (Table 4). Other terpenes including limonene, linalool, neral, and geranial were also identified. The biopathway derivation of these compounds has been previously established for tomato [9].

CH2OH

Myrtenol

Cadinene

A series of aromatic compounds including benzyl alcohol and cumic alcohol (para-{isopropyl)benzyl alcohol) were identified in fresh and cooked tomatillo, respectively. The latter compound exhibits a caraway-like odor and is presumed to be produced via hydroxylation and aromatization of a-terpinene. In comparison to the volatile profile of fresh tomato, notably absent in volatiles identified from tomatillo were the amino acid-derived volatiles including /^o-butylthiazole, nitrophenylethane, and phenylacetonitrile. These compounds play a considerable role in tomato flavor and are also present in the mature green stage of tomato development [9]. A summary of identified flavor compounds which are unique to tomatillo is listed in Table 5. 4.5. Determination of Primary Odorants in Tomatillo via Concentration/Threshold Ratios Many of the identified compounds in Table 4 have minimal significance for re-creation of tomatillo flavor. From this list, a more pinpointed subset of key aroma compounds was desired for a better understanding of key odorants which contribute to tomatillo. GColfactometry techniques such as aroma extract dilution analysis (AEDA) [17] or Charm analysis [18] are often appUed to elucidate the key odorants in flavor isolates. However, these techniques are somewhat time-consuming and require repetitive analyses. Our concern was that relative concentrations of flavorants could change during the time interval required for multiple analyses.

309 Table 5. Flavor Volatiles Unique to Tomatillo Esters Methy l-(2-OH-3 -Me)-valerate Methyl-2-OH-/^6>-valerate (cooked) z^o-Butyl acetate Butyl acetate (Z)-3 & (E)-2-Hexen-l-yl acetate Methyl hexanoate Ethyl octanoate

Aldehydes Octanal 2-Decenal Dodecanal

Aromatics Benzyl alcohol Methyl benzoate Ethyl benzoate Methyl salicylate Cumic alcohol (cooked)

Terpenes a-Terpinolene Terpinen-4-ol Myrtenol Cadinene

Acids Decanoic acid

One approach which has been successfully used to determine key aroma compounds in tomato utilizes the calculation of odor units (UQ). This technique requires access to published threshold values for specific compounds, or for newly identified compounds, the determination of individual odor thresholds in water. Many of the volatiles identified in our tomatillo study have been previously reported [10,19]. The odor unit is defined as the ratio of a flavor compound's concentration divided by its odor threshold: Compound Concentration U. = Odor Threshold The logarithm of the odor threshold (log UQ) is calculated to represent changes in concentration which are significant for olfactory discrimination. Odor activity follows a sigmoidal dose-response curve in that significant aroma responses require order-of-magnitude changes in concentration. Consequently, logarithmic functions more significantly represent meaningful sensory differences. Aroma unit values >1 are indicative of compounds present at a concentration that greatly exceeds their thresholds, and therefore are likely to contribute significant flavor impact. A comparison of flavor significant volatiles in fresh tomatillo with tomato is shown in Table 6. The key volatile compounds are listed in descending rank order, (Z)-3-hexenal being the most significant. Of interest is that the aldehydic components (Z)-3-hexenal, nonanal, hexanal, decanal, and (F)-2-hexenal are present in tomatillo at order-of-magnitude higher concentrations than in tomato, and thus should contribute considerable influence on the green.

310 Table 6 Major Flavor-Significant Volatiles in Fresh Tomatillo vs. Tomato Tomatillo Compound

Odor Thresh. (ppb in H20)^

0.25 (Z)-3-Hexenal 0.007 P-Ionone 0.07 (£,^-2,4-Decadienal 1 Nonanal 4.5 Hexanal 0.002 P-Damascenone 1 Decanal 0.07 (£,Z)-2,4-Decadienal 2 Hexyl acetate 17 (E)-2-Hexenal 500 1-Hexanol 70 (Z)-3-Hexen-l-ol 50 6-Me-5-hepten-2-one 65 isO'Butyl acetate 350 Benzaldehyde 13 (E)-2-Heptenal 60 Geranyl acetone 210 Limonene 1 (E)-2-0ctenal Methyl-(2-OH-3-Me)-valerate 2 1 -Nitro-2-phenylethane 3.5 /^o-Butylthiazole 13 Guaiacol 1,000 2-Phenylethanol

Cone. (ppb)

Log OdorU^

21,000 49 160 2,000 5,400 1 320 22 250 2,000 26,000 2,700 240 240 540 "

4.9 3.8 3.4 3.3 3.1 2.7 2.5 2.5 2.1 2.1 1.7 1.6 0.7 0.6 0.2

90 270 tr. 820

0.2 0.1

Tomato Cone. (ppb)

Log OdorU^

1,600 46 190 180 1,200 1 70 740 120 160 22,000 4,300 670

3.8 3.8 3.4 2.3 2.4 2.7 1.8 4.0 1.8 1.0 1.6 1.8 1.1

400 " 1,300 380 700

0.1 1.3 0.3 2.9

110 140 450 390

1.7 1.6 1.5 -0.4

^ Values obtained from R. G. Buttery (1993), Reference 10; H. Maarse (1991), Reference 19. b Logarithm of compound Concentration divided by its Odor Threshold.

311 Table 7 Major Flavor-Significant Volatiles in Fresh vs. Cooked Tomatillo Fresh Compound

Odor Thresh. (ppb in H20)^

0.25 (Z)-3-Hexenal 0.007 p-Ionone 0.07 (£,E)-2,4-Decadienal 1 Nonanal 4.5 Hexanal 0.002 p-Damascenone 1 Decanal 0.07 (^,Z)-2,4-Decadienal 2 Hexyl acetate 17 (£r)-2-Hexenal 500 1-Hexanol 70 (Z)-3-Hexen-l-ol 50 6-Me-5-hepten-2-one 65 iso-Butyl acetate 350 Benzaldehyde 13 (£)-2-Heptenal 60 Geranyl acetone 210 Limonene 1 (F)-2-0ctenal Methyl-2-OH-/5'o-valerate Methy l-(2-OH-3 -Me)-valerate

Cone. (ppb)

Cooked

Log OdorU^

21,000 49 160 2,000 5,400 1 320 22 250 2,000 26,000 2,700 240 240 540 "

4.9 3.8 3.4 3.3 3.1 2.7 2.5 2.5 2.1 2.1 1.7 1.6 0.7 0.6 0.2

90 270 tr.

0.2 0.1

Cone. (ppb)

Log OdorU^

3,300

4.1

700 820 3 300

2.8 2.3 3.2 2.5

160 7,300 800

1.0 1.2 1.1

2,200

0.8

a

820

300 32 320 510

0.2 2.6

^ Values obtained from R. G. Buttery (1993), Reference 10; H. Maarse (1991), Reference 19. b Logarithm of compound Concentration divided by its Odor Threshold.

312 leafy, fatty-soapy flavor character of tomatillo. Comparatively, (£,Z)-2,4-decadienal, geranyl acetone, and (F)-2-octenal are significantly higher in tomato than tomatillo. A similar comparison for fresh vs. cooked tomatillo is presented in Table 7. The aldehydic components (Z)-3-hexenal, nonanal, hexanal, (£)-2-hexenal and the alcohols 1-hexanol and (Z)-3-hexen-l-ol are considerably reduced in cooked vs. fresh tomatillo. Alternatively, benzaldehyde and methyl-2-hydroxy-/5'o-valerate are aroma-significant components which are enhanced during the cooking process.

5. CONCLUSIONS The flavor profile of fresh tomatillos was found to be dominated by organic acids, especially citric and decanoic acids, which contribute to its characteristic acidic taste. Aldehydes and alcohols including (Z)-3-hexenal, (£',^-2,4-decadienal, nonanal, hexanal, hexanol, and (Z)-3-hexen-l-ol provide a dominant "green flavor" impact on tomatillo flavor due to their high log Odor Unit values. While other classes of volatile compounds were identified as similar to those in fresh tomato, the tomatillo aroma profile did not contain characteristic key tomato volatiles such as /5'o-butylthiazole, nitrophenylethane, or phenylacetonitrile. Compounds unique to tomatillo flavor included hydroxy esters, aromatic esters, 8- to 12-carbon aldehydes, decanoic acid and terpenes. The volatile profile of cooked tomatillos exhibited a 7-fold reduction of aldehyde levels, with concurrent generation of a valeryl hydroxy-ester and cumic alcohol. The relative flavor significance of these compounds in cooked tomatillos will need to be clarified by further investigations.

6. ACKNOWLEDGMENT The authors thank Dr. Marlene A. Stanford for initiating the project idea, and providing pooled samples with carefully controlled heating profiles.

7. REFERENCES 1 M. Cantwell, J. Flores-Minutti, and A. Trejo-Gonzalez, Scientia Horticulturae 50 (1992) 59. 2 H. D. Tindall, Vegetables in the Tropics, Avi Pub., Westport, CN, 1983, pp. 359, 378. 3 C. B. Heiser, Of Plants and People, Univ. Oklahoma Press, Oklahoma City, Oklahoma, 1975, pp. 129-136. 4 S. Uhl, Food Technology, 7 (1996) 79. 5 A. Juttelstad, Food Formulating, 3 (1997) 46. 6 M. Petro-Turza, Food Rev. Int. 2 (1986-87) 309. 7 B. R. Thakur, R. K. Singh, and P. E. Nelson, Food Rev. Int., 12 (1996) 375. 8 R. G. Buttery, R. Teranishi, R. A. Flath and L. C. Ling. In: R. Teranishi, R. G. Buttery and F. Shahidi, (eds.). Flavor Chemistry: Trends and Developments, No. 388, American Chemical Society Symposium Series, Washington, DC, 1989, pp 213-222.

313 9 R. G. Buttery and L. C. Ling. In: R. Teranishi, R. G. Buttery and H. Sugisawa (eds.), Bioactive Volatile Compounds from Plants, No. 525, American Chemical Society Symposium Series, Washington, DC, 1993, pp 23-34. 10 R. G. Buttery. In: T. E. Acree and R. Teranishi (eds.), Flavor Science: Sensory Principles and Techniques, ACS Professional Reference Book, American Chemical Society, Washington, DC, 1993, pp 259-286. 11 K. Karmas, T. G. Hartman, J. P. Salinas, J. Lech and R. T. Rosen. In: C-T. Ho and T. G. Hartman (eds.). Lipids in Food Flavors, No. 558, American Chemical Society Symposium Series, Washington, DC, 1994, pp 130-143. 12 AOAC Official Method 982.14, Official Methods of Analysis of AOAC Int., 16th ed., Arlington, VA, 1995, Chapter 32, p. 30. 13 H. Klein and R. Leubolt, J. Chromatogr., 640 (1993) 259. 14 F. Ulberth, J. AOAC Int., 77 (1994) 1326. 15 W. A. Gould, Tomato Production, Processing and Technology, 3rd ed., CTI Publications, Inc., Baltimore, MD, 1992, p. 437. 16 W. Friedrich, Vitamins, Walter de Gruyter, Hawthorne, NY, 1988, p. 94. 17 W. Grosch, Trends Food Sci. Technol., 4 (1993) 68. 18 T. E. Acree. In: T. E. Acree and R. Teranishi (eds.), Flavor Science: Sensory Principles and Techniques, ACS Professional Reference Book, American Chemical Society, Washington, DC, 1993, pp 1-18. 19 H. Maarse, Ed., Volatile Compounds in Foods and Beverages, Marcel Dekker, New York, NY, 1991.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

Effect of thermal treatment compounds of tomato juice.

315

in

the

headspace

volatile

M. Servili, R. Selvaggini, A.L. Begliomini and G.F. Montedoro

Istituto di Industrie Agrarie , University of Perugia, via S.Costanzo, 1-06126 Perugia - Italy.

Abstract Volatile compounds of tomato juice include compounds of the original fruit, such as terpenes, and other substances that are originated during processing by lipoxygenase activity, carotenoid cooxidation and Maillard reaction. This paper reports: a) a comparison between solid phase microextraction (SPME) and traditional static (SHSA) and dynamic headspace (DHSA); b) optimization of SPME; c) evaluation of the volatile compounds of tomato juice under different conditions of thermal treatment. Results were analyzed by multivariate statistical analysis. One hundred ninety and 219 volatile compounds were sampled using SPME and traditional SHSA and DHSA, respectively; these compounds belonged to the following chemical classes: ketones, aldehydes, alcohols, esters, ethers, hydrocarbons, sulfur, nitrogen and oxygen compounds, phenols, oxygencontaining heterocyclic compounds, free acids and lactones. The thermal treatment mainly modified saturated and unsaturated Cg alcohols and aldehydes, terpene and carotenoid derivatives.

1. INTRODUCTION Fresh and processed tomato contains a large quantity of volatile compounds, that are included in various chemical classes such as ketones, aldehydes, alcohols, esters, ethers, hydrocarbons, sulfur, nitrogen and oxygen compounds, phenols, oxygen-containing heterocycHc compounds, free acids and lactones [1-6]. The most important compounds in tomato flavor are originated during processing by enzymatic reactions. In fact, saturated and unsaturated C^^ and C9 alcohols and aldheydes, that are impact compounds of fresh tomato, are originated by lipoxygenase activity, while terpene and carotene derivatives can be released from odorless glycosidic compounds by glycosidase activities [7-9]. The presence and the quantity of volatile compounds in tomato fruit and tomato derivatives

316 depend on the cultivar, fruit ripening and storage conditions [1, 10, 11]. However, in processed tomato, a critical point for flavor modification is the thermal treatment that is employed to inactivate endogenous enzymes during the process of blanching. As a consequence of this, the endogenous oxidoreductases, such as lipoxygenase, peroxidase and polyphenoloxidase, catalyze the degradation of color (due to p-carotene, lycopene and phenoUc compound cooxidation), while the hydrolases, such as pectinases, can hydrolyze pectins causing loss of consistency during processing [12]. Thermal treatments may cause changes in sensory and nutritional characteristics of tomato and tomato derivatives due to cooxidation reactions of carotenoids and Maillard reaction [13 - 15]. The present work reports: a) a comparison between solid phase microextraction (SPME) and traditional static (SHSA) and dynamic headspace (DHSA); b) optimization of SPME; c) the evaluation of volatile compounds of tomato juice under different conditions of thermal treatment.

2. MATERIALS AND METHODS 2.1. Materials. Tomato samples (cultivar FM 6203) were grown in experimental fields of Perugia Agricultural University during 1996. Authentic reference chemical compounds were obtained from reliable commercial sources. 2.2. Experimental procedure 2.2.1. - Comparison of SPME with the traditional SHSA and DHSA, and its optimization. One hundi'ed grams of tomato fruit were homogenized for 60 sec at 25 °C, the homogenized juice was stirred for 15 min at 25 °C to activate endogenous enzymes, then 100 ml of a saturated solution of CaCl2 (1:1 v/v) were added on and the mixture was used to evaluate volatile compounds as reported below. 2.2.2. - Thermal treatment. The following experimental trials were developed using a microwave oven: cold break: 65 °C for 3, 15 and 40 min; warm break: 70 °C for 3, 10 and 30 min and 80 °C for 3, 10 and 20 min; hot break: 95 °C for 1, 3, 5 and 10 min. Immediately after thermal treatments the samples were freezed at 0 °C, added with saturated solution of CaCl2 (1:1 v/v) and used to evaluate volatile compounds. 2.3. Instrumental analysis. 2.3.1. - Solid-phase microextraction. Five grams of tomato juice were put in a 20 ml vial and thermostated for 15 min; a SPME fibre (65 |Lim Carbowax/divinylbenzene) (Supelco, Inc., Bellefonte, PA, USA) was used for

317

sampling volatile compounds. To compare SPME with traditional SHSA and DHSA, the fibre was exposed, after thermostatation, in the headspace of tomato juice for 30 min at 35 °C. In the optimization study of the analytical conditions of SPME analysis the following times of fibre exposition to the tomato juice headspace 5, 10, 20, 30, and 35 min and temperatures 20 °C, 23 °C, 30 °C, 37 °C and 40 °C were tested according to the Central Composite Design strategy [16]. For desorbing the volatile compounds the fibre was inserted into the GC injector set at 250 °C in splitless mode using a spHtless inlet finer of 0.75 mm ID for 10 min. 2.3.2. - Static headspace analysis. Twenty-five grams of tomato juice were put in the thermostated syringe (adjusted to an internal volume of 2 litres) for 35 min at 35 °C to obtain the equilibrium between the vapor and the condensed phases; then the vapor phase was pushed, at atmospheric isobaric conditions, in 30 min into the trap containing Tenax GC set at a temperature of 20 °C. The volatile compounds were desorbed at 280 °C and sent to the GC injector set in split mode (20:1) [17]. 2.3.3. - Dynamic headspace analysis. Five grams of tomato juice were put in a glass vessel, thermostated at 35 °C, and the extraction was performed using a nitrogen flow of 200 mL/min for 30 min. The volatile compounds were adsorbed in the trap containing Tenax GC set at the same temperature as above mentioned, desorbed at 280 °C and sent to the GC injector set in spfit mode (30:1). 2.3.4. - GC-MS analysis. A GC Varian 3600 equipped with a split/spfitless injector coupled with a mass spectrometer Varian Saturn 3 (Varian, Walnut Creek, CA, USA) was used. A fused-silica capiUary column DB-Wax, 50 m, 0.32 mm ID, 1 |am film thickness (J & W Scientific, Folsom, CA, USA) was employed. The column was operated with helium at a pressure of 15 psi with a flow rate of 2.2 mL/min and a linear velocity of 30.7 cm/sec at 35 °C. The GC oven heating was started at 35 °C, this temperature was maintained for 8 min (10 min for SHSA and DHSA), then increased to 45 °C at a rate of 1.5 °C/min, increased to 150 °C at a rate of 3 °C/min, increased to 180 °C at a rate of 4 °C/min, increased to 210 °C at a rate of 3.6 °C/min where it was held for 14.51 min (12.51 min for SHSA and DHSA); the total time of analysis was 80 min. The injector was always maintained at 250 °C. The temperature of the transfer fine was fixed to 220 °C. Mass spectrometer was operated in the mass range of 10 - 350 a.m.u. at a scan rate of 1 sec/scan and a manifold temperature of 180 °C. The identification of volatile compounds was made comparing mass spectral data with those of some pure analytical standards and with those of the NIST-92 library. 2.4. Statistical analysis. 2.4.1. - Principal components analysis (PCA). Two PCA models first to study the effects of time and temperature in the SPME second to analyze the influence of different thermal treatment compounds of the processed tomato. The chemometric package

were built: the adsorption, the on the volatile "SIMCA - S v.

318 5.1", Umetri AB, Umea, Sweden was used. The analytical data were put in a matrix with the rows corresponding to the samples (n objects) and the columns corresponding to the analytical parameters (k variables). The raw data were normaUzed, with the subtraction of the mean, and autoscaled, dividing these results by the standard deviation. The number of significant components has been found applying the cross validation. The results of PCA modeUing are presented in the graphical form [18]. 2.4.2. - Optimization by Response Surface Modelling (RSM). RSM was performed with the chemometric package "MODDE - v. 2.1", Umetri AB, Umea, Sweden. This analysis was carried out to define the optimal working conditions for the adsorption of the volatile compounds with SPME. A preliminary PCA was performed, as above reported, with data collected in accordance with a Central Composite Design for selecting few variables (volatile compounds) among those with the highest loadings and opposite in the loading-plot [16]. For the optimization, the original data, expressed as peak area (Y), were transformed in a desirability function (d^) using a Hnear transformation so to obtain a range of values of desirability between 0 and 1: ,

I

— 1 mill

I max— / 1

where Yj^^^ and Y^^,^, corresponded to the minimum and the maximum value of peak area, respectively. The overall desirability (D) was calculated as the geometric mean of the individual d^ values: D-Vdi*d2*..*dn The partial least squares analysis (PLS) was employed for developing the model [19].

3. RESULTS AND DISCUSSION 3.1, - Comparison between SPME and traditional SHSA and DHSA. Figure 1 reports the GS-MS chromatograms obtained using SPME, SHSA and DHSA while the volatile compounds identified in tomato juice are reported in Table 1. The volatile compounds identified were 190 and 219 using SPME and traditional headspace analysis, respectively. These compounds belong to the following chemical classes: ketones, aldehydes, alcohols, esters, ethers, hydrocarbons, sulfur, nitrogen and oxygen compounds, phenols, oxygencontaining heterocyclic compounds, free acids and lactones. The main differences

319 between SPME and traditional headspace were observed for the apolar and polar volatile compounds with a low molecular weight, that were not well adsorbed by the Carbowax/divinylbenzene fibre. All the methods allow the evaluation of the most important volatile compounds of tomato flavor such as saturated and unsaturated Cg and C9 aldehydes and alcohols, carotenoid derivatives and "offflavor" such as furans and sulfuric compounds. 3.2. - Optimization of SPME. To optimize the analytical conditions of SPME, PC A was preventively applied to the raw data. The score-plot of the first two components of PCA reported in Figure 2 (that explains 59 % of the total variance with two components) shows a discrimination of the samples according to the time of sampling along the first component, while a low influence of the temperature was observed along the second component. Eight compounds with the highest absolute value of loadings for the two components, in particular, hexanal, dimethyl disulfide, ethylbenzene, 3-methyl-l-butanol, divinylbenzene, cis-citral, ethyl-benzaldehyde and benzyl alcohol were chosen for the optimization by RSM. Response surface, built using the PLS with these variables transformed in overall desirability, is reported in Figure 3. The model, that explains 59 % of the total variance with two components, shows that the optimal sampling condition for SPME was 27 min at 29 °C. 3.3. - Thermal treatment. PCA model applied to the 190 volatile compounds (evaluated using SPME), to study the effect of time and temperature of thermal treatment, explains 68% of the total variance with three components. The score-plot of the first two components shows a good discrimination along the first component between the samples treated and the control, the second component discriminates the samples in relation to the temperature while the third component is related to the time of treatment (Figures 4 and 5). The loading-plot of the first two components shows that the temperature of treatment strongly modifies several compounds related to the tomato flavor such as saturated and unsaturated alcohols and aldehydes, aromatic aldehydes, terpenoids, citrals, P-ionone, 6-methyl-5-hepten-2-one and pseudo-ionone. Also some compounds responsible of the "off-flavor" such as furfural, dimethyl disulfide, 2-isobutyl-thiazole and 3-methyl-butanal, are influenced by the temperature of treatment. The loading-plot of the first vs the third component shows that the samples treated at 80 °C and the time of thermal treatment is highly correlated with 2-methylfuran, 2-ethylfuran, 3-(4-methyl-3pentyl)-furan, 2- or 3-ethyl-thiophene, 2-methyl-2-thiazoline, cis- and trans-2hexenal, 2-octanone and 6-methyl-5-hepten-2-ol. The volatile compounds having a positive loading along the first component decrease according to the thermal treatment, variables with positive loadings in the second one have a minimum at 65 °C for 40 min of processing, while volatile compounds that show positive loadings in the third component have higher concentration at 80 °C for 10 min (Table 2).

320 Solid Phase MicroExtraction 32

56 ^ 59 67

rmm

l_ULl

\})kjJ

16.60

66.6G

timo

Static Head Space 32

86

L Dynamic Head Space 32

56

67

.iijyi w

vJLiwI Li^J' UJJ

66.66

t.imo

Figure 1. Capillary GS-MS analysis of volatile compounds of tomato juice evaluated using SPME, static and dynamic headspace (see Table 1 for peak references).

321 Table 1. Headspace volatile compounds found in tomato juice References

References

Alcohols 2 4 13 21 27 •^1 38 42 •'>4 59 61 67 74 81 83 84 85 90 91 93 99 100 101 104 106 113 115 116 122 125 126 128 150 155 160 162 164 177 1«2 163

methanol ethanol 2-inethyl-l-propanol l-penten-3-ol 2-inethoxy-ethanol 3-niethyl-l-butanol 1-pentanol 2-ethoxy-ethanol cis-2-penten-l-ol 1-hexanol 3-hexen-l-ol 3 - h e x e n - l - o l (i) 2,2-dimethyl-l-hexanol 7-octen-4-ol 1-heptanol 6-methyl-5-hepten-2-ol 4-isopropyl-l-niethyl-2cyelohexen-1-ol 6-niethyl-l-heptanol 2-decen-l-ol 2-niethyl-eyclohexanol linalool cis-l-niethyl-4-isopropyl-2c y e l o h e x e n - 1 -ol 1-octanol 4-methylen-6-hepten-2-ol o r 4-octyn-2-ol 2-propyl-l-heptanol terpinen-4-ol 2-octen-l-ol 2 - o e t e n - l - o l (i) 1,2-ethanediol 1-nonanol 4-niethyl-5-decanol 2-butyl-l-octanol 1-decanol o r 3 , 7 - d i m e t h y l - l octanol nerol geraniol benzyl alcohol nerolidol 1-tridecanol 2-ethyl-l-dodecanol phenyl-ethyl alcohol

185 e u g e n o l o r i s o e u g e n o l 188 3 , 7 , 1 1 - t r i m e t h y l - l - d o d e c a n o l 190 f a r n e s o l

Ketones 4 10 4 2

6 8 24 26 56 1,2 39 3, 2, 9 46 48 4 49 2, 9 152 3, 2,1, 10 153 4 112 3, 1,9,10 158 156 4 165 4 168 173

2, 9, 9 3, 9, 9

3-pentanone l-penten-3-one 5-niethyl-2-hexanone cyclopentanone 6-niethyl-5-hepten-2-one 5-methyl-3-heptanone 2-octanone 4-octen-3-one 3-hydroxy-2-butanone 2-hydroxy-acetophenone geranylacetone trans-6-niethyl-3,5-heptadien-2-one alpha-ionone nerylacetone beta-ionone 4-(dimethylamine)-3-niethyl-2-butanone 2-niethyl-cyclohexanone

172 2 - c y c l o h e x e n - l - o n e 176 p s e u d o - i o n o n e 181 p s e u d o - i o n o n e (i) 187 4-hydroxy-2- o r 3 - n i e t h y l - a c e t o p h e n o n e J 37 m e g a s t i g m a t r i e n o n e

2, 1, 10

3, 5, 2, I 4 4 4 4 2, 10,9 4 4 3, 2, 1, 9

2 S,2 3

Esters 2, 9 4 4

3, 9 3, 9 3, 9, 9 4

2, 1, 9, 9 3, 2, 10 4

43 52 40 94 108 120 154 147 148

hexyl acetate 3- o r 4 - h e x e n y l a c e t a t e methyl 3-hexenoate 1,2-ethanediyl d i - f o r m a t e isobornyl acetate 1,2-ethanediyl m o n o - f o r m a t e 2-hydroxy-ethyl-benzoate methyl salicylate propylene carbonate

180 * * * *

triethyl-1,1,2-ethane-tricarboxylate methyl benzoate ethyl benzoate methyl formate methyl acetate

4

Hydrocarbons 14 e t h y l - b e n z e n e 16 p - x y l e n e 18 o - x y l e n e

9 4 4

322

Table 1. (continued)

*

2-methyl-3-buten-2-ol

References 4

Aldehydes 3 7 9 12 25 28 32 47 '^3 ^^

S-methyl-butanal pentanal hexanal 4-pentenal or 2-pentenal heptanal cis-2-hexenal trans-2-hexenal octanal traiis,trans-2,4-hexadienal 2-heptenal

69 nonanal 76 2,4-hexadienal (i) 78 2-octenal 86 92 98 110 119

trans,trans-2,4-heptadienal 2,4-heptadienal (i) benzaldehyde trans,cis-2,6-nonadienal 2,6,6-trimethyl-l-clohexen-lcarboxaldehyde 123 trans-2-decenal 131 cis-citral 134 2,4-nonadienal

References 20 2,5-diinethyl-l,6-octadiene 23 m-xylene 29 alpha-phellandrene

30 propyl-benzene 35 gamma-terpinene 36 1,2,4- or 1,2,3-triinethyl-benzene 41 p-cymene 44 m-cymene 3, 5, 2, 10 45 1,2,3- or 1,2,4-triinethyl-benzene 3, 5, 2, to 50 1,4- or 1,3-diethyl-benzene 51 butyl-benzene 4 57 l-niethyl-4-propyl-benzene r>, 2, I 60 2-ethyl-l,3-diniethyl-benzene or 1,2,3,4S,2 4 3, 5, 2, 1 3,2

9

3 4 4

62 66 72 77 79 80 82 95

^ '^

^ 3> 9 4 '^ 4

tetramethyl-benzene 2-ethyl-l,3-diniethyl-benzene (i) 1,2,3- or 1,2,4-triniethyl-benzene ^^, 2, 1 2,6-dimethyl-2,6-octadiene_or 3,3,6-triniethyl1,4-heptadiene 3-ethyl-2-niethyl-l,3-hexadiene 1,2,4,5-tetraniethyl-benzene ^ 2-butenyl-benzene cyclopentene or 1,4-pentadiene 2,5,5-triniethyl-l,3,6-heptatriene

4 4

97 l-isopropyl-2,3- or 4,5-diniethyl-cyclopentene 109 1,2-dihydro-naphthalene 129 l-(cyclohexyl-methyl)-4-(l-niethyl-ethyl)-

5,2,9

133 l,2-diniethyl-3-(l-isopropenyl)-cyelopentane_

cyclohexane 127 phenylacetaldehyde 121 132 136 138 142 141 144 149 179 * * * * *

4-inethylbenzaldehyde 2-hydroxybenzaldehyde ethyl-benzaldehyde trans-citral 2-undecenal 2,5- or 2,4-dimethylbenzaldehyde 2,4-decadienal 2,4-decadienal (i) 3-phenyl-2-propenal prop anal 2-niethyl-propanal 2-propenal butanal 2-butenal

Ethers 11 hexyl-octyl-ether

or l-niethyl-4-niethylen-cycloheptane naphthalene (2-ethyl-l-methyl-butylidene)-cyclohexane divinyl-benzene 1-nonyne or 1-decyne 3-dodecyne or 3-tetradecyne styrene (vinyl benzene)

4 4 9 4 4 9

143 140 107 135 34 183

10 10

* * * * * * * * * *

3,4-dimethyl-hexane 1,1-diniethoxy-propane 2-methyI-l,3-butadiene 1-methoxy-hexane 2,4-hexadiene 2,4-dimethyl-heptane or 2,4-dimethyl-hexane 2- or 4-octene camp bene '^ 2-methyl-pentane 2,4-diniethyl-heptane

*

2,2-diniethyl-3-ethyl-pentane

4 4 4 4

"^

'^

323

Table 1. (continued)

References

111 d i e t h y l e n e g l y c o l m o n o methyl-ether 118 d i e t h y l e n e g l y c o l e t h y l - e t h e r o r diethyl-ether 139 2 - h y d r o x y e t h y l e t h e r 171 t r i e t h y l e n e g l y c o l

dimethyl disulfide 3- o r 2 - e t h y l - t h i o p h e n e 2-isobutyl-thiazole 2-methyl-2-thiazoline 2-thiophene-inethanamine dimethyl sulfide carbon disulfide

acetic acid propionic acid 3-methylbutyric acid hexanoic acid 2-hexenoic acid octanoic acid nonanoic acid

Phenols 63 37 71 159 174 186 175

2,3,5-trimethyl-phenol 3,5-dimethyl-phenol 2,3,5,6-tetramethyl-phenol 2-methoxy-phenol phenol 2- o r 3- o r 4 - e t h y l - p h e n o l 4-ethyl-2-methoxy-phenol

Lactones

*

1,1-dimethyl-cyclopentane

58 1-nitro-pentane 15 1- o r 2 - n i t r o - p r o p a n e S,2 5 2, 9 3 4

65 146 151 * *

2-ethenyloxy-ethanol 3-ethoxy-propanal or propylene oxyde etheniloxy-isooctane 2-nitro-butane hexanenitrile

Oxygen-containing heterocyclic compounds 1 5

Free acids 87 102 130 157 170 178 184

l,3,5-tris-(methylen)-cycloheptane

Nitrogen- and oxygen-containing compounds

Sulfur compounds 10 22 73 89 70 * *

References

*

4 10,9 2 4 4 4

2-methylfuran 2-ethylfuran

17 19 33 64 76 88 96 103 105

3,4-dihydro-2H-pyran butyl-oxirane 2-pentyl-furan ^' ^ 1,4-dioxane o r 1,3,6-trioxane 4 3-(4-methyl-3-pentenyl)-furan furfural ^ 5,6- o r 3 , 4 - d i h y d r o - 2 H - p y r a n - 2 - c a r b o x a l d e h y d e 6-ethoxy-dihydro-2(3H)-furanone 5-methyl-furfural ^> ^

114 117 145 161 166 167 169 189 *

5-ethyl-2(5H)-furanone 2-methyl-l,3-dioxolane 3,4-dihydro-2H-pyran-2-carboxaldehyde 2-methyl-benzofuran 2,2'-bi-l,3-dioxolane 2-methanol-l,3-dioxolane 2-hydroxy-methyl-tetrahydro-pyran 2-methoxy-l,3-dioxolane furan

124 b u t y r o l a c t o n e

Halogen compounds *

10 4

trichloroethylene

* Volatile c o m p o u n d s sampled only with static and dynamic headspace

4

324

Scores; til l/tl2l

N•*=• •2ax:20f

•3(fC2£f

•3ao5'

•no/

Kwoy

•4ffX:2ff

•3ax2(y

-10

t[1]

LoadinBs;p|l|/p|2| .65-161

•107 •136

02

174 •146

•139

0.1

•52

•16

•154

- ^ . m .,„

' -.4?" • 59 0.0

- .105

•*^'JK

^•91--.fi2-^"»U

•KX. .•,f'''^^'a.3.r«p47 >]*/R

•1*91 -0.1

•3^

•1777

•15 -0.05

•171

•9 0.00

.99

. ^

•^'issijas

..r -•% -^ 0.C5

P[11

Figure 2 - Score-plot and loading-plot of the first two principal components of PCA of tomato juice sampled with SPME at different times and temperatures of adsorption (see Table 1 for variables).

325

Response Surface of Desirability

^^(mn)

Contour of Desirabflitv

-0.175

-0.062r

35

0.060

-0.063

20

10

15

20

25

30

35

"nme(nnin)

Figure 3 - Response Surface Modelling RSM and contour plot obtained using the PLS built to optimize the time and temperature of volatile sampling using SPME in tomato juice.

326

Scores; tHl/tl21 10-r

95tC-10'

•Control • 95°05'

•95°C-1' 80fC1^'70PG10'

S 0

•ro°xMi' •65^015'

•65°G40'

-~\— -2D

-15

-10

10

tli]

Loadings: p[ll/p[2|

•3ll^ • 104

0.10

•3-

r^Jil.. •IS'i itdji •116

•100 • U

• Ift53 •141

,,

• Btf*'-'* P[2],

^ii64

•Tl^ •<58

, ' S i n s •84 • 2 •1*?08 •98 %1,^

-0.10

-0.10

-0.05

0.00

0.05

0.10

P[11

Figure 4 - Score-plot and loading-plot of the first two principal components of PCA with tomato samples processed at diS'erent times and temperatures of thermal treatment, modelled using 190 volatile compounds (see Table 1 for variables).

327

Scones: tm/tr31 sofoic

10

• 80°O20'

2 •70PC-10'

0

•95' 'OlO'

-2H

,I^_^^.70OG-30'

• Qmtixil

• 95°C5' -4 -2D

-15

-10

-5

10

t[1l

Loadings; p[ll/p|3|

28

, 117?"^

is?

^ 3

-m^^

•W47

•190

•m •m^

•1M5««

'HO • 176

•'*?,

0.00 P[3]

• 171 '6.'r'-"

*it>8

•'04

I

^'9^ •-1flW2 •"^•10 'm^ ^ ,,,„

• l(»124*i1§6

.

'1' .« • 179

•n9 • 146

•^.l •82

-0.10

WIOS •183

•IS

-0.05

0.00

0.05

0.10

Pl1]

Figure 5 - Score-plot and loading plot of the first vs the third principal components of PCA with tomato samples processed at different times and temperatures of thermal treatment, modelled using 190 volatile compounds (see Table 1 for variables).

328

Table 2. Loadings of the first three components of SIMCA model of selected volatile compounds important for the tomato juice flavor.* SIMCA model

_JllU_

Pt2]

_£l3L

1 5 33 76 88 103 105 1 114

2-ninthylfiiran 2-ethylfuran 2-i)entyl-furan 3-(4-mRt.hyI-3-pentenyl)-furan furfural 5-etlioxy-
-0.072 0.008 -0.038 -0.031 0.059 -0.106 0.087 -0.104

0.026 0.014 0.047 -0.058 0.107 0.062 0.072 0.064

0.140 0.162 -0.006 0.176 -0.040 -0.023 0.006 -0.042

Sulfur compounds

10 22 68 70 73 1 89

(limctthyl (lisuindR 3-nthyl-thioplionR / 2-ethyl-thiophenn dimnthyl trisulfide 2-thioph(;nn-mothanamine 2-isobutyl-thiazole 2-methyl-2-thiazoline

-0.030 -0.065 -0.065 -0.052 -0.052 -0.055

-0.121 -0.087 -0.065 -0.092 -0.134 -0.031

-0.055 0.121 -0.076 0.053 -0.030 -0.149

Aldehydes

3 7 9 28 32 25 47 55 69 1 78

3-mc5thyl-butanal pnntanal hexanal cis-2-hexenal trans-2-hexenal hoptanal octanal 2-heptonal nonanal 2-ootenaI

0.004 -0.100 -0.096 -0.046 -0.010 -0.107 -0.099 -0.109 -0.093 -0.109

-0.150 0.054 -0.073 -0.112 -0.143 0.006 0.070 0.034 0.061 0.031

-0.062 0.048 0.069 0.130 0.113 0.066 -0.009 0.043 -0.003 0.039

Alcohols

59 1-hnxanol 61 3-hexen-l-ol 67 3-hoxon-l-ol (i) 83 1-hnptanol 1 101 l-octanol

-0.108 -0.095 -0.103 -0.107 -0.105

0.064 0.018 0.034 0.038 0.062

-0.015 0.047 0.012 -0.039 -0.026

Ketones

6 3-pentanone 8 l-penten-3-one 1 46 2-octanono

-0.093 -0.105 -0.056

0.056 -0.033 -0.017

0.031 0.062 0.172

Terpenoids

95 99 113 153 165 156 160 186 1 190

0.083 0.073 0.036 -0.054 -0.099 -0.114 -0.106 -0.108 -0.053

0.055 0.017 0.017 -0.132 0.027 -0.006 0.050 0.019 -0,040

0.021 0.070 0.076 0.093 0.033 0.015 -0.038 -0.045 0.064

-0.051 -0.010 0.076 -0.112 0.067 -0.098 0.086 0.042

-0.133 -0.070 -0.105 -0.033 -0.004 -0.050 -0.052 -0.117

-0.027 0.156 0.084 0.021 0.072 0.064 0.057 0.042

alpha-terpinone linalool / iRopulegol torpinen-4-ol geranyiacetone nerol nerylaoetone geraniol eugenol / isoougenol farnesol

Volatile carotenoid derivatives 56 6-m«»thyl-5-hepten-2-onfi 84 131 138 158 165 176 1 181

6-inothyl-5-hopten-2-ol cifi-citral trans-eitral alpha-iononfi bnta-ionone pseudo-iononc pKoudo-ionone (i)

* The model was constructed using all the 190 volatile compounds found.

329 4. CONCLUSIONS The volatile compounds sampled in tomato juice were 190 and 219 using SPME and traditional SHSA and DHSA, respectively. The most important differences between these methods regard the sampling of apolar and polar volatile compounds with a low molecular weight. The optimal time and temperature of SPME adsorption were 27 min at 29 °C. Thermal treatment used to inactivate endogenous enzymes during the tomato processing strongly modified the volatile composition of tomato juice.

5. ACKNOWLEDGEMENT This investigation was supported by Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford U.K.

6. R E F E R E N C E S 1 2 3 4 5 6 7 8 9 10 11 12

R.G. Buttery, R. Teranishi and L.C. Ling, J. Agric. Food Chem., 35 (1987) 540. R.G. Buttery, R. Teranishi, L.C. Ling and J.G. J. Agric. Food Chem., 38 (1990) 336. R.G. Buttery, R. Teranishi, R.A. Flath and L:C. Ling, J. Agric. Food Chem., 38, (1990) 792. M. Petro-Turza, Food Reviews International, 3 (1987) 309. R.S.T. Linforth, I. Savary, B. Pattenden and A. J. Taylor, J. Sci. Food Agric, 65 (1994) 241. S. Porretta and C. Ghizzoni, Industria delle Conserve, 69 (1994) 37. C.S. Charron, D.J. Cantliffe and R.R. Heath, Horticultural reviews, 17 (1995) 43. R.G Buttery, G Takeoka, R. Teranishi and L.C. Ling, J. Agric. Food Chem., 38 (1990) 2050. C. Marlatt, C. Ho and M. Chien, J. Agric. Food Chem. 40, (1992) 249. E.A. Baldwin, M.O. Nisperos-Carriedo, R. Baker and J.W. Scott, J. Agric. Food Chem., 39 (1991) 1135. R.E. McDonald, T.C. McCoUum and E.A. Baldwin, J. Amer. Soc. Hort. Sci. 121(1996)531. A.L. Begliomini, G F . Montedoro, M. Servili, M. Petruccioli and F. Federici, J. FoodBiochem., 19 (1995) 161.

330

13 14 15 16 17 18 19

P. Schreier, F. Drawert and S. Bhiwapurkar, Chemie Mikrobiologie und Technologie des Lebensmitteln, 6 (1979) 90. Z. Jiang and B. Ooraikul, Journal of Food Processing and Preservation, 13 (1989) 175. R. L.Rouseff, M.M. Leahy (eds), Fruit flavors Biogenesis Characterization and Authentication, American Chemical Society, Washington D.C. (1993). G.E.P. Box, W.G. Hunter and J.S. Hunter (eds), Statistics for Experimenters, John Wiley & Sons, Inc, New York (1978). M. Bertuccioli, Industria delle bevande, (1979) 106. B.R. Kowalski, (Ed.) Chemometrics, Reidel, Dordrecht (NL), 1984. S. Clementi, G. Cruciani, G. Giulietti, M. Bertuccioli and I. Rosi, Food Quality and Preference, 2 (1990) 1.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

331

Fresh-cut pineapple {Ananas sp.) flavor. Effect of storage.* A. M. Spanier^, M. Flores^, C. James^, J. Lasater^, S. Lloyd^ and J. A. Miller^ ^USDA, ARS, Southern Regional Research Center Food Processing & Sensory Quahty 1100 Robert E. Lee Blvd. New Orleans, LA 70124 ^ Dixie Produce & Packaging, Inc., 5801 G. Street, P.O. Box 23647, Harahan, LA 70183

Abstract Fresh-cut fruits are the fastest growing market in today's produce business. However, once a fruit is cut it becomes a different product from what it was in its uncut form. Thus, produce marketers must ensure their product's flavor and texture as well as the product's safety. We examined the effect of storage (4°C for 3, 7, and 10 days) on the flavor volatile profile of freshly-cut pineapples. Volatiles of fresh-cut and stored pineapple chunks were examined by gas chromatography (GC), GC olfactometry (GC-0), GO mass spectroscopy (GC-MS), and microbiological testing. GC-0 data using dynamic headspace sampling techniques indicated that pineapple-like flavors increased very slightly during storage while unpleasant odors and volatiles such as fermented, cheesy, sour dough, alcohol, oily, etc., showed dramatic increases and masked the more desirable pineapple flavor. The large increases in the level of low boiling alcohols (as determined by GC and GC-MS) in stored pineapple suggest that fermentation events were ongoing. Yeast were confirmed as the source of the fermentation derived alcohols. No other microbes (aerobic plate counts, total coliforms, E. coli, and mold) were found above the range acceptable to the fresh-cut produce industry.

1. INTRODUCTION Fresh-cut fruit is one of the fastest growing markets in today's produce business. According to a recent report by the International Fresh-cut Produce Association (IFPA), sales of fresh-cut produce in the United States are projected to increase from their $5.8 billion market in 1994 to $19 billion by 1999, with the majority of this increase in fresh-cut fruits. Fresh-cut produce has become so ^Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. All communication should be with Dr. Arthur M. Spanier Phone: 504/286-4470 Fax: 504/286-4419 E-mail: [email protected]

332

popular with consumers because it represents a value-added, ready-to-use commodity that satisfies their requirement for nutritional value, naturalness (no preservatives added), and safety. However, these requirements impose a challenge for the producer and packager who must physically transform the fruit into an entirely new product, yet maintain the fruit's original flavor characteristics, quality and safety. This study was designed to gain a better understanding of storage-induced changes in fresh-cut pineapple volatiles. The earliest composition work on pineapple volatiles was reported by Haagen-Smit et al. in 1945 [10, 11]. Considerable information has been reported since then concerning flavor of whole pineapple [2-5, 7, 8, 15-17]. However, no literature is currently available on the flavor and quality changes that occur in fresh-cut pineapple during storage. Information gathered from research directed towards understanding the changes in flavor associated volatiles of fresh-cut and stored fresh-cut pineapple should be an important first step in developing packaging and storage methods that will meet consumers' expectations and requirements for high quality, nutritious, safe freshcut produce. 2. METHODS AND MATERIALS Pineapples: Fresh-cut pineapple (chunks) were provided within two hours of processing from a local produce and packaging business. The chunks were packaged for consumer purchase in clear plastic containers (CPC; 12 cm top i.d. x 9 cm bottom i.d. x 11 cm high) with a tight banded lid. Pineapples were of the Chempaka variety grown in Central America. Each pineapple shipment was obtained from statistically selected acreage that had pineapples sampled to meet visual yellow color and Brix specifications. Brix values were a mean of 12 with a lower limit of 10.0 and a high limit of 13.5. 2.1 STORAGE STUDY: Sample preparation and volatile collection: Four to five repetitions (replicates = new shipping lots) were used in this study. Each replicate was divided into four groups of three (3) CPC each. Each group represented the date of the storage time (days), i.e., 0 (fresh-cut), 3, 7, and 10 days. One chunk each from the top, middle, and bottom layer of the CPC were removed, minced, and mixed to ensure homogeneity of each experimental sample. At least two experimental samples from different CPCs were run from each group (date) from at least four replicates. Twenty grams of the mixed and minced chunks were placed into a 200 mL roundbottomed flask connected to a 300 cm long Graham condenser via a 24/40 ground glass with a Teflon-lined sleeve insert. A Tenax trap was adapted to the other end of the condenser. The volatile trap was composed of 400 mg of Tenax 60/80 in a P5rrex glass tube plugged at both ends with 100 mg of glass wool. The trap was placed into an external closed-inlet device

333

(ECID, from SIS, Inc. Metairie, LA) [18, 19] where it was conditioned prior to sample collection at 250 °C for 1 hour with a nitrogen stream of 40 mL/min. The sample in the round-bottomed flask was stirred and maintained at 50°C; volatiles were trapped for precisely 20 minutes by dynamic purging under helium at a flow rate of 20 mL/min. Gas chromatography (GO. flame ionization detection (FID), and GC-olfactometrv (GC-0): The Tenax trap was placed into the ECID and the volatiles desorbed and cryofocused onto the head of the glass SPB5 capillary column (0.75mm i.d. x 60m long; Supelco, Bellefonte, PA) for 5 minutes at 50°C. Gas chromatography was performed using an HP-5880 with flame ionization detector (FID). The oven was programmed to desorb the volatiles from the head of the column as follows: column held at 1°C for 5 min. Then a 3°C/min temperature ramp was run to a final temperature of 250°C. The column was held for an additional 15 minutes at 250°C. The column was split at the terminal end at a 1:1 ratio. One end of the split went to the FID detector and the other end to an attached, heat-shielded olfactory or snifier-port located just on top of the instrument. Olfactometry was performed by three resident scientists in our program; each evaluator was adept and experienced at odor description. Gas chromatography (GO and Gas Chromatography - Mass Spectroscopy (GO MS): GC/MS analysis of the pineapple volatiles was run slightly differently than the GC-olfactometry. The 4 mm (i.d.) glass lined stainless steel tube was packed with 100 mg of Tenax (60/80) plugged at both ends with 100 mg of glass wool. This trap was fastened on to the short path thermal desorption unit (SPTD) of SIS in Ringoes, N J [18, 19]. The sample was directly injected (via tenax desorption) and cryofocused onto the head of a DB5-MS capillary (0.2 mm i.d. x 50 m with 0.33 um film thickness; J&W Scientific, Tolsom, CA) for 2 minutes at 250°C. The DB5-MS used for GC-MS and the SPB5 used for GC-0 have the same polarity. An HP 5890 gas chromatograph was coupled with an HP 5970 with the RTE-A Wiley Library data system (Hewlett Packard, Palo Alto, CA); the mass detector was programmed for 1 min at 0°C followed by a 3°C/min rise to 150°C and then a 5°C/min rise to 280°C. The capillary column was held for an additional 13 minutes at 280°C for a total run time of approximately 90 min. To consider any potential differences between the DB5-MS and the SPB5, all compounds are listed with their Kovats Index (IK). Microbiological Testing: Testing fresh-cut pineapples for total aerobic bacteria, coliforms, E. coli, mold and yeast was performed at the Quality Assurance Laboratory on site at Dixie Produce, and Packaging, Inc. located in Harahan, LA. Pineapples from six separate shipments over an eight month period were examined using fairly standardized methods consisting of using 3M brand Petrifilm plates

334

(3M Health Care, St. Paul, MN) with ready-made medium systems. All samples were run in replicate with appropriate dilutions for each microbial population being examined.

3. RESULTS & DISCUSSION 3.1 STORAGE STUDY The earliest composition work on pineapple volatiles was reported by HaagenSmit et al [10, 11] at the California Institute of Technology who identified 13 compounds fi:-om 2900 kg of trimmed fruit by fi:-actional distillation and derivatization. Later research by Flath and Forrey [51 identified forty-five compounds [21 previously unreported in pineapple] extracted fi'om 9 liters of pineapple essence obtained from fi:'ozen pineapple juice concentrate. Takeoka and colleagues [22]fi:'omthe USDA, Agricultural Research Service's Western Regional Research Center in Albany California were able to definitively identify the volatiles of fresh pineapple using dynamic headspace analysis and vacuum steam distillation-extraction. Their analyses indicated that a three hour concentrate fi-om 400 grams of pineapple pulp contained a diverse assortment of esters, hydrocarbons, alcohols and carbonyl compounds. These methods required concentration of volatiles, use very large sample sizes, and long volatile extraction times. Changes in the flavor volatile profile of commercially processed, fi-esh-cut pineapple chunks were examined after cold storage (4°C) for 0, 3, 7, and 10 days where 0 represents fi:'esh-cut pineapple. Samples were studied by gas chromatography (GC), GC olfactometry (GC-0), GC-mass spectroscopy (GC-MS) as depicted in Figure 1 and microbiological testing. Components of samples analyzed by GO-MS were given tentative identification by comparing their mass spectra to the RTE-A Wiley library. Since standards were not available at the time of the experiment, the identifications are considered tentative. The retention system proposed by von Kovats [23] was utilized to facilitate comparison of GC-MS data with GC-0 data that was performed on slightly different columns and instruments. Thus, compared to earlier research on pineapple volatiles these pineapple chunks were examined by short-time (20 minute), dynamic headspace purging of relatively small (20 gram) samples. Sample collection and chromatographic resolution for GC-0 and GC-FID was based on modification of a method used in this laboratory for another commodity [6]. Over 145 individual compounds were tentatively identified, using an RTE-A GC-MS library to examine the volatiles in the fresh-cut and stored pineapple samples resolved on a widebore (0.75 mm ID) column. These compounds were a diverse assortment of esters, hydrocarbons, alcohols and aldehydes and are similar to those reported by Takeoka and colleagues [22] who identified approximately 160

335

volatiles in pineapple pulp using a capillary column (0.32 mm ID). Among the volatiles observed were several pineapple-specific volatiles first reported and identified by Berger and colleagues (2; see Table 1). Table 1 lists only those compounds (1) that appeared consistently within a date group, i.e. in all 10 day samples as opposed to in only one or two. Resolution of compounds for identification was compounded by the need to use a wide bore column to facilitate aroma identification by GC olfactometry. Thus, for discussion here, we arbitrarily chose to (1) use only compounds having an area greater than 1% of the total area, and (2) those compounds having a Wiley library hit or match of >85%. The retention time of the samples on the SPB5 column are listed along with the Kovats index determined with the DB5-MS column. Whenever possible, the odors listed in Table 1 are presented both as the GC-0 odor we detected and as a previously published odor-description given for the compound (see Table 1 legend). The table also gives corroborating literature references, where available, for those compounds identified in these experiments. Each odor listed represents the odor perceived by the three investigators in the GC-0 experiment. Figure 1 shows the chromatographic profile of the head space volatiles (GCFID) of pineapple chunks stored for 0 (fresh-cut), 3, 7, and 10 days at 4°C. The figure also shows some of the major odors perceived by GC-0 (Table 1). While area counts were made of each peak to determine the change in the components concentration during storage (Table 1), quantitation of each component's concentration for use in determination of 'odor unit' [defined by Guadagni and colleagues, 9, as the ratio of the concentration of the compound and its odor threshold] was not performed since authentic standards were not available to us at the time of the analysis. Even though the use of a capillary column (0.32 mm ID) is a better method for compound identification, it was decided to use the wider bore 0.75mm ID column for GC-olfactometry to ensure that enough sample would be available to truly reflect the aroma expressed. The chromatogram in Figure 1 is presented beginning at 13 minutes and continuing through 37 minutes to show those peaks that otherwise would have appeared hidden because of the scale height of 1,400,000 counts of the pre-13 minute peaks (Figure 2). The literature [2, 22] and this study (Table 1; Figure 1) have indicated that the mainfi:*uityand pineapple flavors comefi:'omthe following volatile compounds: acetic acid ethyl ester (RT = 13.6), acetic acid 1-methylethyl ester (RT = 16.5), propanoic acid 2-methyl ester (RT = 18.1), acetic acid propyl ester (RT = 20.0), butanoic acid methyl ester (RT = 20.6), 1-butanol 3-methyl (RT = 22.1), propanoic acid 2-methyl ethyl ester (RT = 23.1), butanoic acid 2-methyl ethyl ester (RT = 24.4), pentanoic acid methyl ester (RT = 27.4), and hexanoic acid methyl ester (RT = 34.2). Analysis of the peak areas of these compounds (Table 1) indicate that during storage of fresh-cut pineapple some of the pineapple and fi:'uity flavored volatiles increase slightly (acetic acid 1-methylethyl ester, acetic acid propyl ester

*

^

^

cd

(M

o in

C\J O C\J

;£' CD

t CD C

.c: C) (0

CO

CD

S CO

"5, £ CD

CD

n "S TJ

Q)

1

CD

o o

m T3

^^^

to

O

•11 T3 iS'

O)

®E .£ o C3LC0

© ©

©

x: E

E

©

x: o >« £ CVI

• > »

0)

CD

ca.

CD C

5 2

c" to 0) 0)

CO

8 co"

1 (0

3

S-

O O CO

CO

,_, S c: .o S. p a

t^ "^ 0)

CD

E

o CO

1-

o c CO

o CO o

CM

CM CO

06

CD

in rn in CO

in

in

cd

CO

"^

CD

CO

CO -^

CM

CO

T-

CM

CD C) CO

T3 C) CO O

E

>s £ CD

CO TCM


w

§s

336

^

•

0) 03

i

•S

^ •"*

U

'§

^u

icd

'—'

1"o

0 JS

>—' CO

1

S

"w C«

•^ O

E

—. t3

"-S^ •-

^

/~v

"

tf

U

PLH

"

03 0)

* w H

g

gs ffi

o K! H Pc5

fe fo

>

O Pc5 O

3 fc Q ;2:

o 1—1 H 1—1 iX) O CL,

1^

o jj H

3 O > H O

< (1H CQ

^ w ffi

:2;

H

o

o ^

g

w [£H

o H O

w fe fe w o

TH

2cd H

^ P *

C3)

C

CD

•

*

•

o5 in

O

o

o

1 1 k

CL

Xl

r CM

T-

O

^

CD

w

c

^

o

cx

CO -o

a

©

E

CD

j

Q.

C

2

d.

S 2

CL CX

-Jr"

1

•^ -i

, >, ©

in

T-"

CO CO

't

r

§)

> 2

•Dh-

"oa

s P"" S o c

©

COsB

=3 2 o E

«L.- >^ CO

m ^to o S

CO

O) -

^

S c

T-^

^

d

O ©

CM CO

i

cf

-

•s

-

»!: ®

CO

>;

O O

CO O

CO ©

2 >^

^ O)

"©

CO

^ isCD0

©

-^ ^ CO CO

11 i S i

© CO © • > »

•< #

TJ1^ 00

00 h00

E

CM h00

•V

C3) -^ 00

5K

sz •55

CM CM 00

00

©

00

r^

E

N

o

in in CM

CM

CD

00 Tt CM

^

§^

N.

c»

^^

-^ "^ CM

CM-

r^

CO

00

CM T3

© F E •c

©

^ - -gF- Su © -Q CM:S

«OCM

CM

1^

-* Tf CM

X3_ O

CO ^ CM m

CM '
CM

£ E

^

00

t

2g

E II ^1 ^5

W

.^ S-

"^ O) c^ CM P

.n o

O

o

r«^

00 c» hs. CM -^ in

T-

g £

£ i, I

Q.

i^ C\J "^ "? 9

o

E o CO

3 CD" Q. QL

C3 CD C

c» 1-

CO ^

•«

00 CD '^ T 9

S 5 S^ o

~

CD

o g o q d

a a.

^ ©

2

E

^

CO

'"

•

2> O)

ifT

6

t

>

^

rs. 1 ^ r^ rs. rs.

CM in o o

00

r

in o o

do

-x-

Q-Q.

E

(D (D Q O cfl W 0)

o

§

2

75

E

2 t>

Q. C

<M cvi CO

.E o

CO T-^ CO

•"

^ ^

CO

.5

^ ^

a> —

t

B

^ 5

«

o 9 9

©

00 CO

Tf

o

0> CO

'*

O -^

^

-r-^

^

o 6 cvi

Tt

C>4 Tf

CO

CO Tt

c \ j u > p c o i n < o < o c o i o

r^ CO

''f

CVI

'^

<0

CO

4

in

CO

r^

'^

337

.-i= -s

"trt

5^23

n-i

S;, o

2 "a

o

9

"^ S» S

o O

§ ?

iZ.'^

alcohol, ketone, aldehyde (27.4)

pineapple/banana/apple (24.1) pineapple/banana/apple (24.6) greenynarsh/plastic/musty/astrinc, ent, sour metallic/some sulfury(24.8) |:

1-butanol, 3methyl (22.1)

pineapple/apple/banana (21.8)

QJ P(

H H

^> CD P

;;^ 2. o pj B: ^

5" ^ §•

C= C«

sis

>^ ol ^ ^

8 las'

2 o 2.'

sulfury, onion-like, metallic (36.8)

oily, greas)ji cucumber ^^'^)

fruity/floral/candy/chocolate (33.2)

fruity: apple-like, pineapple (28.7)

2-heptenolc acid methyl ester (36.7)

hexanoic acid, methyl ester 34.2)

butanoic 3-methyl ethyl ester (29.2)

acetic acid 2-methyl propyl ester (7&10d; 24.2}; butanoic i-methyiethyl ester (24.3) ; hexane 2,2,5trimethyljflOd; 24.4) hexanal (25.5) buanoic acid ethyl ester (7& lOd; 25.9)

propanoic acid, 2-methyl ethyl ester (23.1)

butanoic acid, methyl ester (20.6)

acetic acid propyl ester (20.0)

propanoic acid, 2-methyl ester (18.1)

acetic acid 1-methylethyl ester (16.5)

acetic acid ethyl ester (13.6)

Compound {GC-MS)

pineapple (20.9)

strong pineapple (18.1)

pineapple (16.4)

fermented peach (13.6) ketone-like (14.1) alcohol (14.6) mild fruity to fruity (14.8) cheesy, sour, dough-like (15.2

Odor perceived (GC^O)

<^ » S.G hexanoate, bleachy (28.4)

1^ V

^ !^ ^ I

Hit

8o

cr ^ ^ o - ^^ 1 ", a >-^ o

a S 02.

*3 P

^ § -^ :^'

2^ ^ ^ S

338

339 and 1-butanol S-methyl acetate) while others either decrease shghtly (acetic acid ethyl ester, butanoic acid methyl ester, 1-butanol 3-methyl, butanoic acid 2-methyl ethyl ester, and hexanoic acid methyl ester) or show a variable response (butanoic acid 2-methyl methyl ester and pentanoic acid methylester). The chromatographic procedure used in this investigation is well suited for the analysis of unsaturated hydrocarbons that result from enzymatic and/or oxidative degradation [6, 20,21], The major storage dependent change in the chromatograms is the increase in the concentration of enzymatic, fermentative, and oxidativedegradation products, such as hexanal. While peak heights as determined by only one means of detection, i.e. FID, do not conclusively prove a change in flavor, previous studies have correlated such increases in these lipid compounds with an increase in oflF-flavor development [19-21]. Thus, as seen in Figure 1 and Figure 2, it is possible, and perhaps likely, that this increase in volatiles associated with "offflavors" masks the desirable flavor impact of the pineapple-flavored volatiles leading to a diminution of the overall flavor quality of the product. _ 1 if 1

'^2

o (3 Q ^ 1,400,000

>»

1,200,000

1,000,000

800,000

ifll

^°^^^

m • L \

m 7day

n

•

igKi>\

O

3 day

600,000

400,000

200,000

0

1 2 3

T

> . « , » < , . 0 1 1 1 2 1 3

1

Retention Time (minutes)

L > o p So 1 a.

«

«

(s ca

Figure 2: Gas chromatograph (GC) of stored fresh-cut pineapple chunks. GC olfactometry (GC-O) determined aromas are listed in the column on the left. Compounds identified by GCMS library matches are listed in the right column.

340

The peaks observed during the initial 13 minutes (Figure 2) are primarily low molecular weight alcohols, acetic acid methyl esters, etc. These products are indicative of microbial fermentation. Figures 2 and 3 show that with increasing storage time there is not only an increase in the area of the aldehyde peaks such as pentanal, hexanal, and heptanal with their strong green and painty aroma, but also large increases, particularly at 7 and 10 days, in the 1 and 2 methylpropyl esters of acetic acid with their raw green bean, grassy note. Also increasing were acetic acid, butanoic acid ethyl ester with its rancid, raw vegetable aroma (mostly at 7 and 10 days), hexanoic acid ethyl ester with its green, leafy, and somewhat sulfury aroma, acetic acid ethyl ester (ethyl acetate), alcohols such as ethanol, and acetone. Many of these, if not products of lipid oxidation or trans-esterification, are products of microbial metabolism and fermentation processes. The latter suggested that yeast might be a source of fermentation derived alcohol production. This was confirmed by the observation of increased yeast plate counts with storage (Figure 3). No other microbes (aerobic plate counts, total coliforms, E. coli, yeast and mold) were found above the range acceptable to the produce industry.

150,000

^

100,000

yeast mold coli coliforms aerobic

Storage Time (Days) Figure 3. Microbial populations in commercially processed, fresh-cut pineapple chunks held in grocer-style opentop refrigerators at 7.0°C ± 0.4 for two weeks. The numbers above each bar represent the mean microbial load of 6 separate preparations examined over an 8 month period.

341

In summary, GC-FID data indicated that peaks associated with known pineapple flavor aromas showed a fair degree of variability in their response to storage; however, the relative change in the slope (area/storage time) of these peaks was minimal. On the other hand, there were major increases in the type and content (area) of volatiles associated with undesirable flavors such as fermented, grassy, cheesy, and sour dough. The large increases in the level of low boiling alcohols in stored pineapple (GC-FID and GC-MS) suggested that fermentative events were occurring in stored, fresh-cut pineapple; the latter gained support by the observation of increasing yeast plate counts during storage. No other microbes (aerobic plate counts, total coliforms, E. coli, yeast and mold) were found above the range acceptable to the fresh-cut produce industry. In that most fruits contain a significant number of microbes on their outer surface [14], it is reasonable to suggest that a key step to extending the shelf-life of many fresh-cut fruits is the use of good manufacturing practices (GMP) and proper sanitation procedures which minimize microfloral counts. This current research supports the recommendation by Nguyen-the and Carlin [14] on the microbiology of minimally processed fresh fruits and vegetables. Nguyen-the and Carlin indicate that microbes may be reduced by washing intact whole fruit in cold water containing 100-200 ppm chlorine and a pH 7.0 adjusted with citric acid and then rinsing it with water. This is typically not performed on pineapple. Therefore, it seems reasonable to suggest that an external wash with low levels of chlorine bleach (100 ppm) or other GRAS approved material may be appropriate to minimize some of the yeast contamination of the pineapple. In addition to rinsing the fruit prior to cutting, it seems suitable to rinse and wash all of the processing equipment, e.g., cutting blades and surfaces to maintain good GMP facility practices. It is also appropriate and acceptable to maintain cutting instruments as sharp as possible. In addition to suggesting the need for external rinsing of the produce, knowledge of microbial growth and ecosystem analysis should be considered as a necessary requirement for any packaging plant as this information will lead to the development of the best means of preparing and packaging produce of high flavor quality that is nutritionally sound and safe to eat.

4. ACKNOWLEDGMENTS The authors would like to gratefully acknowledge the Spanish government's Formacion de Personal Investigador I Ministerio de Educacion y Ciencia (FPI/MEC) for funding Monica Flores as a postdoctoral fellow in our laboratory. We would also like to acknowledge the assistance of Ms. Edith Garrett, President of the International Fresh-cut Produce Association (IFPA) for her direct introduction of A. M. Spanier to the staff of Dixie Produce and Packaging, Inc. Grateful acknowledgement is also given to Elaine Champagne, Edith Garrett, Casey Grimm,

342

Cynthia Mussinan, for their helpful comments and suggestions regarding this manuscript. 5. REFERENCES 1. Aldrich Chemical Company In Flavors & Fragrances. 1997 Catalogue 2. Berger, R. G.; Drawert, F.; KoUmannsberger, H.; Nitz, S.; Schraufstetter, B., Novel Volatiles in Pineapple Fniit and their Sensory Properties. J, Agr. Food Chem. 33(1985)232-235. 3. Chang, H. T. Jr.; Chenchinn, E.; Vonnahame, P., Nonvolatile Acids in Pineapple Juice. J. Agr. Food Chem. 21 (1973) 208-210. 4. Dirinck, P.; De Pooter, H.; Schamp, N., Aroma Development in Ripening Fruits. In, Flavor Chemistry: Trends and Developments. Teranishi, R., Buttery, R.G. and Shahidi, F., eds., ACS Books, Washington, D.C. (1989) pp. 23-34. 5. Flath, R. A.; Forrey, R. R., Volatile Components of Smooth Cayenne Pineapple. J. Agr. Food Chem. 18 (1970) 306-309. 6. Flores, M.; Grimm, C. C ; Toldra, F.; Spanier, A.M., Correlations of Sensory and Volatile Compounds of Spanish "Serrano" Dry-Cured Ham as a Function of Two Processing Times. J. Agr. Food Chem. (1997) IN PRESS 7. Gortner, W.A., Chemical and Physical Development of the Pineapple Fruit. IV. Plant Pigment Constituents. J. Food Set. 30 (1965) 30-32. 8. Gortner, W.A.; Singleton, V. L., Chemical and Physical Development of the Pineapple Fruit. III. Nitrogenous and Enzyme Constituents. J. Food Sci. 30 (1965) 24-29. 9. Guadagni, D. G.; Buttery, R. G.; Harris, J., Odour Intensities of Hop Oil Components. J Sci. Food Agric. 17(1966)142-144. 10. Haagen-Smit, A. J.; Kirchner, J. G.; Deasy, C. L.; Prater, A. N., Chemical Studies of Pineapple (Ananas sativus Lindl). II. Isolation and identificaton of sulfur-containing esters in pineapple. J. Amer. Chem. Soc. 67 (1945b) 16511652. 11. Haagen-Smit, A. J.; Kirchner, J. G.; Prater; A. N.; Deasy, C. L., Chemical Studies of Pineapple (Ananas sativus Lindl). I. The Volatile Flavor and Odor Consituents of Pineapple. J. Amer. Chem. Soc. 67 (1945a ) 1646-1650. 12. Heath, H.B., In, Source Book of Flavors. AVI Books, Van Nostrand Reinhold, NY(1981) pp. 863. 13. Maarse, H., In, Volatile Compounds in Foods and Beverages. Marcel Dekker, Inc. NY (1991) pp. 764. 14. Nguyen-the, C ; Carlin, F., The Microbiology of Minimally Processed Fresh Fruits and Vegetables. Crit. Rev. Food Sci. Nutr. 34 (1994) 371-401. 15. PauU, R. E., Pineapple and papaya. In, Biochemistry of Fruit Ripening. Seymour, G., Taylor, J., and Tucker, G., eds. Chapman & Hall London (1993) pp. 291-323.

343

16. Singleton, V.L., Chemical and Physical Development of the Pineapple Fruit. I. Weight per fruitlet and other Physical Attributes. J. Food Sci. 30 (1965) 98104. 17. Singleton, V. L.; Gortner, W. A., Chemical and Physical Development of the Pineapple Fruit. II. Carbohydrate and Acid Constituents. J. Food Sci. 30 (1965) 19-23. 18. Spanier, A. M.; Grimm, C. C ; Miller, J. A., Sulfur-Containing Flavor Compounds in Beef: Are they Really Present or are they Artifacts? In, Sulfur Compounds in Foods. Mussinan, C. J. and Keelan, M.E., eds. American Chemical Society Books. Washington, D.C. (1994 ) pp 49-62. 19. Spanier, A. M.; St, Angelo, A. J.; Grimm, C. C ; Miller, J. A., The Relationship of temperature to the production of lipid volatiles from beef. In, Lipids in Food Flavors. ACS Symposium Series #558. Ho, C.T. and Hartman, J. (eds.) Washington D.C. Chapter 6 (1994) pp 78-97. 20. Spanier, A. M.; St. Angelo, A. J.; Shaffer, G. P., Response of Beef Flavor to Oxygen Depletion and an Antioxidant/Chelator Mixture. J. Agr. Food Chem. 40 (1992) 1656-1662. 21. Spanier, A. M.; Vercellotti, J. R.; James, C , Jr., Correlation of sensory, instrumental, and chemical attributes of beef as influenced by structure and oxygen exclusion. J. Food Sci. 57 (1992) 10-15. 22. Takeoka, G.; Buttery, R. G.; Flath, R. A.; Teranishi, R.; Wheeler, E. L.; Wieczorek, R. L.; and Guentert, M., Volatile Constituents of Pineapple (Ananas Comosus [L.] Merr.). In, Flavor Chemistry: Trends and Developments. Teranishi, R., Buttery, R.G. and Shahidi, F., eds., American Chemical Society, Washington, DC (1989) pp 223-237. 23. von Kovats, E., Gas-chromatographische Characterisierung Organischer Verbindungen. Teil 1: Retentionsindices Aliphatischer Halogtenide, Alkohole, Aldehyde und Ketone. Helv. Chim. Acta 41 (1958) 1915-1932.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

345

GC-MS analysis of volatile compounds in durian (Durio zibethinus Murr.) J. Jiang^ S.Y. Choo\ N. Omar^ and N. Ahamad^ ^ Singapore Productivity and Standards Board, Food Biotechnology Centre, 1 Science Park Drive, Singapore 118221. ^ Malaysian Agricultural Research and Development Institute, Peti Surat 12301, Pejabat Besar Pos, 50774, Kuala Lumpur, Malaysia.

Abstract Volatile compounds were extracted from durian by vacuum distillation followed by dichloromethane extraction and GC-MS analysis. A total of 108 compounds were identified, including 49 esters, 18 sulfur compounds, 16 carbonyl compounds, 11 alcohols, 7 hydrocarbons, and 7 miscellaneous components. Of the identified compounds, 52 have not been previously reported as durian flavor components. Esters, ketones and sulfur compounds were the major compounds. Acetoin, ethyl 2-methylbutanoate and diethyl disulfide were predominant, accounting for 34.1-37.5%, 20.7-21.9% and 5.6-6.1% of the total volatiles, respectively. The esters and sulfur compounds are obviously responsible for durian's strong fruity and sulfury odors while ketones, especially acetoin, most likely contribute to its creamy flavor.

1.

EVTRODUCTION

Durian {Durio zibethinus Murr.) is one of many tropical fruits native to South East Asia. The capsule fruit is usually large (up to 4 kg) and is characterized by a green to brownish skin fully covered by numerous thick spikes. Its edible aril is soft and creamy with white or yellowish color. The aril has a sweet taste and strong odor dominated by fruity (estery) and unique sulfury notes. Due to its unique and highly characteristic flavor, durian is deemed as a delicacy and even regarded as the King of Fruits by local people. Volatile composition of durian fruit had been studied by a number of researchers. Baldry et al.^ identified 26 volatile compounds in durian fruits from Malaysia and Singapore, including 12 esters, 7 sulfur-containing compounds, 4 alcohols, 2 aldehydes, and 1 aromatic hydrocarbon. The predominant flavor compounds of Thai durian were identified by Moser et al^ as 1,1-diethoxyethane, ethyl 2-methylbutanoate, ethyl acetate, hydrogen sulfide, ethyl hydrodisulfide and several dialkyl polysulfides. Fischer et al^ investigated Malaysian durian and reported that 17 sulfur-containing compounds and 33 non-sulfury components which were thought to contribute to the sulfury and fruity notes, respectively. Apriyantono and Septina'*

346 reported that of the 10 positively identified flavor compounds, 1,1-diethoxyethane, ethyl 2methylbutanoate, ethyl (Z)-4-decenoate and acetoin were the major volatile components of Indonesian durian. A total of 63 volatile components, mostly esters, sulfur compounds, alcohols and ketones, were identified in 3 clones of Malaysian durian by Wong and Tie.^ Ethyl 2-methylbutanoate, ethyl propanoate and acetoin were the predominant components. Weenen et al^ identified 18 sulfur-containing compounds and 18 non-sulfur components in three varieties of Indonesian durian. Acetoin, ethyl 2-methylbutanonate and hexadecanol were the most prominent components. All the published papers reported generally similar results, but significant differences did exist among different reports with regard to both qualitative and quantitative data on the major flavor components of durian fruits.

2.

MATERIALS AND METHODS

Ripe fruits of Malaysia durian were purchased from a local market in Singapore. The fruits showed no signs of over ripeness, such as splitting of the capsule and wateriness of the aril. Extraction of volatile components was performed immediately after the fruits were brought back to the laboratory. A blended mixture of durian aril (60 g) and deionized water (lOOmL) was distilled under vacuum (20 mBar, 40°C) until the mixture was almost dry. The vapor was condensed in a cooling coil (-3°C) and a liquid nitrogen trap and collected in a 200mL flask. The distillate (127mL) collected in the flask was transferred to a 250mL separating funnel. Residue in the cooling coil and trap was rinsed with 50mL of dichloromethane (DCM) into the separating funnel. A magnetic bar was carefully put into the separating funnel. After the funnel was firmly capped, the content in the funnel was stirred on a magnetic stirrer for 15 minutes at a speed that enabled the distillate and DCM layers to be constantly mixed. After the aqueous and DCM layers were fially separated, DCM layer was collected in a 200mL conical flask. The extraction was repeated twice following the same procedure. The combined DCM extracts were concentrated to 0. ImL under a flow of pure nitrogen. The concentrated extract was analyzed using a Unicam Automass 150 GC-MS system (Unicam Automass, Argenteuil, France). The components of the extract were separated using a 40m X 0.25mm x 0.3 |im DB-Wax column (J&W Scientific, Folsom, CA, USA). Column temperature was increased from 40 to 250°C at a rate of 3°C/min and maintained for 15 min. Injector temperature was set at 250°C. MS parameters were as follow: EI mode with an ionization voltage 70 eV, ion source temperature 150°C and scan range from 20 to 350 amu. Analysis of the extract was also performed using a 40m x 0.18mm x 0.4 jim DB-1 column (J&W Scientific, Folsom, CA, USA) and identical GC and MS conditions. Unknown compounds were identified either by comparison of their spectra with those in an NIST Mass Spectra Library or by comparison of both spectral and retention data obtained from the durian sample and authentic chemical standards.

3.

RESULTS AND DISCUSSION

A total of 108 compounds were identified from the durian extract, including 49 esters, 18 sulfur compounds, 16 carbonyls (aldehydes/ketones), 11 alcohols, 7 hydrocarbons, and 7

347

miscellaneous components. Of the 108 identified compounds in this study, 52 had not been previously reported as durian volatile components and most of them were detected in extremely low concentrations (Table 1). Esters and ketones each accounted for approximately 40% of the total volatiles while sulfur-containing compounds accounted for nearly 8%. Presence of these volatile flavor compounds in relatively large quantities explained the characteristic durian odor with strong fruity (ester-like), creamy and unique sulfiiry notes. 3.1 Ester Compounds Esters were found to contribute significantly to durian volatile composition both in terms of quantity and the total number of detected components. These compounds are responsible for the strong fruity and estery notes of durian fruits. In this study, a total of 49 esters were identified, covering a wide range of ester molecules, mainly ethyl, methyl and propyl esters of C2-10 carboxylic acids. Of the 49 identified esters, 19 have not been previously reported as durian volatile components. Of the five esters of unsaturated carboxylic acids, propyl (E)-!methyl-2-butenoate and ethyl (Z)-4-octenoate were for the first time identified in durian. All the five unsaturated esters were previously reported in various fruits except propyl (E)-2methyl-2-butenoate which had only been detected in camomile oil.^ Esters accounted for about 40% of the total volatiles in the durian variety studied in our laboratory. In a previous study, esters were found in a much higher proportion (49.25-57.88%) in 3 Malaysian durian varieties.^ In our study, majority of the identified esters were detected in very small quantities except seven of them with a percentage above 1%. Ethyl 2-methylbutanoate was the predominant ester, accounting for 20.73-21.87% of the total volatiles. Its predominance has been reported in Malaysian durian by Baldry et al.^ and Wong and Tie^, in Thai durian by Moser et al.^ and in Indonesian durian by Weenen et al^ and Apriyantono and Septiana'*. In some previous studies, other esters were also found as the major components in Malaysian durian, such as ethyl acetate with a concentration over 10%^ and ethyl propanoate with a concentration around 20%^ Both ethyl acetate and ethyl propanoate were detected in our study, but in much lower concentrations, 0.36-0.97% and 2.14-3.11% respectively. In a recent study, Weenen et al^ could not detect ethyl propanoate in the three Indonesian durian varieties studied. 3.2 Sulfur Compounds In this current study, 18 sulfur-containing compounds were identified, constituting the second largest group of durian volatile components in terms of the number of components. Mass spectra of at least 10 other unidentified peaks showed typical characteristics of sulftir compounds, and therefore the total number of sulfur-containing compounds should be more than 18. These sulfiir compounds are obviously responsible for the unique sulfiiry and onionlike odor notes typical to durian fruits. The sulfiir-containing compounds not reported previously in durian included ethylene sulfide, sulfur dioxide, butanethiol, carbon disulfide, butyl ethyl disulfide and dipropyl trisulfide. The majority of the sulfur compounds have been reported in Allium species, various meats and dairy products, but rarely found in fruits and alcoholic beverages. Butyl ethyl disulfide, the newly identified sulfur component in durian, has not been reported in any food material. The identified sulfiar compound in this study account for about 8% of the total volatiles. Diethyl disulfide (5.60-6.13%) and ethyl propyl disulfide

348

Table 1 Durian volatile compounds extracted by vacuum distillation followed by dichloromethane extraction and separated on 2 different columns. % of Total^ ID^ DB-Wax DB-1 Reference'^ Compounds Hydrocarbons pentadecane methylbenzene ethylbenzene 1,2-dimethylbenzene 1,4-dimethylbenzene P-caryophyllene a-caryophyllene

MS+R MS+R MS+R MS MS MS+R MS+R

0.05 0.08 0.36 0.17 0.04 0.19 0.03

Alcohols ethanol l-propanol 2-methyl-1 -propanol 1 -methoxy-2-propanol 1-butanol 2-methyl-1 -butanol 3 -methyl-1 -butanol 2,3-butanediol 2,4-dimethyl-3-pentanol 1,2-pentanediol 3-hexanol

MS+R MS+R MS+R

0.12 2.42 0.02

MS+R MS+R MS MS+R MS MS MS+R

1.68 0.91

Aldehydes propanal butanal 2-butenal 2-methyl-2-butenal

MS+R MS MS MS

Ketones acetone 2-butanone 1 -hydroxy-2-butanone 3 -hydroxy-2-butanone 3 -methyl-2-butanone 2,3-butanedione 2-hydroxy-3 -pentanone 3 -hydroxy-2-pentanone 1 -ethoxy-4-methyl-2-pentanone 4-hydroxy-3 -hexanone 2,6-dimethyl-3-heptanone l,2-dioxolan-2-one

MS+R MS+R MS+R MS+R MS+R MS+R MS MS MS MS MS MS

0.44

0.11 0.06 0.21 0.03 0.22

0.11 0.98 0.07 1.40 0.50 0.11 0.14 0.45

1

1 1,3,5 3,5,6 5 5 1,3,5 3,5

0.01 0.04 1

0.09 0.01 0.02 0.01

5

0.13 0.06 34.09 0.19 1.75 1.69 t 0.04 0.01

0.04 0.02 37.49 0.03 0.11 1.29 0.04 0.08 0.01

3, 4, 5, 6 3,5 5,6 5 5

349 Table 1: continued. Esters actetate, methyl acetate, ethyl acetate, butyl propanoate, methyl propanoate, ethyl propanoate, propyl propanoate, butyl propanoate, pentyl 2-methylpropanoate, methyl 2-methylpropanoate, ethyl 2-methylpropanoate, propyl (iS')-2-hydroxypropanoate, ethyl 2-methyl-2-propenoate, ethyl butanoate, methyl butanoate, ethyl butanoate, propyl 2-butenoate, ethyl DL-3-hydroxybutanoate, ethyl 2-methylbutanoate, methyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl {E )-2-methyl-2-butenoate, ethyl 2-methylbutanoate, propyl 2-methylbutanoate, butyl 2-methylbutanoate, 2-methylpropyl 2-ethyl acrylate, methyl (£')-2-methyl-2-butenoate, propyl butanedioate, diethyl pentanoate, ethyl 2-methylpentanoate, ethyl 3 -methyl-2-oxo-pentanoate, methyl 4-methyl-2-oxo-pentanoate, methyl hexanoate, methyl hexanoate, ethyl hexanoate, propyl 3-hydroxyhexanoate, ethyl heptanoate, ethyl heptanoate, propyl octanoate, methyl octanoate, ethyl octanoate, propyl (Z)-4-octenoate, ethyl

MS+R MS+R MS+R MS+R MS+R MS MS MS MS MS MS MS MS MS+R MS+R MS+R MS MS+R MS+R MS+R MS MS+R MS MS MS MS MS MS MS MS MS MS+R MS+R MS MS MS+R MS MS MS+R MS MS

0.05 0.36 0.02 0.33 3.11 2.38

0.73

0.97 0.01 0.55 2.14 1.08 0.02 0.05 0.09 0.62 0.06

1 1, 2, 3, 5, 6 5 1,5,6 1,3,5, 1,3,5 5 3 1,3,5 5

t 0.10 0.44 0.12 0.04 0.37 2.25 20.73 0.07 0.20 5.58 t 0.01 0.07 t 0.03 t 0.08 0.01 0.08 1.49 0.19 0.01 0.12 t 0.50 0.02 0.02

0.02 0.04 2.25 0.23

1 5 1,3,5 3,5 5

2.01 21.87

1,5,6 1,2,3,4,5, 6 5 5 1,3,5

0.24 4.99 0.02 0.01 0.09 0.06

5

0.08

1.71

5 5,6

0.16

5

0.05 0.56

5 5,6

0.03

350 Table 1: continued. nonanoate, ethyl decanoate, ethyl dodecanoate, ethyl hexadecanoate, methyl hexadecanoate, ethyl carbonate, dimethyl carbonate, diethyl

MS+R MS+R MS+R MS+R MS+R MS MS

t 0.08 0.01 0.04 0.09 t 0.02

0.02 0.13

Sulfur compounds hydrogen sulfide ethylene sulfide sulfur dioxide methanethiol ethanethiol propanethiol butanethiol ethanethioate,«?-ethyl ethanethioate, tS'-(2-methylbutyl) carbon disulfide disulfide, diethyl disulfide, dipropyl disulfide, methyl ethyl disulfide, ethyl propyl disulfide, butyl ethyl trisulfide, dipropyl trans -3,5-dimethyl-1,2,4-trithiolane cis -3,5 -dimethyl-1,2,4-trithiolane

MS MS MS MS MS+R MS+R MS MS MS MS MS+R MS+R MS MS MS MS+R MS MS

0.01

0.01 0.01 0.02 t

Miscellaneous 5-ethyldihydro-2(3H)-fiiranone ethylene oxide trimethyloxirane 2-butyl-2-ethyl-1,3-dioxolane hydroperoxide, 1-methylbutyl hydroperoxide, 1-methylhexyl peroxide, bis(l-methylethyl)

MS MS MS MS MS MS MS

0.01 t 0.07 0.21 0.01 0.17 t 5.60 0.09 0.19 1.05 0.01 0.01 0.09 0.24 0.04 0.08 0.01 0.01 0.12 0.02

3,5 5 6 5 1,2

1 1,5 1,5

0.12 0.01

5,6

6.13 0.04 0.17 1.26

1, 2, 3, 5, 6 3 3,5,6 2, 3, 5,

t 0.06 0.14

3,5,6 3,5,6

0.01 0.06

0.04 0.19

MS = based on mass spectral data; MS+R = based on mass spectral and retention data. Percentage = 100(individual peak area/total peak area); t = trace (<0.01%). Reference in which a component was reported; compounds without a refererence number have not been previously reported.

351 (1.05-1.26%) were the major sulfur compounds in this study while the remainder of sulfurcontaining compounds were detected only in extremely low levels. Diethyl disulfide has also been found as a major sulfur compound in Malaysian durian by Baldry et al.^, Fisher et al^ and Wong and Tie^ as well as in Thai durian by Moser et al^ In another study, ^S-ethyl thioacetate was found in 3 Indonesian durian varieties as the major sulfur compound with the highest concentration among the 18 identified sulfiir compounds.^ 3.3 Carbonyl Compounds Carbonyl compounds, especially ketones, were also a prominent group of durian volatile components, with a total percentage similar to that of esters (40%). However, the total number of ketones/aldehydes identified in this study was much less than that of esters. The most abundant individual component in this study was acetoin which accounted for 34.0937.49% of the total durian volatiles in this study. Two other hydroxyketones (2-hydroxy-3pentanone and 3-hydroxy-2-pentanone) were also found in relatively high concentrations, compared to the rest carbonyl compounds. Acetoin had been previously reported as the predominant component in the 3 Malaysian durian varieties studied by Wong and Tie^ and in the 3 Indonesian varieties studied by Weenen et al^. This predominant component is most likely responsible for durian's creamy note, as indicated in another study on Malaysian durian by Fisher et al^ 3.4 Miscellaneous Compounds In addition to the three major groups of compounds, a number of alcohols, hydrocarbons and miscellaneous compounds were also identified in this study. Most of theses components were found in small or trace amounts except propanol and butanol which had relatively higher concentrations. In some previous studies, ethanol^ and propanol^ were found as the major durian volatiles. Two interesting hydrocarbons, a-caryophellene and p-caryophellene, were for the first time identified as durian volatile components in this study. These two compounds had been frequently found in many essential oils and some fruits.^ A common lactone, 4hydroxyhexanoic acid lactone or 5-ethyldihydro-2(3H)-furanone, was identified as a durian component in this study. In a previous study, another lactone, y-butyrolactone, was also detected in Malaysian durians.^ Careful examination of the results from this study and previous studies revealed some generally agreeable information on the volatile flavor composition of durian fruits. Esters, sulfur-containing compounds and carbonyls were the major groups of volatile constituents of durian fruits. Other types of compounds were also reported as the major durian volatiles, such as 1,1-diethoxyethane^''* and hexadecanol^, but these compounds were not detected by any other researchers. It was also generally agreeable that ethyl 2-methylbutanoate, diethyl disulfide and acetoin were the major individual components. Despite the similarities, significant differences were obvious among the results obtained by different researchers, especially on the qualitative and quantitative data of the major volatile flavor components. Such differences could have been due to different varieties and maturities of durian fruits and different techniques applied for the extraction and analysis of durian volatile compounds, as discussed by Wong and Tie^ The results reported by Wong and Tie^ and Weenen et al.^ revealed obvious varietal differences in the volatile composition of durian fruits from the same

352

country. Durian lovers clearly know that durian fruits from different countries show distinctive flavor characteristics. For example, Malaysian durians generally have a stronger aroma than Thai durians. What makes such differences? Unfortunately, the answer is still not known because there are no comparative data available on the qualitative and quantitative differences in the volatile composition of durian fruits from different countries. It would be very interesting and challenging to search for the answer.

4. REFERENCES 1 2 3 4 5 6 7

J. Baldry, J. Dougan, G.E. Howard. Phytochemistry, 11 (1972) 2081. R. Moser, D. Duvel, R. Greve. Phytochemistry, 19 (1980) 79. N. Fischer, F.J. Hammerschmidt, E.J. Brunke. Dragoco Rep. Flavor Inf Serv. (1994) 77. A. Apriyantono and E.E. Septiana. Proceedings of EURO FOOD CHEM VIII, Vienna, Austria September 18-20, 1995, Vol. 3, 745. K.C. Wong and D.Y. Tie. Flavor and Fragrance J., 10 (1995) 79. H. Weenen, W.E. Koolhaas and A. Apriyantono. J. Agric. Food Chem., 44 (1996) 3291. H. Maarse, L.C. Willemsens and M.H. Boelens (eds.). Volatile Compounds in Food, TNO-CIVO Food Analysis Institute, Zeist, The Netherlands, 1989.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

353

The effect of drying treatment on the flavor and quality of Longan fruit C. Y. Chang", C. H. Chang", T. H. Yu", L. Y. Lin^ and Y. H. Yen" ^Department of Food Engineering, Da-Yeh University, Chang-Hwa, Taiwan, R.O.C. *^Department of Food Nutrition, Hungkung Institute of Technology, Taichung, Taiwan, R.O.C. Abstract The volatile compounds of the Longan fruit (Euphoria longana, Lamarck) were isolated using vacuum distillation and dichloromethane extraction. Samples were analyzed by GC and GC-MS to study the changes of the flavor of the fruit during the drying process. A total of 102 different volatile compounds were identified from the fruit samples. Based on the fiinctional groups, these compounds can be grouped into 9 classes: hydrocarbons, alcohols, acids, phenols, ketones, aldehydes, furans, esters, and miscellaneous compounds. Ocimenes were found to be the predominant and characteristic volatile compounds in the raw and dried fruits. After drying, the amounts of ketones and alcohols decreased whereas the amounts of furans and esters increased. Overall, the total amount of the volatile compounds decreased slightly after drying. The Aw of the fruit decreased after 18-hrs drying. During drying, the Hunter L and b values of the flesh of the fruits decreased while the a value increased in the early stage and then decreased. The amount of total reducing sugars and total free amino acids decreased after drying. Based on the results of the volatile and quality changes of the fruit during drying process, it can be deduced that theft)rmationof the volatile compounds of the fruit was a result of reactions between reducing sugars and amino acids in the flesh of the fruits.

1. INTRODUCTION Longan (Euphoria longana, Lamarck) is a sub-tropical fruit, which grows in the southern area of Mainland China. It is widely cultivated in Kuangtung, Kuanghsi, Shichuan, and Fuchien provinces. The total area in Taiwan devoted to the growth of Longan fiuit is around 12,000 hectares. The fruit is mainly cultivated in Tainan, Nanto, Taichung, Changhwa, Kaoshiung, and Chiayi counties (Yen, 1993). Taiwan's climate permits Longan fruit to be one of the major fruits produced in summer. Longan fruit is rich in sugar and very nutritious. It has been used as a dietary supplement in China since ancient time, and is widely applied in herb medication for benefiting the mind and spleen. Longan fruit can be served fresh or processed as dried fruit, jam, and wine. The dried

354

fruit is highly desired by consumers because of its unique flavor (Hwang, 1987). There are significant differences in flavor and eating quality of fresh and dried Longan fruits. These differences begin to develop from fertilization through harvesting of Longan fruit. The growth of fruit flesh results in a reduction in acidity and an increase in sugar content. A mature Longan fruit is plump and juicy with a brownish fruit shell and a yellowish flesh (Yen and Chang, 1991). Ocimene has been identified as the predominant and characteristic volatile compound in the raw fruit (Kuo et al, 1985) whereas other volatile compounds contribute to the flowery and sweet odor of the fruit (Liu, 1993). Theflavorof dried Longan fruit might come from the products of Maillard reaction and/or caramelization during the drying process. Changes of flavor and quality of Longan fruit during the drying process have not been studied yet. In this research, a convection oven was employed to dry fresh Longan fruits to simulate the process of commercial production. The raw fruits were dried in batch and the products were sampled during the drying process. The volatile compounds and proximate composition of each sample were analyzed to understand the development of changes of quality due to changes in volatile compound profiles. The relationship between the development of the volatiles and the changes of quality of dried Longan fruit was also investigated.

2. EXPRIMENTAL PROCEDURES 2.1. Materials The raw Longan fruits used in this study (Figure 1) were cultivated and harvested from the same Longan tree in a mountainous area of Changhwa county. The Longan fruit stems were cut off and the shells were kept intact.

Figure 1. The fresh Longan fruits used in this study.

355

2.2. Sample Preparation Raw Longan fruits (6,000 g) were put into a convection oven and dried at 70 °C. During drying, the fruits were turned over and over for one min every one hour to let the temperature of the center point of each fruit attain around 55-60°C. Six batches of Longan fruit with different degrees of drying (0, 6, 12, 18, 24, and 36 hrs) were prepared. The volatile compounds, chemical compositions, and physical properties of these samples were analyzed.

2.3. Analysis of the Volatile Compounds of Longan Fruits Isolation of the Volatile Compounds The flesh (350 g, on a dry basis) of Longan fruit sample was placed into a 1000 mL flask and 500 mL of distilled water was added. The sample flask was sealed with aluminum foil and stirred for 1 min at 30-min intervals for a total of 2 hrs. The material in the flask was transferred to a blender and homogenized for 1 min. During blending, 5 mL of heptanone stock solution [0.886 mg/mL of dichloromethane (CH2CI2)] was added as the internal standard. The solution was vacuum-distilled in a water bath at 30-40 mbar and 45 °C. After distillation, 100 mL of CH2CI2 and a small amount of NaCl were added to the distillate. The mixture was stirred for 2 hrs with a magnetic stirrer. The CH2CI2 layer was collected from a separatory funnel and dried over anhydrous sodium sulfate. The CH2CI2 extraction procedure was repeated and the extractants pooled. A final concentrated volume of 0.05 mL was accomplished by purging the sample with a stream of nitrogen. Gas Chromatography Analysis and Quantification of Volatile Compounds A Hitachi G3000 gas chromatograph equipped with a fused silica capillary column (50 m x 0.32 mm id.; 1 |im, DB-Wax, J&W Inc.) and a flame ionization detector was used to analyze the volatile compounds. The operating conditions were as follows: injector temperature, 250 °C; detector temperature, 270 °C; nitrogen carrier flow rate, 1.2 mL/min; temperature program, 40 °C (5 min), 2 °C/min, 240 °C (99 min). A split ratio of 50:1 was used. The content of volatile compounds, expressed as ppb, was estimated by computing the GC area against that of the area of the internal standard. Peaks of different volatile compounds with the same area were assumed to have the same quantity. Quantitation of the volatile compounds were based on one determination. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis The concentrated sample was analyzed by GC-MS using a Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett-Packard 5971 MSD containing the same column as that for the gas chromatography. Operating conditions were as follows: injector temperature, 250 °C; detector temperature, 270 °C; helium carrier flow rate, 3 mL/min; temperature program, 50 °C (1.5 min), 2 °C/min, 200 °C (99 min); the temperature of interface, 200 °C. Mass spectra were obtained by electron ionization at 70 eV and an ion source temperature at 200 °C. Tentative Identification of the Volatile Compounds Tentative identification of the volatile compounds in the Longan fiiiit isolate was based mostly on GC-MS. The structural assignment

356 of volatile compounds was accomplished by comparing the mass spectral data with those of compounds available from the Browser-Wiley computer library, the EPA/NIH data base (Heller and Milne, 1978; 1980), the NBS computer library, TNO (1988), or previously published literature (Yu et al., 1989; 1993; 1994). The retention indices were used for the confirmation of structural assignments.

2.4. Analysis of the Physical Properties of Dried Longan Fruits Relative Weight and Moisture Content The relative weight of the dried fi\iits was expressed by assigning the weight of the fresh finit as 100. The flesh (2-3 g) of the fruit sample was dried to a constant weight in an oven at 105 °C. The moisture content of Longan finit sample was measured using the AOAC oven-heating method (1983). Water Activity (Aw) Aw of the flesh (5.0 g) of Longanfixiitsample was measured using a Rotronic-Hygroskop DT water activity measuring instrument (Rotronic Instrument Co., Switzerland) at 25 °C. The Aw measurements were triplicated for each fiuit sample. Color The Hunter color L, a, and h values of the flesh of the sample were directly measured using a Hunter colorimeter (Color Analyzer, Color Mate OEM, Milton Roy Co., USA). Three determinations were conducted randomly on the surface of each sample. Measurements for each sample were triplicated.

2.5. Determination of the Free Sugars and the Free Amino Acids of Longan Fruits Free Sugars Ten grams of Longan finit flesh was added with 200 mL of 80 % ethyl alcohol. The material was then refluxed in a water bath at 95 °C for 1 hr. At which time, the sample was filtered. Thefi-eesugar content of thefiltratewas analyzed using HPLC (Jasco PU980, Japan). The operating conditions for HPLC were as follows: mobile phase, acetonitrile/H20=75/25 (VA^); column, Sphereclone 5|i NH2 250 X 4.6 mm; flow rate, 0.5 mL/min; detector, RI (Jasco 830, Japan) (Lo, 1987). Free Amino Acids Twenty-mg of the Longanfiaiitfleshwas added with 500 \xL of citrate buffer. The mixture was centrifiiged and the clear supernatant solution was collected. The residue was extracted five times with citrate buffer to recover its free amino acids. The clear supernatant solution were combined and filtered afl;er centrifiigation. Free amino acid content of the filtrate was determined using an amino acid analyzer (Beckman 6300, USA).

3. RESULTS AND DISCUSSION 3.1. Changes in the physical properties of Longan fruits during the drying process The change in the relative weight of Longan finits during drying process is shown in Figure 2. The relative weight of the finits dropped rapidly during drying. The relative weight of

357 110 100 H b

90

I 80 « 70

>

5 60 H Pi

50 -I

40 -I 30

_l 6

^ ^ ^ 12 18 24 Drying Time (hr)

Figure 2. Change in the relative weight of Longan fruit dried at 70 °C. The rapid reduction in fruit weight during the drying process was caused by the evaporation of water from the fruit shell, flesh, and seed. The change in the moisture content of the fruit flesh during drying process is shown in Figure 3 which reveals that the weight reduction of the fruit during drying was due mainly to the loss of water from the fruit flesh.

1 6

1 1 1 12 18 24 Drying Time (hr)

1 30

1 36

Figure 3. Change in the moisture content of Longan fruit flesh dried at 70 °C.

358 Water activity (Aw) is recognized as a reliable indicator of food perishability. A high Aw value in fresh Longan fruits (0.99, Figure 4) suggests that they would easily deteriorate during storage. Figure 4 depicts the effect of drying time on the Aw of the fruits. It shows that the value of the Aw dropped dramatically after drying for 24 hrs and remained 0.61 after the process. At the Aw of 0.61, most microorganisms cannot grow, even the osmophilic yeasts (Beauchat, 1981). Because of its low Aw, dried Longan fiaiits can be stored at room temperature for a long period of time.

1.0 H

0.9 H ^

0.8

< ^

0.7 A 0.6 H 0.5

I 6

I I 12 18 24 Drying Time (hr)

30

36

Figure 4. Change in the water activity of Longan fiiiit flesh dried at 70 °C.

The Hunter L, a, and b values of Longan fiiiit flesh after drying for different period of time are shown in Figure 5. The onset of the decrease of Hunter L value of the fiaiit flesh apparently was about at the drying time of 6 hrs. The Hunter h value also had a decreasing trend during drying process; the Hunter a value increased only slightly from the beginning period of drying and then decreased after 30 hours. The visual browning of the fruit flesh suggested that there were Maillard reactions that may have occured between the sugars and amino acids in the fiuit flesh during the drying process.

359

18

24

36

Drying Time (hr)

Figure 5. Change in the Hunter L, a, and b values of Longan fruit flesh dried at 70 °C.

3.2. Changes of the content of the free sugars and the free amino acids in Longan fruits during drying process Changes in free sugar content of Longan fruits during drying are shown in Table 1. The amount of free sugars in the flesh of raw fruit is presented in the order of glucose, maltose, sucrose, xylose, and fructose. After drying for 36 hrs, sucrose became the major sugar in the flesh of diied fruit, and maltose and xylose were not detectable. During di-ying process, the amount of glucose decreased throughout the dr5dng process, especially in the first 6 hrs. The amount of maltose also decreased rapidly and was not detectable after drying for 18 hrs. The amount of fructose increased and then decreased since drying for 18 hrs whereas the amount of sucrose had an opposite trend as that of fructose. The change in free sugar content during drying process reveals that the decrease in glucose may have been caused by its isomerization to fructose as seen by the increase in fructose in the early stage of drying. The decrease in glucose might also be due to the Maillard reaction and/or carameUzation, which would result in the formation on new volatile compounds and imparted a brownish color to the flesh of Longan fruits.

360 Table 1. The free sugar content of Longan fruit dried at 70 °C. Content ( g / lOOg Longan fruit flesh, based on dry weight)

0* 6 12 18 24 30 36

xylose

fructose

glucose

maltose

sucrose

total sugar

2.94 N.D.** N.D. N.D. N.D. N.D. N.D.

2.30 7.28 11.90 21.76 18.46 11.79 12.62

44.69 22.17 15.84 12.54 9.77 13.75 11.79

15.00 5.41 4.09 N.D. N.D. N.D. N.D.

5.37 3.37 4.23 13.14 14.73 14.02 24.65

67.35 38.24 35.26 47.44 42.96 39.56 49.06

* Drying time (h.3urs). ** N . D . : Not Detectable.

Table 2 shows the change in free amino acid content in Longan fruits during drying. It is observed that the predominant free amino acids in the flesh of raw fruits is glutamine and then follows by proUne, alanine, aspartic acid, tyrosine, serine, leucine, isoleucine, valine, and glycine. Some of these amino aicds, such as proline and leucine, significantly decreased in the amount after drying. The total amount of free amino acids decreased throughout the process. This result of decrease in the amount of free amino acids after drying is similar to that of free sugars. It suggests that the decrease in the amount of free amino acids might be due to Maillard reaction between the amino acids and reducing sugars. This might also explain the generation of the volatile compounds and the formation of brownish colors (see below).

3.3. C h a n g e s in volatile c o m p o n e n t s of L o n g a n fruits d u r i n g drying process The gas chromatogi-ams of the flavor isolates from the flesh of raw and 36hr-dried Longan fruits are presented in Figure 6. It shows clearly visible differences between the gas chromatograms of the two fruit isolates. A total of 102 compounds (Table 3) were identified from the Longan fruit flesh isolates. Based on the functional groups, these flavor components are grouped into 9 classes: hydrocarbons, alcohols, acids, phenols, ketones, aldehydes, furans, esters, and miscellaneous compounds.

361

Table 2, The contents of free amino acids in the flesh of Longan fruits dried at 70 ^C. Content ( 0* 10.06 5.06 31.25 25.91 1.42 20.14 1.54 1.79 2.39 8.42 N.D. 0.55 108.53

Asp Ser Glu Pro Gly Ala Val

ne Leu Tyr Lys NH3 Total

mg / lOOg Longan fruit flesh, based on dry weight) 6 12 24 18 30 36 8.74 11.08 6.59 4.19 2.04 3.38 4.64 2.57 3.28 1.87 1.16 0.38 18.97 18.73 20.18 11.56 7.79 2.55 20.73 17.42 16.97 11.15 7.09 6.85 1.16 1.55 0.96 0.54 N.D.** 0.38 12.85 15.54 13.05 8.69 4.90 3.31 0.67 1.26 1.00 0.54 0.36 N.D. 1.07 1.79 1.07 0.64 0.64 0.47 2.04 1.71 1.57 0.64 0.86 0.32 9.42 5.16 5.53 3.16 2.31 2.74 N.D. N.D. 0.05 0.05 N.D. 0.16 0.46 0.35 0.51 0.18 0.09 0.16 79.07 78.87 70.71 43.42 28.75 18.98

* Drying time (hours). ** N.D. : Not Detectable. CO CO

'^

« 1 un

Ico 1

1 ooco

CZ3 CD

o

L-j

Raw

oo

^ ^

t—

1

Lju-Jlj.JlLJuuJUiJuuJiJLL UiX....j..-jL lLjLJLIL.>-^----L c/a

cj» '«S«.-.CM

^ "^

r— C^3

~


—<'«r

"

Q Q

" 1

1 , 1

1.

jJJ

"

D

r

i

d

1

.UlllJj ..4,11'^wiUL/^t-

50

e

o>

1 1

100

Figure 6. The gas chromatograms of the flavor isolates from the flesh of raw and dried Longan fruits.

362

Table 3 Volatile compounds identified fi'om the flesh of Longan samples. Peak No.

CAS No.

Formula

M.W.

2-methyl-2-butene a-phellandrene a-terpinene P-phellandrene a-ocimene trans-P-ocimene p-mentha-l,5,8-triene (isomer I) cis-l,3-pentadiene trans-sabinene hydrate 2,4,6-trimethyl-1,3,5-trioxane 5-3-carene p-mentha-l,5,8-triene (isomer 2) l,2-dimethoxy-4-[2-propenyl]-benzene a-cedrene 1,2-dimethoxy-4-[ 1 -propenyl]-benzene

513-35-9 99-83-2 99-86-5 555-10-2 502-99-8 3779-61-1 21195-59-5 504-60-9 546-79-2 123-63-7 13466-78-9 21195-59-5 93-15-2 469-61-4 93-16-3

C5H10 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14 C5H8 C10H18O C6H1203 C10H16 C10H14 C11H1402 C15H24 C11H1402

70 136 136 136 136 136 134 68 154 132 136 134 178 204 178

2-methyl propanal trans-2-hexenal furfural benzaldehyde 5-methyl furfural benzeneacetaldehyde

78-84-2 6728-26-3 98-01-1 100-52-7 620-02-0 122-78-1

C4H80 C6H10O C5H402 C7H60 C6H602 C8H80

72 98 96 106 110 120

2-methyl-3-buten-2-ol isobutyl alcohol 2-pentanol 1-butanol isoamyl alcohol 3 -methyl-3 -buten-1 -ol 4-heptanol 4-methyl-1 -pentanol 2-heptanol 3 -methyl-2-buten-1 -ol 3 -methyl-1 -pentanol 1-hexanol 2-hexanol 3 -ethoxy-1 -propanol cis-3-hexen-l-ol l-octen-3-ol linalool oxide (isomer 1)

115-18-4 78-83-1 6032-29-7 76-36-3 123-51-3 763-32-6 589-55-9 626-89-1 543-49-7 556-82-1 589-35-5 111-27-3 626-93-7 111-35-3 928-96-1 3391-86-4 5989-33-3

C5H10O C4H10O C5H120 C4H10O C5H10 C5H10O C7H160 C6H140 C7H160 C5H10O C6H140 C6H140 C6H140 C5H1202 C6H120 C8H160 C10H18O2

86 74 88 74 70 86 116 102 116 86 102 102 102 104 100 128 170

Compounds

Hydrocarbon 1 11 13 15 18 19 39 40 52 53 69 83 84 92 93

Aldehyde 2 16 42 50 54 63

Alcohol 5 6 7 9 14 17 21 23 24 25 26 28 29 31 32 37 38

363 (Table 3 continued) Peak No. 41 43 51 56 58 60 61 61 64 66 71 72 76 78 79

CAS No.

Formula

M.W.

562-74-3

C10H18O

154

5989-33-3 22564-99-4 562-74-3 35376-39-7 88125-84-2 ~ 98-00-0 16721-38-3 14049-11-7 142-50-7 106-24-1 100-51-6 60-12-8

C10H18O2 C10H18O C10H18O C10H18O C7H120 C10H16O C10H16O C5H602 C10H18O C10H18O2 C15H260 C10H18O C7H80 C8H10O

170 154 154 154 112 152 152 98 154 170 222 154 108 122

2-propanone 2,3-butanedione 2-heptanone 3 -hydroxy-2-butanone 4-hydroxy-4-methyl-2-pentanone E-6-methyl-3,5 -heptadien-2-one 5-methyl-3-hexen-2-one isopulegone pulegone 3,4-dihydro-8-hydroxy-3 -methyl-1Hbenzopyran-1-one

67-64-1 431-03-8 110-43-0 513-86-0 123-42-2 16647-04-4 5166-53-0 29606-79-9 89-82-7 17397-85-2

C3H60 C4H602 C7H140 C4H802 C6H1202 C8H120 C7H120 C10H16O C10H16O C10H10O3

58 86 114 88 116 124 112 152 152 178

methyl cis-2-butenoate methyl 2-hydroxy-3-methyl butyrate ethyl 3-hydroxy-3-methyl butyrate ethyl 2-hydroxy-3-methyl butyrate methyl 3-hydroxy butyrate methyl 2-hydroxy-3-methyl pentanoate methyl 2-hydroxy-5-methyl benzoate methyl 4-methyl benzoate methyl 2-methoxy benzoate ethyl 2-methoxy benzoate methyl hexadecanoate methyl 2-amino benzoate

4358-59-2 17417-00-4 18267-36-2 2441-06-7 1487-49-6 41654-19-7 22717-57-3 99-75-2 606-45-1 7335-26-4 112-39-0 134-20-3

C5H802 C6H1203 C7H1403 C7H1403 C5H10O3 C7H1403 C9H10O3 C9H10O2 C9H10O3 C10H12O3 C17H3402 C8H9N02

100 132 146 146 118 146 166 150 166 180 270 151

Compounds 4-methyl-l-[l-methyl ethyl], 3cyclohexen-1-ol linalool oxide (isomer 2) 3,7-dimethyl-1,6-octadien-3 -ol 4-terpineol cis-p-2-menthen-1 -ol [5-methylcyclopent-1 -enyl] methanol p-mentha-trans-2,8-dien-1 -ol p-mentha-trans-2,8-dien-1 -ol 2-furan methanol cis-piperitol epoxylinalool d-nerolidol trans-geraniol benzenemethanol benzeneethanol

Ketone 3 4 10 22 30 55 59 75 91 100

Ester 33 34 35 36 44 46 81 85 88 90 94 95

364 (Table 3 (continued) CAS No.

Formula

M.W.

ethyl 3-hydroxy butyrate methyl 3,7-dimethyl-2,6-octadienoate cis-3-hexenyl butyrate phenylethyl acetate ethyl hexadecanoate methyl 9,12-octadecadinoate

5405-41-4 2349-14-6 16491-36-4 103-45-7 628-97-7 2566-97-4

C6H1203 C11H1802 C10H18O2 C10H12O2 C18H3602 C19H3402

132 182 170 164 284 294

dihydro-2-methyl-3 [2H]-furanone 2,5-dihydrofuran 2-acetylfuran 3,6-dimethyl-2,3,3a,4,5,7ahexahydrobenzofliran dihydro-2[3H]-furanone 5 -ethyldihy dro-2 [3 H] -fliranone 2-furancarboxylic acid 2,3-dihydrobenzofuran

3188-00-9 1708-29-8 1192-62-7 70786-44-6

C5H802 C4H60 C6H602 C10H16O

100 70 110 152

96-48-0 695-06-7 88-14-2 496-16-2

C4H602 C6H1202 C5H403 C8H80

86 114 112 120

116-53-0 124-07-2 65-85-0 143-07-7 57-10-3

C5H10O2 C8H1602 C7H602 C12H2402 C16H3202

102 144 122 200 256

108-95-2 90-05-1 2785-89-9 106-44-5

C6H60 C7H802 C9H1202 C7H80

94 124 152 108

108-50-9 1122-62-9 95-16-9 1072-83-9

C6H8N2 C7H7NO C7H5NS C6H7NO

108 121 135 109

Compounds

Peak No. 48 67 73 74 96 101

Furan 20 45 47 49 62 68 70 97

Acid 65 87 98 99 102

isovaleric acid octanoic acid benzoic acid dodecanoic acid hexadecanoic acid

Phenol 12 77 86 89

phenol 2-methoxyphenol 4-ethyl-2-methoxyphenol 4-methyl phenol

Miscellaneous 27 57 80 82

2,6-dimethylpyrazine 2-acetylpyridine benzenethiazole 2-acetylpyrrole

365 The major volatile compounds found in the hydrocarbon grouping are the ocimenes. Ocimenes have been identified as the predominant volatile compounds in the raw fruit (Kuo et al., 1985). The major volatile compounds in the aldehyde grouping are furfural and 5-methyl furfural. These two compounds were beUeved to be generated fi'om sugars through carameUzation or thermal degradation. Most of the compounds in the alcohol grouping are short-chain alcohols. These alcohols, including isoamyl alcohol, linalool oxide, trans geraniol, and benzenemethanol, contribute to the flower smell and to the wine taste of the raw and dried fruits. Pulegone, the major volatile compound of the ketone grouping, was beheved to contribute a mint flavor. In the ester group, ethyl hexadecanoate is the representative compound; it contributes a mild, sweet taste. The compounds of the furan group, including 2-acetylfuran, 2-furancarboxyHc acid, and 2,5dihydrofuran, were beUeved to be generated from the sugars through thermal degradation. In the miscellaneous group of compounds, 2-acetylpyrrol, represents a compound with baked or roasted flavor and was beheved to be generated fi'om Maillard reaction. Table 4 shows the temporal change in the content of volatile components in Longan fruit during drying process. The alcohol grouping is the major class of volatile compounds in raw fruit flesh, with a value of 32,177.5 ppb. The other grouping, including esters, hydrocarbons, and ketones, have the values of 31,177.7, 30,261.1, and 26,842.4 ppb, respectively. After 36-hr drying, the major group of the volatile compounds of the dried fruit flesh shifted from the alcohols to the esters group, with a 36 hour value of 54,210.4 ppb. The other groups, including hydrocarbons, alcohols, andfurans, had the values of 28,588.6, 13,885.4, and 13,838.3 ppb, respectively after 36 hours of drjang. Table 4. The contents of volatile compounds, classified by the functional groups, in the flesh of Longan fruit dried at 70 °C. Concentration (ppb, based on dry weight) 0*

6

12

24

Compound Type Phenol Furan Acid Aldehyde Ketone Alcohol Hydrocarbon Ester Miscellaneous

956.9 551.6 440.7 399.2 961.7 629.7 901.9 1,512.5 393.2 2,468.4 959.8 1,718.7 4,320.1 13,838.3 3,938.8 2,287.3 996.6 1,559.8 1,173.6 990.0 3,409.5 1,870.8 312.7 950.0 1,893.8 1,749.6 2,780.1 3,438.5 26,842.4 1,848.7 420.0 3,157.0 1,154.1 1,785.8 4,741.9 32,177.5 14,439.8 9,200.2 6,303.4 3,216.1 6,001.4 13,885.4 30,261.1 18,005.9 14,080.2 10,589.5 45,805.0 45,288.0 28,588.6 31,177.7 33,263.8 21,403.3 24,143.8 17,969.6 44,363.5 54,210.4 1,943.9 861.5 416.5 281.5 625.9 248.6 374.6

Total

130,681.6

Drying time (hours)

71,964.5

50,234.3

18

51,984.5

30

36

73,425.5 106,977.0 121,212.8

366 The data in Table 4 show that during drying process, the total amount of the volatile compounds decreased sharply in the first 6-hr drying. This reduction in the total amount might be caused by the evaporation of the volatile compounds in the raw fruits. During the 6-18 hour drying period, the total amount of the volatile compounds varied a small range, this may be due to the balance between the evaporation and the generation of volatile compounds. In later stages of the drying period (24-36 hrs), the total amount of the volatile compounds increased to a value of 121,212.8 ppb. This might be a result of the prosperous generation of many volatile compounds in the groups, including phenols, furans, aldehydes, ketones, alcohols, and esters.

3.4. C o m p a r i s o n of t h e C o n t e n t s of t h e Volatile C o m p o u n d s in t h e F l e s h of Raw and Dried L o n g a n Fruits Figure 7 depicts the volatile compound content placed in functional groups in the flesh of raw and 36-hr-diied fruits. It is found that the furans grouping increased to a great extent through drying. The esters grouping had the same trend as the furans grouping. Inversely, the concentration of ketones and alcohols groupings decreased after di*ying process. The volatile compounds of other groupings didn't changed significantly in the quantity after di*ying process. The total amount of the volatile compounds in the 36-hr-dried fruit flesh was smaller than that of the raw fruit flesh.

1.4e+5 ^

1.2e+5

t, •o

l.Oe+5

Raw 36 hrs-Dried

G

° 8.0e+4 -\ o. a. ^ 6.0e+4 e "c

O

4.0e+4 2.0e+4 O.Oe+0

T—^

n- -T

1

Figure 7. Contents of volatile compounds in the flesh of raw and 36 hrs-dried Longan fruits

367 By comparing the changes of the free sugars, free amino acids and volatile compounds of Longan fruits before and after drying process, it was found that the difference between the fresh and dried Longan fruits in flavor was due to the higher contents in furans and esters in dried Longan fruits. The volatile compounds of furans and esters groupings were most likely generated from the sugars through many reactions, especially Maillard reaction and thermal degradation and complexation.

4. ACKNOWLEDGEMENTS This work was supported by a grant (NSC85-2321-B-212-002) from the National Science Council, Executive Yuan, Repubhc of China.

5. REFERENCES 1. L. R. Beauchat, Cereal Food World. 26 (1981) 345. 2. S. R. Heller and G. W. Milne, A. EPA/NIH Mass Spectral Data Base (Vol. 1), U.S. Government Printing Office: Washington, DC, 1978. 3. S. R. HeUer and G. W. Milne, A. EPA/NIH Mass Spectral Data Base (Suppl. 1), U.S. Government Printing Office: Washington, DC, 1980. 4. B. C. Hwang, Longan. In Farmers Guide, Harvest Farm Magazine issued. Taipei, ROC. 1987, 701 . 5. M. C. Kuo, C. C. Chen and M. C. Wu, Research Report of Food Industry Research and Development Institute, No. 380, 1985. 6. S. E. Liu, Flavor Chemistry of Taiwanese Food, Part IV: The Volatile Components of Longan. Food Industry Research and Development Institute, Hsinchu, Taiwan, 1993. 7. S. H. Lo, Food Industry (Taiwan), 19, 1987, 35. 8. TNO. Compilation of mass spectra of volatile compounds in food; Central Institute of Nutrition and Food Research-TNO: Zeist, The Netherlands, 1988. 9. C. R. Yen and J. W. Chang, J. Chinese Soc. Hort. Sci. 37, 1991, 21. 10. T. H. Yu, C. M. Wu and Y. C. Liu, J. Agric. Food Chem. 37, 1989, 725. 11. T. H. Yu, C. M. Wu and C.-T. Ho, J. Agric. Food Chem. 41, 1993, 800. 12. T. H. Yu, C. M. Wu, R. T. Rosen, T. G. Hartman, and C.-T. Ho J. Agric. Food Chem. 42, 1994, 146.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

369

Effect of processing conditions on volatile composition of apple jellies and jams M. Moldao-Martins, N. Moreira, I. Sousa, and M.L. Beirao da Costa, Instuto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa Codex PORTUGAL

Abstract When producing fruit jellies and jams it is intended to preserve as far as possible the presence of the aromatic characteristics of the fresh fruit. In the present work, the influences of sugar content - 6 to 55°Brix - and type of pectin - low methoxyl and medium methoxyl pectin on the volatile composition of apple jellies and jams is studied. Volatile compounds were extracted by Clevenger distillation for 180 min and collected in w-hexane. All extracts were analysed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GCMS). The influence of jelly structure on flavor release was evaluated by measurement of objective texture. It was observed that the studied parameters quantitatively affected the chromatographic profile, keeping the composition similar. Main compounds identified were 3methylbutanol, 2-methylbutanol, 1-hexanal, 2-hexanal, hexyl propionate and estragole. The decrease observed in volatile release from jellies and jams seemed to be related to the retention of volatiles in the gel matrix and to sugar bonding.

1. INTRODUCTION The quality of apple fruit and related products depends, among other things, on the flavor characteristics of the raw material, which is linked to the cultivar and affected by cUmate, harvesting time, and storage conditions [1]. With regard to the harvesting date, the lack of volatiles in the immature apple fruit is probably due to the low level of volatile precursors and enzyme forming systems [2]. This is very important for apples intended to be stored in controlled atmosphere (CA), since the fruits in this case are usually picked earlier than those for immediate consumption or refrigerated storage. Studies of volatiles in apples have shown that between 20 to 40 volatiles are responsible for apple aroma. These compounds fall into three classes: the "apple-peel smelling" esters like ethyl 2-methylbutanoate, the lipid oxidation products like (E)-2-hexenal and the terpenoids like P- damascenone [3]. In the processed fruit products changes in flavor may also occur. Changes in the contents of the volatile compounds of fresh apple juice afl;er heat pasteurisation have been reported [4]. The perception of flavor is related not only to the chemical interaction between the flavors and the matrix, but also to the physical properties of each kind of food. For instance, flavor perception is lower in gels than in viscous solutions [5]. Many studies have reported that hydrocolloids not only modify viscosity, but also reduce intensities of odor, taste and flavor [5].

370

Pectin is the gelling agent generally used in the fmit preserves industry to produce jellies and jams. Traditionally, high methoxyl (HM) pectin are used. This type of pectin needs boiling temperatures and high contents of sugar to produce acceptable products. Low methoxyl pectins (LM) are used in products of lower sugar content; in this case the strength of gels varies essentially with concentration of calcium ions. The present work is a study of the influence of processing conditions, namely the type of pectin and the sugar content (which also influences the texture) on the volatile composition of apple jellies and jams produced from Golden Delicious apples stored at controlled atmosphere.

2. MATERIAL AND METHODS 2.1. Materials Golden Delicious apples stored for six months at controlled atmosphere (% CO2 - 1,5 %; O2 - 2 %; Temperature - 0.5°C) were used. Low methoxyl pectin, SBI Unipectine LM325 - LM Medium methoxyl pectin, LM 325 and H&F AF 602 - MM Sugar 01 commercial grade. All other reagents are analytical grade and standards are GC grade. 2.2. Jellies and jams preparation Jellies were produced from apple peels and seeds and jams prepared with apple pulp, based on the recipe Diese (Portuguese Company) NS/047 with different additions of sugar and type of pectin (Table 1). In the case of LM pectins an extra supply of Ca++ ions of 0.5 g / g pectin was added and for the MM pectins the pH was adjusted to 3.0. The sugar content quoted in this work is the total sugar (apple natural sugar + added sugar) as determined by refractometry (°Brix). Table 1. Jellies and jams main differences in compositic n 3?
J2 J3

1

1

2.3. Aroma extraction methods Distillation on a modified Clevenger apparatus (CLEV) was conducted for 180 min at atmospheric pressure. Aroma compounds were collected in 2 mL of w-hexane. w-Hexane solutions were concentrated to 50 |aL by gently blowing nitrogen gas over the «-hexane surface. 2.4. Analytical methods Analyses were conducted both on raw material (apple pulp and peel) and prepared jellies and jams.

371 The essential oil was analysed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS). GC analysis was performed on a Hewlett-Packard 5890 gas chromatograph equipped with an FID and an HP-5 column (cross-linked 5% biphenyl, 95% dimethylsiloxane) 50 m x 0.32 mm i.d., film thickness 0.17 |im. GC conditions. Oven temperature: programmed 60°C for 10 min, followed by a slope of 2°C/min to 180°C; 10°C/min to 200°C and a plateau at 200°C/30 min. Injector and detector temperatures were 200 and 250°C, respectively. Carrier gas, N2 was adjusted to a linear velocity of 1 mL/min. The samples were injected using the split mode (split ratio 1:8) using a 0.2 |iL injection volume. The quantification of the components was made by internal standard methodology. The GC-MS unit consisted of a Hewlett-Packard 5970 mass selective detector operating in the electron impact mode (70 eV) coupled to a Hewlett-Packard 5890 gas chromatograph. A capillary column Supelco Wax 10; 30 m x 0.25 mm i.d., film thickness 0.25 |j,m was used. Analytical conditions. Oven temperature: programmed at 80°C for 10 min, followed by a slope of 2°C/min to 180°C ; lOX/min to 200 °C and a plateau at 200°C/30 min. Injector temperature was 200°C. Samples were injected using the split mode (split ratio 1:19) using a 0.2 |LIL injection volume. Carrier gas, He, was adjusted to a linear velocity of 0.89 mL/min. 2.5. Texture analysis Textural parameters were calculated from a penetration test [6] using a TA-TX2 (Stable Microsystems) texturometer with a 38 mm diameter cylinder probe. The probe was set to penetrate the gel 10 mm deep, at a speed of 2 mm s"V The jellies and jams were contained in 100 mL cylindrical glass flasks with 60 mm diameter at 35 mm height.

3. RESULTS AND DISCUSSION Table 2 shows the main volatile compounds identified in fresh apple peel and pulp. The compounds of each class included 7 esters, 3 aldehydes, 3 alcohols and 2 terpenoids. The solvent stripping procedure outlined removed some volatile compounds such as acetaldehyde. It is interesting to note that the same compounds were identified in the peel and in the pulp although at different amounts. The largest volatile constituents of fresh fruit are aldehydes and esters. The most abundant volatile compounds in the peel are C13 aldehyde (20.196 ppm) and hexyl hexanoate (3.963 ppm). The latter compound is absent in the pulp. Estragole, an important terpenoid in apple flavor, was found to be present both in pulp (1.423 ppm) and in peel (1.137 ppm). The volatile compounds of fresh peel and jellies are shown in Figure 1. It can be seen that most of the flavor changes in jellies reflect a substantial decrease in the content of aldehydes and esters. The most dramatic change is the decrease in aldehydes content (from around 20 ppm to 0.3-0.4 ppm). On the other hand, alcohols show a much smaller decrease (from 0.7 ppm to 0.3 ppm).

372

Table 2. Volatile compounds of fresh Golden Delicious fractions (ppm).

ESTERS

Batyliasrelate J-lli9Eeft?l*eef««cC^^V i Eesi^l fe»(js|rte„, , , , ^ Metier! d«ci^»Mi^e

ALDEHYDES

i Z^ Wi^^ h^t^H^fv^^ i i Iie9i^lp^l0ot»te i Mk^i^m^ \2-Bs3mas&tia:am&^

B[e3^|iHES»B€»^

Cj^Aid^^e ALCOHOLS

i^Besiai^ i 2<Mi!^i^iiil3insd i 3rS»i^l^1tabm(]i

[ISstri^e M^m^^hflk0t^w^

[ TERPENOroS \

PEElc 1

FIIIJ^ 0.597 0.606

0.252 0.850 1.285 1.113 3.963 0.747 0.565 0.368 0.248 20.196 1

0.155

0.202 0.432 0.376 0.775 0.357 0.277 0.323 1.423

0.278 0.194 0.210 1.137 0.333

-

1

The volatile compounds of fresh pulp and jams are shown in Figure 2. Most of the flavor changes in jams is due to a decrease in the aldehyde content. Table 3 Texture parameters Brittleness (mm)

Gel Strength (g)

Average value

S.E.

2.5

9.811

0.001

-

nm

-

9.976

0.024

nm

0.5 -

nm

-

Jl

88.1

3.7

9.811

0.001

J2

109.8

11.4

10.000

0.000

J3

79.5

3.7

9.975

0.025

J4

73.4

3.3

9.976

0.024

Average value

S.E.

Gl

81.1

G2

nm

G3

42.9

G4

nm - not measurable For jams the gel strength results can be grouped into two classes: Jl, J2 and J3, J4 as a function of the pectin degree of esterification [9]. Brittleness was considered to be related with the flavor release [7] of the gel materials. From Table 3 the results allow for two classes of brittle values Jl and J2, J3, J4. These two classes can also be clearly identified on figure 2 for the content of volatile compounds with the exception of the esters class of compounds, where this tendency is not so clear. This is in agreement with the reported [7] direct relation of brittleness with flavor release.

373

1,8

20.812

1,6

2.0200

8.77

V a,2 0,6826

o

H,4 0,2

0

I Figure 1. Volatile compounds of fresh peel and jellies (ppm).

Figure 2 - Contents of volatile compounds of fresh pulp and jams (ppm). The values for gel strength (force at rupture) and brittleness (deformation at rupture) for jellies and jams can be seen in Table 3. The jellies produced with no extra addition of sugar did not reached measurable gelification and these jellies showed a higher release of flavor as it

374

is shown in Figure 1. It is known L^^^J that the building up of the gel matrix will reduce the release of flavor. As expected, L^J the gel strength is determined by the nature of the gelling agent. The jellies produced with LM pectin showed values of gel strength of the order of 80g and with MM pectin the value was about 40g. There was no significant difference (P<0.05) in the brittleness values for jellies. ACKNOWLEDGMENTS We acknowledge the kind supply of apples by FRUTUS, S.A..

REFERENCES 1. N. Paillard, In: P. Schreier, (ed.), Flavor'8J, Berlin, Walter de Gruyter & Co(1981). 2. E.M. Yahia, T.E. Acree and , F.W Liu, Lebensm.-Wiss. U. Technol, 23, (1990), 488-493. 3. D.G. Cunningham, T.E. Acree, J. Barnard, R. Butts, and P. Braell, Food Chemistry, 19 (2), (1986), 137-147. 4. N. Kakiuchi, S. Moriguchi, N. Ichimura, Y. Kato and Y. Banba, {Nippon Shokuhin Kogyo Gakkaishi, 34, (2), (1987), 115-122. 5. R.M. Pangborn, I.M. Trabue, and A.S.Szczesniak, J. Texture Studies, 4, (1973), 224. 6. I.M.N. Sousa, E.C. Matias, and O. Laureano, The texture of low calorie grape juice jelly, Z Lebensm. Unters. Forsch. A, 205, (1997), 140-142. 7. E.R. Morris, In: K., Nishinari and E. Doi, (ed.). FoodHydrocolloids, Structures, Properties and Functions, Plenum Press, New York (1993). 8. A. Nussinovitch, G. Kaletunc, D. Normand, and M. Peleg, Journal of Texture Studies, 21, (4) (1990), 427-438. 9. E. Guichard, S. Issanchou, A. Descourvieres, and P. Etievant, J. Food Sci., 56 (6), (1991), 1621-1627.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

375

The relationship between ethylene and aroma volatiles production in ripening climacteric fruit S. Grant Wylliei, J.B. Golding^ W.B. McGlasson2 and M. Williams^.

1 Centre for Biostructural and Biomolecular Research, ^ School of Horticulture, University of Western Sydney, Hawkesbury, Richmond, NSW, Australia.

3 Department of Chemistry, University of Western Sydney, Nepean, Kingswood, NSW, Australia.

Abstract This work discusses the influence of the ethylene antagonist 1-methylcydopropene (1-MCP) and the timing of its application on the ripening related parameters in bananas such as ethylene, CO2 and volatiles production, volatiles composition and skin color. The preclimacteric application of 1-MCP to banana fruit delayed the onset of the common parameters associated with ripening, namely ethylene and aroma volatiles production, respiration and skin color changes. The extent of these delays depended on the developmental stage of the fruit at application and on whether the fruit was subsequently challenged with the ethylene analogue, propylene. The application of 1-MCP to bananas continuously challenged with propylene delayed the onset of ripening and volatiles production by two days. When preclimacteric mature green bananas were allowed to naturally ripen in the absence of propylene, 1-MCP delayed the onset of ripening by nine days. Hence 1-MCP application offers a convenient means of examining the role of ethylene in ripening and offers an excellent opportunity to differentiate and clearly study the biochemical and physiological interactions occurring in ripening fruit.

376

1. INTRODUCTION Although the role of ethylene in initiating the ripening process in many fruit is well established, the details of the mechanism of its action particularly in relation to volatiles production are not well understood. A better comprehension of the role of ethylene in fruit ripening is crucial to our understanding of the ripening process and to provide better selection of pre and / or postharvest fruit treatments to maximize fruit quality. Numerous changes are associated with fruit ripening. These characteristic ripening changes are the result of a cascade of genetically controlled biochemical changes. Some of these changes do not begin immediately after the onset of ripening. The continued production of ethylene is believed to be required for the integration of these many biochemical events. It is not dear whether some of the biochemical changes associated with ripening are directly dependent on the presence of ethylene or whether they are mediated by some other event once ripening is initiated. Bananas (Musa sp. [AAA group, Cavendish subgroup] cv. WiUiams) were chosen for these series of experiments as they are a 'classical' climacteric fruit which behave in a highly predictable manner during ripening. Bananas are one of the most studied fruits (1), and good supplies of mature green bananas are readily available from the markets throughout the year. Bananas and other climacteric fruit produce a characteristic climacteric rise in respiration after harvest. Unripe bananas show a constant but very low level of ethylene production until the onset of ripening. Ethylene production then increases and this is followed by a rise in the respiration rate as indicated by the increasing evolution of CO2. The most obvious feature of banana ripening is the change in skin color from green to yellow. This color change is largely due to a destruction of chlorophyll which unmasks the carotenoids present in the unripe banana (2). A wide range of volatile compounds are produced by ripening bananas and these have been extensively studied (3,4,5). Over two hundred and twenty volatiles have been isolated and identified in bananas (6). Esters account for about 70% of the total volatile compounds and acetates and butyrates predominate within this fraction (4). It is generally considered that volatile production is coincident with ethylene during the climacteric (4,7), however the precise biochemical relationship in the production of these compounds with the ethylene dimacteric remains undear. Propylene is an active analogue of ethylene which enables the measurement of endogenous ethylene production during the ensuing increase in respiration. McMurchie et al (8) first used propylene to study the role of ethylene in ripening bananas. 1-Methylcydopropene (1-MCP) is a volatile irreversible inhibitor of ethylene action (9,10). 1-MCP inhibits ethylene action when plants are treated at concentrations as low as 0.5 nL/L(9) and has been successfully applied to

377

cutflowers (11,12) and potted flowering plants (9) to inhibit the action of ethylene. There has been little work conducted on the postharvest effects of 1-MCP application on fruits. Sisler et al (13) showed that 1-MCP was effective in protecting bananas and mature green tomatoes from ethylene. Abdi et al (pers comm.) have shown with Japanese-type plums that the postharvest application of 1-MCP was effective in delaying the onset of ripening. The changes associated with the ripening in plums, such as volatile production and the completion of color development, were significantly affected by the application of 1-MCP. The postharvest application of 1-MCP can therefore be used to explore those changes that are ethylene dependent and those which are ethylene independent. Hence 1-MCP application offers a convenient means of examining the role of ethylene in ripening and offers an excellent opportunity to differentiate and clearly study the biochemical and physiological interactions occurring in ripening firuit. This work discusses the influence of 1-MCP and the timing of its application on the ripening related parameters in bananas such as ethylene, CO2 and volatiles production, volatiles composition and skin color.

2. MATERIALS AND METHODS 2.1 Fruit Mature green bananas (Musa sp. [AAA group. Cavendish subgroup] cv. Williams) were obtained from commercial agents, prior to ethylene treatment. 2.2 Fruit Treatment The bananas from a single hand were used for the experiments. Individual bananas were dipped in fungicide (prochloraz) at the recommended rate. The bananas were sealed in airtight 2L respiration containers with a flow rate of water saturated air at about IL/h. Where required, propylene was continuously applied at 500 jiL/L in the humidified air stream to the appropriate bananas. According to Burg and Burg (14) this concentration is equivalent to 5 |iL/L of ethylene, an amount previously shown to give optimum advancement of endogenous ethylene production in tomato fruit (15). Treated bananas were fumigated with 1-MCP in 6 L air tight containers where the concentration of 1-MCP was equivalent to 6.2 jaL/L air. After exposure for 6 hours at 20°C the individual bananas were placed in their respective respiration containers. 1-MCP was sjnithesised according to the method of Majid et aL (16).

378

2.3 Ethylene and CO2 analysis Ethylene and carbon dioxide were measured daily on a gas sample from the effluent of the respiration containers as described by Jobling et al (17). Ethylene was analysed on a GowMac Model 580 gas chromatograph fitted with an alumina column and FID with nitrogen carrier gas at 25mL/min. Carbon dioxide was determined by pxdse analysis using an IRGA (Horiba Model PIR-2000, Kyoto, Japan) with nitrogen carrier gas. 2.4 Volatiles analysis Glass gas chromatograph injector liners packed with Tenax TA (40mg) were used as volatile collection traps. They were connected to the individual respiration chamber outlets and the effluent collected untU a known volume of gas had passed through each trap. Each trap was then placed in the injector of a programmable temperature vaporiser (OPTIC 1, Ai, Cambridge) connected to a Hewlett Packard 5890A gas chromatograph fitted with a 25m x 0.22mm i.d.x 1.0 nm fflm thickness BP-1 fused siUca capillary column( SGE), an FID detector and a spUt/splitless injector operating at a spUt ratio of 20:1. The analysis was initiated by programming the injector temperature from 40°C to 220°C at 16°C/s, where it remained for the rest of the analysis. The column was maintained at 40°C for 5 min then programmed at lO^CAnin-^ to 200°C. The FID detector was maintained at 240°C. Data was collected using a Hewlett Packard Chemstation 3365 data processing package. To compare the volatile production of each banana, a volatile production index was used, where the total volatile area units were divided by the volume of headspace collected. 2.5 Color Determination After the respiration and volatile measurements were conducted, the respiration containers were opened and the peel color was assessed using a 1 - 8 scoring system (18). Where the bananas in color stage 1 were green hard and rigid, color 5 was yellow with green tips, and color stage 8 was yellow with increasing brown areas. The individual bananas were terminated upon reaching color stage 7, which were considered fuUy ripe with the skin is yellow with Ughtly flecked brown spots.

3. RESULTS AND DISCUSSION Figures 1, 2 and 3 illustrate the effect of the preclimacteric appUcation of 1MCP on the physiology of bananas which were continuously challenged with the ethylene analogue, propylene. Ethylene production, respiration as measured by

379

—o— 1-MCP —•— Control

1

2

3

4

5

6 7 8 9 10 11 12 13 Time (days)

Figure 1. The effect of predimacteric application of 1-MCP (Gh at 6.1|aL/L) on ethylene production in ripening bananas. Each point is the mean of three bananas with bars indicating the standard deviation.

—o— 1-MCP —•— Control

1

2

3

4

5

6

7

8

9

10 11 12 13

Time (days) Figure 2. The effect of preclimacteric application of 1-MCP (6h at 6.1|aL/L) on carbon dioxide production in ripening bananas. Each point is the mean of three bananas with bars indicating the standard deviation.

380

-o— 1-MCP -•—Control 08 U

O u

s

o O

u^ ^

-

-J

1

2

3

I

I

I

L.

4

5

6

7

8

9 10 11 12 13

Time (days) Figure 3. The effect of predimacteric application of 1-MCP (61i at 6.1|iL/L) on skin color score of ripening bananas. Each point is the mean of three bananas with bars indicating the standard deviation.

1-MCP Control

I 30000 "§ 25000

I 20000 (§ 15000 I 10000 :?

5000 1

2

3

4

5

6

7

8

9 10 11 12 13

Time (days) Figure 4. The effect of predimacteric application of 1-MCP (6h at 6.1|iL/L) on the total volatiles index in ripening biananas. Each point is the mean of three bananas with bars indicating the standard deviation.

381

C02 production and skin color development were are all delayed by two days by this preclimacteric 1-MCP application. However, once ripening had commenced, these parameters of the 1-MCP treated fruit develop at much the same rate and intensity as the untreated controls. The effect of 1-MCP treatment on the total fniit volatiles production is shown in Figure 4. Again the 1-MCP treated fruit showed a two day delay in generating volatiles when compared with control fruit. The activation of the biosynthetic pathways responsible for the formation of volatQes must therefore also be ethylene dependent. Additionally no ethylene independent volatiles were detected during ripening. Thus all four of these important indicators of climacteric fruit ripening are linked directly or indirectly to ethylene production an observation which again reinforces the central role of this plant hormone in fruit development processes. While the preclimacteric application of 1-MCP in bananas significantly delays the onset of the climacteric and associated ripening changes, preliminary indications are that this delay is much greater when the fruit is not continually challenged with propylene. A banana treated with a single six hour dose of 1-MCP (12ppm) thirteen days before the expected onset of its dimacteric and subsequently ventilated with humidified air did not commence ethylene production until nine days after the control fruit. In these control fruit the gap between the start of ethylene production and the commencement of volatiles production was two days. In the 1-MCP treated fruit this interval was extended to five days. A banana treated with a single dose (6hr) of 1-MCP (12ppm) one day before the predicted onset of the climacteric did not begin producing ethylene for a further fourteen days and volatiles for a further nine days. Therefore, when 1-MCP treatment was followed by storage in air rather than challenged with propylene, the application of 1-MCP can be seen to have significantly delayed the onset of ethylene production and extended the interval between this event and the commencement of volatiles production. The appUcation of 1-MCP closer to the beginning of the climacteric appears to extend the time delay before onset of the climacteric in the fruit to a greater extent than earlier application. 1-MCP treatment of Passe-Crassane pears resulted in a complete inhibition of ripening for a substantial period (19) and suppressed ethylene production, ACC oxidase and ACC synthase activities. The volatiles emitted by 1-MCP treated fruit were qualitatively similar to those of control fruit but showed distinct quantitative differences (Figure 5). In particular the group of higher molecular weight esters represented by isoamyl butanoate and isoamyl isovalerate are much less prominent in the volatiles of 1MCP treated fruit. This observation together with the fact that the production of volatiles and skin color development in 1-MCP treated fruit stored in air and propylene environments, suggests the response to ethylene in the flesh of the fruit may be different to that of the skin. This is consistent with previous observations that the ripening of bananas can be separated into changes occurring in the peel and those in the flesh and that they ripen from the flesh out with flesh ripening preceding peel yellowing (20). Recent work has shown that in bananas an ACC oxidase transcript appears in the flesh earher than in

382

the peel but this can be altered by the application of exogenous ethylene (21). This is compatible with our observation that skin yellowing precedes volatiles production in 1-MCP treated fruit that are challenged with propylene but volatiles production lags it in 1-MCP treated fruit ventilated with air. It appears that the quantitative composition of the volatiles formed in the skin differs from that formed in the flesh and this may explain the sensory differences, particularly the fruitiness attribute, found between ethylene ripened and naturally ripenedfiruitat the same color stage, reported by Scriven et al (22).

a

II Ji 1

-J—j—r—f—I

5

I

I

7.5

I

I

I

I

I

10

i

LAi I

I

I

I

[—I

I

t

f

A I

15

125

I

, I

i R

IAJ UK I

I

I

17.5

I

I

I

I

20

a

I 7.6

10

UILL

JLioJ-

L« T

125

15

17.5

20

Retention Time (min.) Figure 5. Volatiles profile of an 1-MCP treated banana maintained in air and a control banana at the same ripening stage.

383

4. CONCLUSION

The application of the ethylene receptor antagonist 1-MCP to predimacteric bananas resulted in a significant delay in the onset of the common indicators of ripening, ethylene and carbon dioxide production, skin color and volatile formation suggesting that all of these parameters are moderated by ethylene. Quantitative differences in the volatUes profile between propylene treated and normal fruit suggest that the biochemical pathways for volatiles production in skin and flesh are also subject to difierential response to ethylene. Further work is currently in progress to explore these possibilities.

5. REFERENCES 1. G.B. Seymour. Banana. In Biochemistry of Fruit Ripening. G.S. Sejmaour, J. Taylor and G. Tucker (eds). Chapman and Hall. London. 1993. 2. J.Gross, M.Carmon, A. Lifshitz and C. Costes, Food Sci. and Tech., 9 (1976) 211. 3. R. Tressl and W.G. Jennings, J. Agric. Food Chem., 20 (1972) 189. 4. C. Macku and W.G. Jennings, J. Agric. Food Chem., 35 (1987) 845. 5. H. Shiota, J. Agric. Food Chem., 41(1993) 2056. 6. H.Maarse and C.A. Visscher, In. Volatile Compounds in Food. Qualitative and Quantitative Data. Volume 1. TNO-CIVO. The Netherlands. 1989 7. J. Song and F. Bangerth, Acta Horticul., 368 (1994) 150. 8. E.J. McMurchie, W.B. McGlasson and I.L. Eaks, Nature, 237 (1972) 235. 9. M. Serek, E.C. Sisler and M.S. Reid, J. Amer. Soc. Hort. Sci., 119 (1994) 1230. 10. E.C. Sisler, E. Dupille and M. Serek, Plant Growth Regulation, 18 (1996) 79. 11. M. Serek, E.C. Sisler, T. Tsipora S. Mayak, HortSci., 30 (1995) 1310. 12. R. Porat, E. Shlomo, M. Serek, E.C. Sisler and A. Borochov, Postharvest Biol. Technol., 6(1995)313. 13. E.C. Sisler, M. Serek and E. Dupille, Plant Growth Regulation, 18 (1996) 169. 14. S.P. Burg and E.A. Burg, E.A., Plant Physiol., 42 (1967) 144. 15. W.B. McGlasson, H.C. Dostal and E.C. Tigchelaar, Plant Physiol. 55 (1975) 218. 16. R.M. Majid, T.C. Clarke and CD. Duncan, J.Org. Chem., 36 (1971) 1320. 17 J. Jobling, W.B. McGlasson and D.R. Dilley, Postharvest Biol. Technol., 1 (1991) 111. 18. Commonwealth Scientific and Industrial Research Organisation, Banana ripening guide. Banana Research Advisory Committee Technical Bulletin 3. CSIRO Melbourne. 1971.

384

19. J-M. Levievre, L. Tichit, P. Dao, L. Fillion, Y-W. Nam, J-C. Pech and A. Latche, Plant Mol. Biol., 33 (1997) 847. 20. F.B. Abeles, W.M. Page, and M.E. Saltveit, (1992) Ethylene in plant biology. 2nd ed. Academic Press. San Diego. 1992. 21. R. Lopezgomez, A. Campbell, J.G. Dong, S.F. Yang and M.A. Gomezlim, Plant Sci. 123 (1997) 123. 22. F.M. Scriven, O.G. Choo and R.B.H. WiUs, HortSci., 24 (1989) 983.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

385

Sensory characterization of Halloumi cheese and relationship with headspace composition J.R. Piggott, A. Margomenou, S.J. Withers and J.M. Conner University of Strathclyde, Department of Bioscience and Biotechnology, 204 George Street, Glasgow G l IXW, Scotland Abstract Halloumi is a traditional cheese from Cyprus, which is attracting increasing consumer interest. For successful production the factors that determine its properties must be understood, so Halloumi cheeses were analysed for chemical composition and sensory properties. Sensory properties were found to vary between cheeses, and some texture and flavor characteristics were greatly affected by cooking. Volatile compounds were contributed by mint and by milk breakdown products, and it was possible to predict panel mean scores for some flavor notes by partial least squares regression on the headspace volatiles.

1. INTRODUCTION Halloumi is a semi-soft to semi-hard cheese, unripened and without a skin, traditionally preserved in brine until sold to the consumer. It originated in Cyprus, but has since become popular all over the Middle East, an important market for dairy products. It has traditionally been made from sheep's or goat's milk, or a mixture of both, but now it may also be made from cow's milk. After standardization milk is coagulated with rennet, and the whey removed from the curds and heated to 80-90°C for 30 min to coagulate proteins. The curd is cut, pressed, and the resulting fused mass cut into blocks. The blocks are placed in the heated whey, from which the proteins have been removed, for 30 - 60 min during which the curd acquires the firmness of the finished cheese. The blocks are cooled and salted, and fragments of dried mint leaves {Mentha viridis) are often added with the salt to give the finished cheese a slightly speckled appearance and characteristic flavor [1]. Finally, the cheese blocks are packed in brine. Published analyses of Halloumi show a wide range of composition; moisture has varied from 26 - 49%, fat from 20 - 30%, and salt from 2 - 6% [2]. The pH of experimentally produced trial cheeses was 5.9 ± 0.2 [3]. No analysis of Halloumi volatiles has been reported, but there is no maturation so fat breakdown and proteolysis of the caseins must be very limited. Additionally it is cooked at a high temperature so the enzymes and microflora which could accelerate the aging process are destroyed. The type of milk used may also be important, and for

386 example sheep's milk could enhance the development of higher levels of short chain fatty acids than cow's milk. The texture and flavor of Halloumi are considerably affected by the stages of manufacturing [1], but there seems to have been no systematic study to determine the effects on sensory properties of differences in manufacturing details or region of production. The work described here was carried out in order to determine the volatiles of Halloumi cheese, to provide a description of its sensory properties and to understand the correlations between the composition and flavor of the cheese. 2. EXPERIMENTAL Ten brands of cheese were purchased in London. Headspace and sensory analyses were performed on raw cheese, and on cheese cooked by frying and grilling. Moisture, pH, nitrogen, fat, salt and acidity were measured by standard methods. Volatile compounds were determined by employing a GC-MS with a Finnegan- MAT ITS40 using a 25 m x 0.22 mm (df = 0.25 pm) fused siHca BP20 column, with an ionization potential 70 eV, filament emission of 10 pA, helium carrier gas at 250 mm sec"^, and scan range between 40 and 400 m/z. Cheese (1 g) was sealed in a glass vial with a PTFE-lined rubber septum, which was placed in a water bath at 37°C. Headspace was sampled for 30 min with a Supelco SPME fibre coated with 85 pm poly-acrylate. The fibre was desorbed at 240°C for 5 min. The oven was programmed from 60°C for desorption to 200°C at 7°C min"^ then to 240°C at 20°C min'\ with a final isothermal hold for 3 min. Compounds were tentatively identified by comparison with library spectra. A panel of 11 assessors, trained and experienced in the sensory analysis of a variety of products but not specifically trained with Halloumi, used descriptive sensory analysis to profile flavor and texture. The assessors tasted raw, fried and grilled samples of cheese, and suggested terms to describe the texture, odor and flavor. The assessors then used the agreed vocabulary to profile the ten cheeses, initially raw and followed by samples fried and finally grilled. The cheeses were assessed four times over a 5-week period. The assessors scored each descriptor on a continuous scale using the PSA-System for data collection. Data matrices were examined by principal components analysis (PCA) using the Unscrambler, and volatile and sensory data were related by partial least squares regression (PLS) with the Unscrambler.

3. RESULTS AND DISCUSSION Chemical analyses of Halloumi cheese were broadly in accord with previous reports and are not discussed further. Analysis of variance of descriptive sensory data showed that the assessors had used many terms to discriminate between samples, and between raw and cooked cheeses. The first three principal components of the sensory data showed significant differences (p < 0.05) between individual product-cooking combinations (Figure 1), but the fourth component showed only an effect of cooking (p < 0.05). The cheeses were largely separated

387

2 T CM

-•— • c

1

• Raw

t

• Fried

CD C

• Grilled

o

Q.

E o o

-1 t

o c

'

*

•

• • A

•

1 ""

-3 t

1

-4

•

J

1

1

1

-2

0

2

— 1

1

Principal component 1 Figure 1. Sample scores for 10 Halloumi cheeses prepared in 3 ways plotted on the first two principal components from descriptive sensory data.

coarse

0.4 J

0.3 -1^

grainy crumbly •

• •

0.2 +

S 0.1 + Q.

i 0+

sweet

•

•

^ • • oily waxy ^ ^ • • milky 4 buttery creamy

o

i -0.2 + Q.

-0.3 + -0.4 -0.3

sour

•

•4-

-0.2

-0.1

0

-*-l

0.1

0.2

—H

0.3

1

0.4

Principal component 1

Figure 2. Descriptor loadings plotted on the first two principal components from descriptive sensory data on 10 Halloumi cheeses prepared in 3 ways.

388

according to their origin, and the effects of cooking were only seen in component 3. Component 1 was strongly related to flavor (Figure 2), and a contrast of sweet and sour, whereas component 2 contrasted a group of texture terms to the positive end with a milder flavor (milky and creamy). The main features of these cheeses were the minty and herby characters, which were detected in both raw and cooked samples. The assessors could easily discriminate between the cheeses for saltiness, but that disappeared when the cheese was cooked, whereupon the milky, creamy and fatty character became more noticeable. Some texture attributes were affected by cooking (coarse, rubbery, tough and squeaky), but the hardness seemed to be reduced. The interaction of sample and cooking process affected leathery. Crumbly, grainy, artificial, bitter, buttery and waxy showed no effects. Table 1 Analyses of variance of headspace volatiles of Halloumi cheese Compound

Product

Process

Sample

Pentanol Ethyl acetate Nonanone Acetic acid Pulegone Undecanol Unknown 2 Unknown 3 Unknown 11 Carvone Butanoic acid Unknown 4 Decanol Dodecanol Phenol Unknown 5 Hexanoic acid Terpene Unknown 6 Lactone Heptanoic acid Unknown 7 Unknown 8 Octanoic acid Unknown 9 Unknown 10

*** *** ** *** *** ** *** *** *** *** * * * ** * * *

** *** * * * *** ** * * * ** -

* * *** *** *** *** ** * * * *** ** -

where: * p < 0.05; ** p < 0.01; *** p < 0.001

Product X process * * * -

Retention time 3:20 4:42 7:34 9:17 11:04 12:00 12:11 12:52 14:21 14:29 16:23 16:40 16:47 18:19 18:49 19:19 19:37 20:09 20:14 20:42 21:07 21:30 22:07 22:33 22:40 23:44

389

40 T

r = 0.70 • Raw • Fried • Grilled

S2 o o CO M—

C/D —I

30 +

DL

• •

•

•

• •

• A •

CM

•

O O

(0 •D 0)

"o

20 +

1

• •

•

^ i "A

•

•

A A

A

10 J 10

1 20

— 1 —

1

30

40

Mean panel score Figure 3. Predicted scores for minty flavour calculated from 2 PLS factors plotted against panel mean scores for 10 Halloumi cheeses prepared in 3 ways.

ou •

£2 o -I—• o CO

H—

C/D _J

r = 0.66 • Raw • Fried A Grilled

40 -

CL 0

•

i—

O

o

(0 •D

•

30 -

O

"o

A A

CD

— ^ CL

PO 20

M^ 1

—1

30

40

1

50

Mean panel score

Figure 4. Predicted scores for milky flavour calculated from 2 PLS factors plotted against panel mean scores for 10 Halloumi cheeses prepared in 3 ways.

390 Headspace analysis showed that the cheeses varied substantially, and there were also effects of the cooking process. Virtually all 26 compounds quantified showed significant differences due to the cheese or to the cooking process (Table 1). The volatiles found in this type of cheese, even though it is classified as a white brined cheese, do not have much in common with the rest of the cheeses in the group. This cheese does not undergo maturation, there are not always starter cultures in use in order to help the breakdown of the milk components, and it undergoes cooking in production, so much of the microflora is killed and subsequent breakdown is likely to be very slow. Consumption of the cheese is normally very soon after production, so there is very little time for any changes in storage. However, a number of volatile acids and other compounds characteristic of fat breakdown were found, including an unidentified lactone [4]. Carbonyl compounds, though none was identified in this case, would also be expected. Some alcohols and acids may arise from amino acid breakdown, and aroma compounds including phenol have been reported from amino acid breakdown [5,6]. PLS regression analysis was used to predict panel mean sensory scores for flavor terms from the headspace data. Only those flavor terms which showed significant differences between samples were used. The panel mean score for minty plotted against the predicted score from 2 PLS components is shown in Figure 3. The compounds particularly associated with this flavor were pulegone, two unidentified peaks, "mint terpene" (an otherwise unidentified compound with a mass spectrum characteristic of a terpene, whose origin was assumed to be in the mint leaves or other herbs used in the cheese) and carvone. It was encouraging that minty could be predicted successfully by PLS from the volatiles; this could have been expected but was a useful indication that the data were reliable. Other sensory characteristics were more or less successfully predicted (milky, creamy and fatty), but were less obviously related to a single ingredient or component. Prediction of the fatty flavor note was least satisfactory, and only two compounds were positively related to it (acetic acid and an unidentified compound), while phenol and a further unidentified peak were loosely negatively related. The predictions of creamy and milky were somewhat better; these flavors were closely correlated and were generally characteristic of the raw cheeses. As an example, the predicted scores for milky from 2 PLS components are shown in Figure 4. This flavor characterised primarily those cheeses that contained cow's milk, and both these flavors were much reduced when the cheese was grilled or fried. Compounds associated with this flavor were mainly two unidentified peaks, but others including nonanone and phenol were more loosely associated. 4. CONCLUSIONS Halloumi cheeses were found to have essentially the same composition as previously reported. Sensory properties were found to vary between cheeses, and some texture and flavor characteristics were greatly affected by cooking. Volatile compounds were contributed by mint and by milk breakdown products. It was possible to predict panel mean scores for some flavor notes (minty, milky, creamy.

391 fatty) by partial least squares regression on the headspace volatiles. 5. REFERENCES 1 2 3 4 5 6

R.K. Robinson and A.Y. Tamime, Halloumi cheese - the product and its manufacture. Blackie, Glasgow, 1991. E.M. Anifantakis and S.E. Kaminarides, Australian J. Dairy Technol., 38 (1983) 29. R.R. Shaker, J. Lelievre and M.W. Taylor, New Zealand J. Dairy Sci. Technol., 22 (1987) 181. J. Bakker and B.A. Law, Understanding Natural Flavours (J.R. Piggott and A. Paterson, eds.), Blackie, Glasgow, 1994. J. Adda, J.C. Gripon and L. Vassal, Food Chem., 9 (1982) 115. D. Hemme, C. Bouillane, F. Metro and M.J. Desmazeaud, Sci. Aliments, 2 (1982) 113.

Acknowledgments The authors wish to acknowledge the valuable assistance and advice of Adnan Tamime. The UK Biotechnology and Biological Sciences Research Council and The Chivas and Glenlivet Group provided financial support.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

393

Comparison study of UHT milk aroma L. Hashim and H. Chaveron Laboratoire Biophysicochimie et Technologie Alimentaires, University de Technologie de Compi^gne, B.P. 20529, 60205 Compiegne-France. Abstract The flavor of milk may change when the product is submitted to thermal treatments. The heating of milk causes the formation of volatile compounds from milk components. Volatile compounds of UHT whole milk, UHT semiskimmed milk, and UHT skimmed milk were analysed u s i n g gas chromatography. The odor intensity of different molecules was realized by a sniffing test of the volatile compoimds of different milks at the outlet of the gas chromatography-capillary column. Sensory analysis of different milks was conducted by a trained taste panel. It showed t h a t milk flavor was affected by thermal t r e a t m e n t and milk composition. According to the different analytical techniques described, relevant differences were found among the studied milks. 1. EWRODUCTION The consumer acceptance and preference for milk as a beverage is influenced by its flavor more than any other attribute (1). Flavor is a property detected by the senses, in particular taste and smell, and thereby requires taste panel work for its evaluation. Milk has a pleasant mouth-feel, determined by its physical nature, i.e. an emulsion of fat globules in a colloidal aqueous solution, and a slightly salty and sweet taste due to the presence of salts and lactose (2). The flavor of fresh milk, although characteristic, is normally of a low intensity. Heated milk should have acceptable flavor characteristics, the milk from which it is processed m u s t meet appropriate physical, chemical, microbiological and sensory quality standards. When milk is heated, changes in flavor occur, the kind and intensity of the flavor depending on the time and temperature of the treatment (3). The term heated flavors is used to include all the flavors which are produced by thermal processing of fluid milk. At least 400 volatile compounds have been detected in milk processed in different ways (4). The flavor of ultra-high-temperature (UHT) processed milk has been described as "cooked, cabbagey, and sulfur". The intensity of this flavor and

394

associated odor has been correlated with the degree of free sulfhydryls (SH) and volatile sulfides liberated via the heat denatioration of the whey protein (5, 6). The lipid components of milk are important contributors to milk flavor. Compounds such as volatile fatty acids, dicarbonyls, and monocarbonyls impart flavor to milk though present only in trace amounts. Various ketones, saturated aldehydes , and unsaturated aldehydes affect flavor though present in concentrations of parts/million or parts/billion (7). Flavor changes in milk arise because of changes in its chemical constituents. The various types of flavor defect in milk have been reviewed (8). Therefore, researchers studying flavors use a combination of taste panel work and chemical analysis (9). The purpose of the this research was to study the differences in flavor profiles of UHT whole milk, UHT semi-skimmed milk, and UHT skimmed milk by gas chromatography-sniflfing tests and sensory analysis. 2. EXPERIMENTAL DATA 2.1 Samples UHT whole milk, UHT semi-skimmed milk, and UHT skimmed milk were purchased in France. These milks were processed by UHT systems and filled in Tetra Brik 1 Liter containers. 2.2 Analjiical analysis Steam distillation-microextraction was used to extract the volatile compounds of milks (10). Samples of 100 mL of each milk were analyzed to obtain the volatile extract. Volatile compounds were separated by capillary column gas chromatography (GC) using a Girdel-30 equipped with a flame ionization detector (FID). A CP Wax 52B (polyethyleneglycol, 50m X 0.32mm) fused silica capillary column (Chrompack) operated with helium as the carrier gas was employed. The column temperature was programmed from 50°C to 220°C at a rate of 5°C/min. Injector and detector temperature were set at 250°C. Chromatographic data were processed with a computing integrator (Shimadzu C-R6A). The odor intensity of different molecules was realized by sniffing different milk volatiles at the outlet of the gas chromatography-capillary column. 2.3 Sensory evaluation Sensory analysis of different milks was done by a trained taste panel using a flavor profile test with different descriptos (10 students from the Technical University of Compiegne were trained to do the tests). A 10-point scale was used. Representative samples were independently and randomly presented for evaluation.

395

3. CHROMATOGRAPHIC ANALYSIS Figure 1 shows the gas chromatogram of UHT whole milk aroma.

Figure 1. Capillary gas chromatogram of UHT whole milk volatiles

The aroma chromatograms of different milks were, in general, very similar. The most important differences between them were the intensities and the areas of certain peaks. Using sniff-test, the odor intensity and odor description was determined for 16 peaks. These peaks show a typical milk aroma. The results are presented in Table 1 and Figures 2, 3, and 4. It should be noted that throughout this study the only property evaluated was the milky aroma. The data shown in Figures 2, 3, and 4 clearly demonstrate that UHT whole milk has the most interesting milky odor. It can be seen from these figures that peaks 1, 2, 5, 7, and 10 in UHT whole milk have odor intensities superior to those in the other milks. These peaks have shown the most interesting and typical milky aromas. Table 1 shows the odor descriptions of various peaks. It can be noticed that the odors vary from buttery, creamy, fruity, chemical, milky, sweet, roasted, and burnt. The two latter aromas have low odor intensities and were detected only at the end of the chromatogram.

396

V3

u o

IS

O

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16

Peak number Figure 2. Odor intensity of UHT Skimmed milk (Peak numbers are as indicated in Figure 1)

0^

o

o

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16

Peak number

Figure 3. Odor intensity of UHT Semi skimmed milk (Peak numbers are as indicated in Figure 1)

397

en

o O

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16

Peak number Figure 4. Odor intensity of UHT Whole milk (Peak numbers are as indicated in Figure 1) Table 1 Odor description of volatile compounds from UHT milks Peak number

Retention timeCmin)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

4.4 5.5 7.1 9.6 10.4 13.4 14.3 17.4 20.5 22.4 24.5 26.3 31.5 34.9 38.2 40.6

L 16

Odor description Buttery 1 creamy 1 creamy creamy | milky milky milky-fruity milky-slightly milky-chemical milky-sweet milky milky milky-chemical milky milky-roasted milky-btirnt

398

4. SENSORY ANALYSIS The flavor of particular interest in heated milk is the "cooked" flavor. This flavor changes rapidly during the early days of storage. The vocabulary used for description is also not straightforward, and terms, such as "cooked", "cabbagey", "sulphury" and "caramellised", are all frequently used (5). One of the best accounts of the flavor changes in milk on heating and during storage is given by Ashton (11). Sensory evaluation provides the most practical method for monitoring the type and intensity of heated flavors. Figures 5, 6, and 7 show the intensity of the milk descriptors studied. The descriptors sweet, creamy, thickness, milky and hedonic (most pleasant) have higher scores and intensity in whole milk compared to other milks. It can be seen that these differences are in linear relation with the composition of different milks. It should be noted that differences between whole milk and semiskimmed milk are lower than differences between semi-skimmed milk and skimmed milk. The flavor profile of whole milk is more acceptable than that of the other milk, even with a few off-flavors.

a 0^

u o C/5

Descriptors

Figure 5. Intensity of different descriptors of UHT skimmed milk

399

V3

a

o

Descriptors Figure 6. Intensity of different descriptors of UHT semi-skimmed milk

0)

o C/5

1 CO

o U

1 u

4>

1

1 <

'3 o

M

^

P^

H

Descriptors

Figure 7. Intensity of different descriptors of UHT whole milk

400

5. CONCLUSIONS Additional research is needed to identify the nature and the intensity of different volatile compounds. These results could be compared to those obtained from dried milks, and other dairy products to establish a method for monitoring the type and intensity of heated flavors. This study shows the use of chromatographic techniques for monitoring the concentration of volatile compounds associated with heated flavors and the sensory evaluation of these compounds using olfactory techniques with trained panelists.

a REFERENCES 1. E.L. Thomas, J. Dairy Sci., 64 (1981) 1023. 2. D.J. Manning and H.E. Nursten, Developments in Dairy Chemistry-3, Fox (ed), London and New York, 1985. 3. M.M. Calvo and L. Hoz, Int. Dairy J., 2 (1992) 69. 4. H.T Badings and R. Neeter, Neth. Milk Dairy J., 34 (1980) 9. 5. R.S. Mehta, J. Food Protection, 43 (1980) 212. 6. A.P. Hansen, K.R. Swartzel and Giesbrecht, J. Dairy Sci., 63 (1980) 187. 7. J.E. Kinsella, Chemistry and Industry, 2 (1969) 36. 8. H.T. Badings, Dairy Chemistry and analysis, Walstra&Jennes (eds), 1984. 9. M.J. Lewis, Modern Dairy Technology-1, Robinson (ed), London and New York, 1986. 10. L. Hashim and H. Chaveron, Food Research International, 6 (1994) 537. 11. T.R. Ashton, J. Soc. Dairy Technol., 18 (1965) 65.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

401

Some Toxic Culinary Herbs in North America Arthur O. Tucker and Michael J. Maciarello Department of Agriculture and Natural Resources, Delaware University, Dover, DE 19901-2277

State

Abstract In recent years, new herbs, particularly from Asia and Latin America, have appeared upon the North American markets. Some of these herbs have accumulated sufficient toxicological literature to question their safety, and caution is advised on their consumption: hoja santalyerba santa acuyo {Piper auritum Humb., Bonpl., & Kunth), California bay [Umbellularia californica (Hook. & Arnott) Nutt.], perilla/5/1/^0 [Perilla frutescens (L.) Britton], pink/red peppercorns (Schinus terebinthifolia Raddi and S. molle L.), and ^ p a z ^ r ^ / w o r m s e e d / c h e n o p o d i u m {Chenopodium ambrosioides L.). Examination of the essential oils by GC/MS revealed many dominant components which are documented to be toxic to mammalian systems. 1.

INTRODUCTION

In the United States, flavor ingredients are regulated by the Food and Drug Administration (FDA) under the Food Additives Amendment, section 409 of the Food and Drugs Act, passed by the 1958 Congress. The Flavor and Extract Manufacturers' Association (FEMA) provides an expert panel to determine those flavors and levels which are excepted from the coverage of section 409 and whose use in food is "generally recognized as safe" (GRAS); this is summarized in Chapter 21, Parts 182 and 184 of the Code of Federal Regulations (C.F.R.). In 1972 the National Academy of Sciences' Food Protection Committee reviewed GRAS chemicals, and since then the FEMA expert panel has reviewed flavor ingredients with periodic publications in Food Technology, In Europe, a classification of Nature-identical is similar to GRAS, and the European Community (EC) is preparing inventories of flavoring materials. Good introductions to food regulation are Hutt and Merrill [1], Stofberg [2],

402

and Burdock [3]; some additional information is provided in Leung and Foster [4]. Fragrance ingredients in the U.S. are regulated by the in-house Research Institute for Fragrance Materials (RIFM) with findings published in Food and Chemical Toxicology, a cross reference list was published in 1992 [5]. On an international scale, the International Fragrance Association (IFRA) has published a Code of Practice with periodic updates from 1974 to the present date in a two-ring binder [6]. While these agencies have provided lists of safe flavors and fragrances, they have, in addition, provided advice and, sometimes, bans against certain products on the market. In recent years, new herbs, particularly from Asia and Latin America, have appeared upon the North American markets and have not been closely examined by these agencies. Some of these herbs have accumulated sufficient toxicological literature to question their safety. 2.

MATERIALS AND METHODS

Vouchers of plants were deposited in the Claude E. Phillips Herbarium, Delaware State University (DOV). Oils were distilled with a neo-Clevenger of Moritz after Kaiser and Lang with the modification of Hefendehl [7, 8]. Mass spectra were recorded with a 5970 HewlettPackard Mass Selective detector coupled to a HP 5890 GC using a HP 50 m X 0.2 mm fused silica column coated with 0.33 jxm FFAP (crosslinked). The GC was operated under the following conditions: injector temp.: 250°C; oven temp, program: 60°C held for one min, then 2.5°C per min to 115°C, then 1.0°C per min to 210°C and held for 30 min; injection size: l|xL (~50% solution in spectroscopy grade n-pentane) split 1:100. The MSD EI was operated under the following conditions: electron impact source 70 eV, 250°C. Identifications were made by Kovats Indices and library searches of our volatile oil library supplemented with those of NBS, NIST, and Wiley.

3.

RESULTS AND DISCUSSION

3. 1. SASSAFRAS AND HOJA SANTA/YERBA

SANTA

ACUYO

An extract or oil of the root of sassafras. Sassafras albidum (Nutt.) Nees, is hepatocarcinogenic in mice, rats, and dogs from the content of 74-85% safrole. Safrole is viewed as a precarcinogen which is

403

metabolized to the carcinogenic T-sulfooxysafrole via T-hydroxysafrole (in mouse liver). Separate from carcinogenicity, ingestion of 5 ml of sassafras oil by an adult or a few drops by a toddler will cause death or induce vomiting, tachycardia, and tremors. Consumption of 10 cups or more per day of sassafras tea will induce diaphoresis in an adult. Sassafras root has been banned by the FDA since 1960 and the Council of Europe since 1974; the FDA ruling was comprehensively reviewed in 1976 and upheld. Sassafras leaves and commercial file powders normally do not contain any safrole but may contain up to trace quantities (average 0.66% of oil). Sassafras leaves are permitted by FDA in foods if safrolefree. Yet, while toxic to humans, safrole does have a market. Safrole is an important raw material for the synthesis of heliotropin for fragrances and piperonyl butoxide, a synergist for insecticides. The principal source of safrole has been the Brazilian sassafras, Ocotea pretiosa (Nees) Mez, but a moratorium on the cutting down of Brazilian sassafras trees in Brazilia is steering buyers to China in search of Cinnamomum species rich in safrole. Sassafras would have a similar market if available in sufficient quantities. Safrole has also been used as a precursor in underground production of methylenedioxymethamphetamine (MDMA, ecstasy, XTC, Adam) [9-14]. Recently, leaves of ''hoja santa" have been recommended for cooking [15-17], and, under the alternate name of ''yerba santa acuyo," these leaves have appeared in some herbal teas marketed in North America [18]. We have identified the hoja santalyerba santa acuyo sold in the U.S. as Piper auritum Humb., Bonpl., & Kunth (DOV13107), not P. sanctum (Miq.) Schlecht. ex Miq., despite the misleading common names. Piper auritum is a common on moist forest edges and open sites from Mexico to Colombia and in some of the islands of the West Indies [19]. Alternate names applied to this species include momo (Tabasco), xmacolan (Yucatan, Maya), acoyo (Veracruz), hoja de la estrella (Costa Rica), santa maria, cordoncillo (Nicaragua), anisillo, monca blanca (Costa Rica), hoja de jute, and juniapra (Guatamela). In Veracruz, the leaves are used for seasoning tamales, while in Costa Rica, the fresh leaves are applied to relieve headaches [20]. Leaves of hinojo sabalero (P. auritum) have traditionally been used to feed, and thereby flavor fish in Panama [21]. Leaves of P. auritum contain 70-77% safrole and caphoradione A and B, two aporphine-type alkaloids of unknown physiological activity [22-25]. Our analysis of a commercial yerba santa acuyo sample reveals 68.89% safrole, while fresh leaves of P, auritum have 36.69±2.76% safrole (Table 1). The safrole of hoja santa is as toxic as safrole of sassafras, and no parts of P. auritum are GRAS.

404

TABLE 1. THE ESSENTIAL OILS OF A COMMERCIAL DRIED YERBA SANTA ACUYO AND FRESH LEAVES OF PIPER AURITUM (DOV13107) Compound Commercial % Fresh Mean ± SD % (N = 3) & Oil Yield 0.22 oil yield 0.54±0.17 0.60 a-pinene 6.48±0.47 0.13 camphene 0.31±0.07 0.51 p-pinene 7.0610.39 0.21 sabinene 0.3810.66 0.17 myrcene 4.2310.10 0 a-phellandrene 0.0310.05 1.14 a-terpinene 9.7310.20 0.10 limonene 1.0010.25 0.13 p-phellandrene 0.2910.29 0.13 (^)-2-hexenal 0 0 (Z)-p-ocimene 1.4810.21 y-terpinene 16.1310.44 4.13 0.4810.27 p-cymene 2.25 11.3510.55 3.91 terpinolene 0 0.4510.02 allo-ocimene 0 0.1910.04 2-nonanone 1.15 0.0910.16 a-copaene 1.09 0 camphor 0 3.1410.12 linalool 0.44 0.3810.31 P-cubebene 0.27 0 bornyl acetate 0.87 0 terpinen-4-ol 2.97 0.1010.05 P-caryophyllene 0 0.44 a-humulene 4.84 0.3010.07 germacrene D 0.0810.02 1.70 a-muurolene 0.1210.03 0 germacrene B 0 1.82 bicyclogermacrene 0.0210.03 0.62 5-cadinene 68.89 36.6912.76 safrole 0 0.0110.02 methyl eugenol 0 0.0710.00 (Z)-nerolidol 0.81 (Z)-methyl isoeugenol 0 0 eugenol 0.0110.02

405

3.2. CALIFORNIA BAY The leaves of California bay, Umbellularia californica (Hook. & Arnott) Nutt., have been marketed in either jars or wreaths by several companies based in California as a substitute of Greek bay, Lauras nobilis L. The leaves of California bay contain a mean of 36-47% umbellulone with little geographic variation but considerable variation during the season [26-29]; our analysis of a commercial California bay oil shows 5.43% umbellulone (Table 2). TABLE 2. A COMMERCIAL CALIFORNIA BAY OIL. Compound % 2.43 a-pinene 1.12 P-pinene 15.31 sabinene 0.13 myrcene 28.16 1,8-cineole 4.17 y-terpinene 0.25 isopentyl butyrate 1.53 /j-cymene 1.47 terpinolene 7.47 trans-s&hinQne, hydrate 0.27 CJ5-sabinene hydrate 1.42 terpinen-4-ol 5.42 umbellulone methyl chavicol 0.11 11.42 a-terpineol a-terpinyl acetate 2.23 0.29 (£)-a-bisabolene geranyl acetate 0.51 methyl eugenol 1.70 elemol 0.64 0.33 (£)-cinnamyl acetate 2.22 (Z)-isoeugenol thymol 1.44 chavicol 0.40

406

Umbellulone is toxic to the central nervous system when ingested and causes convulsive sneezing, headaches, and sinus irritation when inhaled deeply [30]. Contact with the oil or its vapors has resulted in severe headache, skin irritation, and in some cases unconsciousness. Umbellulone can be hemolytic but apparently acts by blocking pulmonary circulation [31]. Leaves of U. californica also contain the alkaloids domesticine, n(9r-domesticine, and isoboldine of unknown toxicity [32]. Hence, no parts of California bay are GRAS. 3.3.

PERILLA/S///SO

TABLE 3. THE ESSENTIAL OIL OF FRESH TOPS (TERMINAL 0.5 m) OF PERILLA FRUTESCENS TIA TO'(DOVI3009). Compound & Oil Yield Mean ± SD % (N = 3) 0.12±0.04 oil yield 1.27±0.29 a-pinene 1.15±0.31 P-pinene 0.33±0.09 sabinene 0.63±0.19 myrcene 0.11±0.19 a-terpinene 30.63+7.01 limonene 0.31±0.11 1,8-cineole terpinolene 0.18±0.06 l-octen-3-ol 0.71+0.27 a-copaene 0.27±0.04 linalool 1.52±0.16 P-caryophyllene 6.0510.06 a-humulene 0.6410.06 a-terpineol 0.3110.06 2.8910.22 germacrene D 4.9810.48 (Z,£)-a-farnesene 0.2010.05 5-cadinene 43.5016.54 perillaldehyde trans-shisool 2.5310.84 perillyl alcohol 0.6510.31

407

Perilla or shiso, Perilla frutescens (L.) Britton, has recently captivated chefs [33] but is very variable in morphology and chemistry [34, 35]. Leaves of perilla may contain 0-94% perilla ketone [36-44], which is a potent lung toxin [45, 46]. Perilla and/or perilla ketone may cause acute pulmonary edema in cattle and sheep, atypical interstitial pneumonia or emphysema in cattle, and restrictive lung disease in horses [47-53] Perilla can also cause "intestinal propulsion" in mice [54]. Many forms of perilla are documented to produce contact dermatitis on prolonged handling [55, 56] In the field, perilla is also allelopathic [57] Recently the Vietnamese cultivar Tia To' has become popular on the fresh herb market in North America. This seed line, which has green upper leaf surfaces and purple under leaf surfaces [58], has no perilla ketone but is dominated by 43.50±6.52% perillaldehyde (Table 3), providing a cumin-like odor. Forms of P. frutescens high in perillaldehyde are popular in Japan as aojiso for suppressing the sardine odor of niboshi soup stock [59]. Perillaldehyde has sedative, antidermatophytic, and allelopathic properties [57, 60-64] and although it was given GRAS status by FEMA in 1978, its full safety still remains to be resolved. No parts of perilla are GRAS [65]. 3.4. PINK/RED PEPPERCORNS {BAIES ROSE DE BOURBON) Pink or red peppercorns are primarily the fruits of Schinus terebinthifolia Raddi, Brazilian pepper tree, but the fruits of S. molle L., the Peruvian or California pepper tree, are also harvested. Both species are in the Anacardiaceae, the poison ivy family, and both species are documented to cause contact dermatitis. Both species are also documented to cause ashthma-like attacks (with as little as one peppercorn, depending upon the individual) when ingested, with accompanying violent headache, swollen eyelids, shortness of breath, chest pains, sore throat, hoarseness, upset stomach, and diarrhea. While the plant parts of these species of Schinus are toxic, the oils of both species contain no unusual constituents and are not toxic. The fruits of both species contain triterpenoids (not isolated in the essential oil), two of which have been characterized as active site-directed specific competitive inhibitors of phospholidase A2. No whole parts of these two species of Schinus are GRAS, and the FDA issued a warning in 1982; only the oil of S. molle is GRAS [66-74].

408

3.5. EPAZOrjS/WORMSEED/CHENOPODIUM Epazote (alias Mexican tea) has been promoted in a number of English books and articles on Mexican cooking as adding an unusual flavor to foods and preventing flatulence from bean dishes [16, 17, 7577]; it is also known as paico in Peru [78]. Epazote, which is derived from the Nahutal epazotl, is Chenopodium ambrosioides L., alias Teloxys ambrosioides (L.) W. A. Weber [79-81]; this is the same species known as wormseed and yields wormseed oil (alias chenopodium oil). Wormseed oil is antifungal and anthelmintic (purgative to intestinal worms and amoebae), but the therapeutic dose is close to the minimum toxic level [82-85]. The active ingredient of C. ambrosioides is 6-100% ascaridole [66, 86-92]. The scant literature indicates that epazote is C. ambrosioides var. ambrosioides and relatively low in ascaridole; C. ambrosioides var. anthelminticum (L.) Gray, which is supposedly higher in ascaridole and thus called wormseed, is differentiated by leaves being more strongly toothed than the typical variety, the lower sometimes almost laciniate-pinattifid with mostly leafless spikes [93]. Our analysis by GC/MS of an epazote from Texas reveals 1.98±0.70% ascaridole, while two commercial wormseed/chenopodium oils have 16.20% and 43.10% ascaridole (Table 4). Chenopodium ambrosioides also contains isoascaridole and ascaridole 3,4-epoxide, which have unknown toxicity [94]. Wormseed oil causes skin and mucous-membrane irritation, headache, vertigo, nausea, vomiting, constipation, tinnitus, temporary deafness, diplopia and blindness, transient stimulation followed by depression of the central nervous system leading to delirium and coma, occasional convulsions, circulatory collapse due to vasomotor paralysis and sometimes pulmonary edema. Wormseed oil is also toxic to the kidneys and liver and haematuria, alburinuria and jaundice have been observed. Wormseed oil is irritating to the skin and not recommended for skin applications by IFRA. Only 1 tsp of wormseed oil has been reported to be fatal to a 14-month old baby, while a two-year old child died after being given 16 minims (0.947 cc) of oil over a period of 3 weeks [6, 73]. Leaves of C. ambrosioides have been reported to be carcinogenic in rats [95]. We note that C. ambrosoides is included in almost every book on poisonous plants [96, 97]. No parts of C. ambrosioides are GRAS; in 1974 the Council of Europe included chenopodium oil in the list of natural flavoring substances not permitted [6, 73]. Chenopodium ambrosioides does have two chemical forms which may be nontoxic. These chemovarieties may have either 26-43% transpinocarveol or 62-65% pinocarvone, giving them a fragrance reminiscent

409

of typical balm-of-Gilead [Cedronella canariensis (L.) P. Webb & Berthel.] [98, 99]. Future exploration of this species as a culinary herb should investigate these selections. TABLE 4. THE ESSENTIAL OIL OF 0.5 m FRESH FLOWERING TOPS OF AN EPAZOTE FROM TEXAS (DOVI9551) AND TWO COMMERCIAL WORMSEED/ CHENOPODIUM OILS. Epazote Wormseei d Chenopdium Compound OU & Oil Yield Mean± SD % (N = 3) Oil oil yield 0.39+0.06 dimethyl sulfide 0 0.01+0.01 0 tricyclene 0 0.22+0.18 0 a-pinene 3.18 0.82±0.72 0 a-fenchene 0 0.02±0.03 0 0 0.14±0.06 0 camphene p-pinene 0 0.18±0.05 0 sabinene 0.01+0.01 0 0 0.0310.06 0 5-3-carene 0 0.01±20.02 myrcene 0 0 a-phellandrene 0.01±0.02 0 0 12.24 a-terpinene 17.54±2.16 12.71 limonene 42.3219.31 11.56 5.73 P-phellandrene 0.0910.02 0 0 0 1,8-cineole 0 6.32 1.16 y-terpinene 0.5810.10 0.09 18.22 12.78 ;7-cymene 8.0910.56 0 terpinolene 0 0.37 0 trans-limonenQ oxide 0.091010 0 0 benzaldehyde 0 0.39 fra/J5-dihydrocarvone 0.0910.15 0 0 0.38 carvone 2.2810.72 0.44 0 fran^-carveol 1.0610.58 0 0 CJ5-carveol 0.2510.43 0 43.10 ascaridole 1.9810.70 16.20 0.15 eugenol 0 0 0.17 thymol 0.0210.04 0.23 0.34 carvacrol 0.0410.07 0.47

410

4. ACKNOWLEDGMENTS Analytical research was supported by the State of Delaware and the Cooperative State Research, Education, and Extension Service (#801-15-010). Thanks are also extended to the Professional Development Committee and the Department of Agriculture & Natural Resources, Delaware State University for support of travel.

5. REFERENCES 1. P. B. Hutt and R. A. Merrill, Food and Drug Law: Cases and Materials. 2nd ed. Foundation Press, Westbury, NY, 1991. 2. J. Stofberg, Perf. Flav., 17(5) (1992) H I . 3. G. A. Burdock (ed.), Fenaroli's Handbook of Flavor Ingredients. 3rd ed. CRC Press, Boca Raton, FL, 1995. 4. A. Y. Leung and S. Foster, Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics. 2nd ed. John Wiley & Sons, New York, 1996. 5. Research Institute for Fragrance Materials, RIFM 1992 Cross Reference List. RIFM, Hackensack, NJ, 1992. 6. International Fragrance Association, Code of Practice. IFRA, Geneva, 1974-date. 7. H. Kaiser and W. Lang, Deutsche Apotheker-Zeitung/Siiddeutsche Apotheker-Zeitung, 91 (1951) 163. 8. E. von Rudloff, Recent Adv. Phytochem., 2 (1969) 127. 9. E. W. Boberg, E. C. Miller, J. A. Miller, A. Poland, and A. Liem, Cancer Res., 43 (1983) 5163. 10. L. G. French, J. Chem. Educ, 72 (1995) 484. 11. D. L. Heikes, J. Chromatogr. Sci. ,32 (1994) 253. 12. D. P. Kamden and D. A. Gage, PI. Med., 61 (1995) 574. 13. A. O. Tucker, M. J. Maciarello, and C. E. Broderick, In "Spices, Herbs and Edible Fungi" G. Charlambous, Ed. Elsevier, Amsterdam, 1994, pp. 595-604. 14. R. W. Wiseman, T. R. Fennell, J. A. Miller, and E. C. Miller, Cancer Res., 45 (1985) 3096. 15. E. Cohen, People, 48(6) (1997) 109. 16. L. Hutson, The Herb Garden Cookbook. Gulf Publ. Co., Houston, 1992. 17. L. Hutson, Fine Card., 23 (1992) 52. 18. Republic of Tea, The Book of Tea & Herbs. Cole Group, Santa Rosa, CA, 1993. 19. W. Burger, Fieldiana, 35 (1971) 1. 20. P. C. Standley, Contr. U. S. Herb., 23 (1961) 1.

411

21. 22. 23. 24.

L. G. Joly, Econ. Bot., 35 (1981) 383. O. Castro C. and L. J. Poveda A., Ing. Ci. Quim., 7(1-2) (1983) 24. J. F. Ciccio, Ing. Ci. Quim., 15 (1995) 39. M. P. Gupta, T. D. Arias, N. H. Williams, R. Bos, and D. H. E. Tattje, J. Nat. Prod., 48 (1985) 330. 25. R. Hansel, A. Leuschke, and A. Gomez-Pompa, Lloydia, 38 (1975) 529. 26. R. G. Buttery, D. R. Black, D. G. Guadagni, L. C. Ling, G. Connolly, and R. Teranishi, J. Agric. Food Chem., 22 (1974) 773. 27. R. J. L. Goralka and J. H. Langenheim, Biochem. Syst. EcoL, 23 (1995) 439. 28. R. E. Kepner, B. O. Ellison, M. Breckenridge, G. Connolly, S. C. Madden, and C. J. MuUer, J. Agric. Food Chem., 22 (1974) 781. 29. B. M. Lawrence, A. C. Bromstein, and J. H. Langenheim, Phytochemistry, 13 (1974) 2009. 30. J. T. MacGregor, J. T., L. L. Layton, and R. G. Buttery, J. Agric. Food Chem., 22 (1974) 777. 31. M. E. Drake and E. T. Stuhr, J. Amer. Pharm. Assoc, Sci. Ed., 24(3) (1935) 196. 32. B. Pech and J. Bruneton, J. Nat. Prod., 47 (1984) 390. 33. M. Bittman, New York Times, May 17 (1995) C3. 34. D. M. Brenner, In "New Crops" J. Janick and J. E. Simon, Eds. John Wiley and Sons, NY, 1993, pp. 322-328. 35. J. F. Morton, In "Progress on Terrestrial and Marine Natural Products of Medicinal and Biological Interest" J. M. Pezzuto, A. D. Kinghorn, H. H. S. Fong, and G. A. Cordell, Eds. Amer. Bot. Council, Austin, 1991, pp. 34-38. 36. N. X. Dung, L. D. Moi, L. D. Cu', and P. A. Leclercq, J. Essential Oil Res., 7 (1995) 429. 37. H. Kameoka and K. Nishikawa, Nippon Nogei Kagaku Zasshi, 50 (1976) 345. 38. Y. Koezuka, G. Honda, and M. Tabeta, Shoyakugaku Zasshi, 38 (1984) 238. 39. B. M. Lawrence, Essential Oils 1988—-1991. Allured Publ. Corp., Carol Stream, IL, 1991. 40. B. M. Lawrence, In "Advances in Labiate Science" R. M. Harley and T. Reynolds, Eds. Royal Botanic Gardens, Kew, 1992, pp. 399-436. 41. P.-H. Yeh, Perf. Essential Oil Rec, 51 (1960 293. 42. S. Zhao, L. Quan, Z. Kang, and Y. Bian, Yaowu Fenxi Zazhi, 12 (1992) 206. 43. S. Zhao, Z. Zhu, Y. Feng, L. Quan, and L. Xue, Tianran Chanwu Yanjiu Yu Kaifa, 5(3) (1993) 8. 44. L. Zhu, Y. Li, B. Li, B. Lu, and W. Zhang, Aromatic Plants and Essential Constituents. Supplement 1. Peace Book Co., Hong Kong, 1995.

412

45. B. J. Wilson, J. E. Garst, R. D. Linnabary, and R. B. Channell, Science, 197 (1977) 573. 46. W. C. Wilson, W. C , J. Simon, and J. E. Garst, J. Animal Sci., 68 1990) 1072. 47. R. G. Breeze, I. E. Selman, and A. Wiseman, Bovine Practitioner, 1978 (1978) 75. 48. R. G. Breeze, W. W. Laegreid, W. M. Bayly, and B. J. Wilson, Equine Veterin. J., 16 (1984) 180. 49. J. W. Coggeshall, P. L. Lefferts, M. J. Butterfield, G. R. Bernard, F .E. Carroll, N. A. Pou, and J. R. Snapper, Amer. Rev. Resp. Dis., 136 (1987) 1453. 50. L. A. Kerr, J. Johnson, and G. E. Burrows, Veterin. Human Toxicol., 28 (1986) 412. 51. R. D. Linnabary, A. Fetcher, and R. Sigler, Modern Veterin. Practice, 65 (1984) 849. 52. R. D. Linnabary, B. J. Wilson, J. E. Garst, and M.A. Holscher, Veterin. Human Toxicol., 20 (1978) 325. 53. W. A. Phillips, and D. Von Tungein, Modern Veterin. Practice, 67 (1986) 252. 54. Y. Koezuka, G. Honda, and M. Tabeta, PI. Med., 1985 (1985) 480. 55. T. Kanzakland, and S. Kimura, Contact Dermatitis, 26 (1992) 55. 56. N. Okazaki, M. Matsunaka, M. Kondon, and K. Okamoto, Skin Res., 24 (1982) 250. 57. J. Harada, In "Proceedings of the 11th Asian-Pacific Weed Science Society Conference, Taipei, Republic of China, November 29December 5" Weed Society of the Republic of China, 1987, pp. 601-605. 58. K. R. Kuebel and A. O. Tucker, Econ. Bot., 42 (1988) 413. 59. K. Kasahara, C. Osawa, and K. Nishibori, Fish. Sci., 62 1996) 838. 60. G. Honda, K. Koga, Y. Koezuka, and M. Tabata, Shoyakugaku Zasshi, 38 (1984) 127. 61. G. Honda, Y. Koezuka, W. Kamisako, and M. Tabata, Chem. Pharm. Bull., 34 (1986) 1672. 62. R. Kang, R. Helma, M. J. Stout, H. Jaber, Z. Chen, and T. Nakatsu, J. Agric. Food Chem., 40 (1992) 2328. 63. C. L. J. Opdyke and C. Letizia, Food Chem. Toxicol., 20 (1982) 633. 64. A. Sugaya, T. Tsuda, and T. Obuchi, J. Pharm. Soc. Jap., 101 (1981) 642. 65. C. L. J. Opdyke and C. Letizia, and A. M. Api, Food Chem. Toxicol., 26 (1988) 273. 66. L. Bauer and G. A. de Assis Brasil e Silva, Revista Brasil. Farm., 54 (1973) 240.

413

67. R. A. Bernhard, Y. Shibamoto, K. Yamaguchi, and E. White, J. Agric. Food Chem., 31 (1983) 463. 68. A. Hicks, Petits Propos Culinaires, 10 (1982) 11. 69. M. K. Jain, B.-Z. Yu, J. M. Rogers, A. E. Smith, E. T. A. Boger, R. L. Ostrander, and A.L. Rheingold, Phytochemistry, 39 (1995) 537. 70. B. M. Lawrence, Perf. Flav., 9(5) (1984) 65. 71. M. Maffei and F. Chialva, Flavour Fragrance J., 5 (1990) 49. 72. J. F. Morton, Econ. Bot., 32 (1978) 353. 73. C. L. J. Opdyke, Food Cosmetics Toxicol., 14 (1976) 659. 74. E. Stahl, Deutsche Apotheker Zeitung 122 (1982) 337. 75. D. Kennedy, The Art of Mexican Cooking. Bantam Books, New York, 1989. 76. L. P. Muniz, New York Times, January 10 (1990) CI, C4. 77. P. Quintana, The Taste of Mexico. Steward, Tabori and Chang, New York, 1986. 78. S. Coe, America's First Cuisines. University of Texas Press, Austin, 1994. 79. R. E. Bond, Petits Propos Culinaires, 34 (1990) 45. 80. G. Palomino H., M. Segura D., R. Bye, and R. Mercado R, SouthW. Naturalist, 35 (1990) 351. 81. W. A. Weber, Phytologia, 58 (1988) 477. 82. W. H. Lewis and M. P. F. Elvin-Lewis, Medical Botany: Plants Affecting Man's Health. John-Wiley & Sons, New York, 1977. 83. S. M. Narva, In "Plants and people" Benes, P., Ed. Dublin Seminar for New England Folklife Annual Proceedings 1995. Boston Univ., Boston, 1996, pp. 78-91. 84. E. Okuyama, K. Umeyama, Y., Y. Saito, M. Yamazaki, and M. Stake, Chem. Pharm. Bull., 41 (1993) 1309. 85. P. W. Pare, J. Zajicek, V. L. Ferracini, and I. S. Melo, Biochem. Syst. Ecol., 21 (1993) 649. 86. J. de Pascula-T., I. S. Bellido, C. Torres, and M. A. Perez, Rivista Ital. Essenze Profumi, 62 (1980) 123. 87. E. Guenther, The Essential Oils. Vol. 6. Van Nostrand Reinhold Co., New York, 1952. 88. J. Rochat, J. Alary, and C. Luu Due, Ann. Pharm. Fran?., 30 (1972) 577. 89. G. Riicker, M. Neugebauer, and A. Kiefer, Pharmazie, 50 (1995) 601. 90. J. Verghese, On Essential Oils. Synthite Industrial Chemicals Private, Kolenchery, India, 1986. 91. G. S. Weiland, L. B. Broughton, and J. E. Metzger, Univ. Maryland Agric. Exp. Sta. Bull. 384 (1935) 1. 92. L. Zhu, Y. Li, B. Li, B. Lu, and N. Xia, Aromatic Plants and Essential Constituents. Peace Book Co., Hong Kong, 1993.

414

93. A. Gray, Manual of the Botany of the Northern United States. 5th ed. Ivison, Blakeman, Taylor and Co., New York, 1867. 94. M. A. Johnson and R. Croteau, Arch. Biochem. Biophys., 235 (1984) 254. 95. G. J. Kapadia, E. B. Chung, B. Ghosh, Y. N. Shukla, S. P. Basek, J. F. Morton, and S. N. Pradhan, J. Natl. Cancer Inst., 60 (1978) 683. 96. J. M. Kingsbury, Poisonous Plants of the United States and Canada. Prentice-Hall, Englewood Cliffs, NJ, 1964. 97. W. C. Muenscher, Poisonous Plants of the United States. Rev. ed. Macmillan Co., New York, 1951. 98. L. Sagrero-Nieves and J. P. Bartley, J. Essential Oil Res., 7 (1995) 221. 99. K. Umemoto, J. Agric. Chem. Soc. Jap., 52 (1978) 49.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

415

Influence of preparation on the aroma compounds of oatmeal porridge. Michael J. Morello The Quaker Oats Company, John Stuart Research Laboratories, 617 West Main Street, Barrington, XL 60010-4199, USA

Abstract Volatile compounds isolated from oatmeal porridge are discussed as a function of preparation. Porridge was prepared by both the traditional stove top and microwave oven procedures. Supercritical carbon dioxide was used to extract the volatiles which were then analyzed by GC/MS and GC-NPD. Relative concentrations of carbonyl and nitrogen heterocyclic compounds were found to change as a function of preparation time and procedure.

1. INTRODUCTION The flavor of oats like other cereal products is developed through heating (i). Unlike other cereals, oats typically undergo hydro-thermal treatment to inactivate lipolytic enzymes prior to use for human consumption (2). This treatment is required due to high relative Upid contents (5-9%; >70% unsaturated) (3) and high activity of Upolytic enzymes (4). Although, hydrothermal treatment inhibits enzymatic oxidation, the unsaturated lipids in oats remain susceptible to autoxidation. Therefore, it is not surprising that many of the investigations of oat flavor have focused on the secondary products of lipid oxidation (5-10). In conjunction with these investigations some authors (7,8) have noted signiJBcant concentrations of Maillard reaction products. Based on these studies the volatile compounds from oats fall into two broad categories: secondary products of Upid oxidation and Maillard Reaction products. In addition, there have been some reports on the contribution of non-volatile compounds to the taste of oats (11,12). In the earliest reports of volatUes from oat flakes Hrdlicka and Janicek (13,14) reported aldehydes, 2-alkanones, and amines. Heydanek and McGorrin reported the volatile compounds in oat groats (dehuUed oats) prior to enzyme inactivation (5), rancid oat groats (6), oat flakes (7) and in porridge (cooked oat flakes). In dried groats they found volatile

416 compounds varied with the isolation procedure. They reported mainly terpenes, alkylbenzenes, and hexanal in dried groats. When groats were hydrated prior to isolation, the levels of alcohols and aldehydes increased significantly. In rancid groats, the authors reported aldehydes, enals, dienals and dienones. They attributed these compounds to autoxidation and suggested pentanal, hexanal, and 2,4-decadienal were indicators of oat oxidation. In porridge, the authors reported that levels of aldehydes, pyrazines, and thiazoles increased relative to the starting oat flakes; however, no quantitative data was given. Fors and Schlich (8) investigated compounds isolated from oils extracted fi'om unroasted and roasted oats both before and after milling. Using Principle Component Analysis they reduced the data set to a few representative compounds which included decadienal and trimethyl pyrazine. Guth and Grosch (9) used aroma extract dilution analysis to identify hexanal, octanal, ^mMS-2,3-epoxyoctanal, iraAis,^mAis-2,4-nonadienal, trans,^rans-2,4-decadienal, iraAis,-4,5-epoxy-irans-2-decenal, 4-hydroxy-2-nonenoic acid lactone, and vanillin as having the highest flavor dilution factors in the volatile isolate of an oat extrusion product. Molteberg et al (10) correlated aldehydes, enals, and pentyl furan to chemical and sensory data for raw and heat-treated oat flours. In the heat treated samples, the highest correlations were found for grass flavor, raw odor and flavor. Importantly, oat odor and flavor were negatively correlated to the volatiles investigated. In addition, nonanal was the compound with the highest correlation to flavors for the heat treated flours. The attributes which nonanal was correlated to were paint flavor, paint odor, acidity, and grass flavor. Motleberg et al also investigated the correlation between phenolic compounds (11) and sensory quality. They determined that jo-coumaric acid, vanillin, p-hydroxybenzaldehyde, and coniferyl alcohol were correlated to high levels of rancidity and bitterness and were negatively correlated to freshness, oat odor, and oat flavor. Biermann and Grosch (12) identified 9-hydroxy-^mn.s,ds-10,12-octadecadienoic-1 '-monoacylglycerol and 13-hydroxy-cis,^raMS-9,11-octadecadienoic -V- monoacylglycerol as imparting bitter taste to oats. The intent of this study was to determine how volatile compounds from oats change as a result of porridge preparation, and to determine how diffierent preparation procedures influence changes in porridge volatiles. Based on the previous work on oat flavor, relative concentrations of hexanal, trans-2y3epoxyoctanal, nonanal, ^mMS,^mMS-2,4-nonadienal, ^ra/is,^mns-2,4-decadienal, 4-hydroxy-2-nonenoic acid lactone, and vanillin would be monitored as being indicative of oxidation; and IC through 4C alkylpyrazines would be monitored as indicative of Maillard reactions. Criteria used for selecting the isolation procedure were that [1] it be mild in order to avoid flavor generation during isolation, [2] it should be ofi^-Une so that isolates could be analyzed by both GC/MS and GC-NPD, and [3] isolation could be done on multiple samples simultaneously. Supercritical fluid

417

extraction met these criteria (IS). Taylor has summarized the steps required to develop a successful SFE procedure (16), and Kerrola has reviewed flavor specific applications of SFE (17).

2. MATERIALS AND METHODS. 2.1. Porridge preparation Oat flakes, 5-minute cooking variety oatmeal, were purchased locally. Stove top porridge was prepared by bringing the water and salt to a rolling boil, adding the oatmeal, returning to a gentle boil for 5 minutes, removing fi:om the heat, and standing covered for 5 minutes. Microwave porridge was prepared by combining the water, salt, and oatmeal and heating on high power (700 watt) for 165 seconds. Proportions of water: salt: oatmeal were 237 gm: 0.43 gm: 42 gm. 2.2. Sample preparation Oat flakes were ground in a high speed blender. Water was added to the ground flakes and the mixture was ground once more. Hydromatrix (Varian; Harbor City, CA) was added to the ground oat-water mixture and the mixture was ground again. The free flowing material was then added to the extraction cells. The proportions of oatmeal: water: Hydromatrix were 3: 1: 1. Equal portions of porridge and Hydromatrix were combined and mixed in a high speed blender. The free flowing powder was then added to the extraction cells. 2.3. Supercritical fluid extraction The extraction system used for this work was a Dionex SFE-703 (Sunnyvale, CA). The extractor had eight parallel horizontal extraction positions. The extraction cells were 16 mL (100 x 14 mm) for oat flakes and 32 mL (200 X 14 mm) for porridge. The extraction medium was carbon dioxide with helium head-pressure (SFC/SFE grade Air Products; Allentown, PA). Extractions were carried out at 200 atm. and 50°C. Linear fused-silica restrictors were heated to 125°C. Volatiles were trapped in methylene chloride (20 mL): methanol (0.2 mL) using Dionex dual chamber vials (18) chilled to 4°C. Oat flake samples were extracted for 100 minutes or roughly 3 cell volumes. Porridge samples were extracted for 180 minutes or roughly 2.7 cell volumes. It was necessary to replenish the trapping solvent with an additional 10 mL after the first 120 minutes of extraction. Extracts from two cells were combined to form one isolate, and all extractions were carried out in triplicate. Post extraction, the dilute analyte (isolates from two cells), along with 20 uL internal standard (2-methoxy-3-methypyrazine (5.43 ng/uL) and 2heptanone (0.42 ug/uL)), were transferred to a micro-concentrator equipped with a three bulb Snyder column. An ebulator was added to the concentrator

418

and the volume was reduced to ca. 1.5 mL. Heating was done with a tube heater (Kontes; Vineland, NJ) held at 90®C. Analytes were further concentrated to 0.1 mL under a stream of dry nitrogen just prior to analysis by GC/MS and GC-NPD. 2.4. Gas Chromatography-Nitrogen Phosphorous Detection The GC was a Hewlett-Packard 6880A equipped with a 7673A ALS and nitrogen phosphorous detector. SpUtless injection, 275°C, with the purge valve activated at 1.0 minutes. The column was a J&W Scientific DB-5ms capillary (30 m, 0.32 mm, film thickness 1.0 ^m) programmed: 40**C for 5 min, S'^C/min to 215®C, 7.0 min, initial flow 1.6 mL/min. The detector was operated at constant column + makeup flow at 275°C. A HP ChemStation Data System (Rev. A.03.03) was used to analyze the data. Pyrazines were identified by comparison to retention times and indices from the literature (J,19) and reference compounds: methylpyrazine, 2,5-dimethylpyrazine, 2,3-dimethylpyrazine, ethylpyrazine, trimethylpyrazine, 2-ethyl-5/6-methylpyrazine, 2-ethyl-3methylpyrazine, and tetramethylpyrazine (Pyrazine Specialties). Concentrations were calculated relative to the 2-methoxy-3-methypyrazine (Pyrazine Specialties) internal standard. 2.5. GC/MS Total ion chromatograms were collected using an HP 5980A GC interfaced to an HP 5988A MS controlled by an HP 59872 RTE-A data system. The column was identical to that used for GC-NPD. Conditions: Injector 275**C; Interface 240°C; Source 200°C; Oven program: 40°C for 3 min, 3°C/min to 160°C, 10°C/min to 270^C. Initial column flow 1.88 mL/min. Split ratio 10:1. Oxidation products were identified by comparing their mass spectra (70 eV) and retention indices to reference standards hexanal, nonanal, trans,trans-2,4nonadienal, ^raMS,^rans-2,4-decadienal, and vanillin; and published data (^rans-2,3-epoxyoctanal and 4-hydroxy-2-nonenoic acid lactone) (9). Concentrations were calculated relative to the 2-heptanone (Aldrich) internal standard.

3. RESULTS AND DISCUSSION 3.1. Results based on the weight of oat flakes Comparing the data from oat flakes and the porridges based on the starting weight of oat flakes indicates two processes are occurring during preparation: volatile loss and volatile generation. Results for oxidation products are given in Table 1, and results for nitrogen heterocyclic compounds are given in Table 2. Comparing the relative concentration of compounds found in the two porridges to those in the starting oatmeal indicates loss and generation of

419 volatiles are different for the two preparation procedures. For porridge prepared on the stove top, hexanal, 4-hydroxy-2-nonenoic acid lactone, methylpyrazine and dimethylpyrazine were lower in relative concentration; while, nonanal, ^raMS,imMS-2,4-decadienal, vanillin, 3C pyrazines, and 4C pyrazines were greater in relative concentration than the starting oatmeal. For porridge prepared in the microwave reductions in relative concentrations of hexanal, methylpyrazine, and dimethylpyrazine were not as great as for the stove top sample; however, there were also reductions in relative concentrations of ^rans-2,3-epoxyoctanal and iraMS,^ra/is-2,4-nonadienal. As with the stove top porridge there were increases in the relative concentrations of trans,trans-2y4' decadienal, 3C pyrazine, and 4C pyrazine in the microwave prepared porridge.

Table 1 Relative Concentration of Oxidation Products ug/gm Oat Flakes Flakes Porridge RI Stove Top 1.115 0.753 800 Hexanal 0.072 0.081 1095 trans-2,3-Epoxyoctanal 0.312 0.779 1101 Nonanal 0.029 0.026 1218 trans,trans-2,4-Nonadienal 0.380 0.123 1319 trans, trans-2,4-Decadienal 0.018 0.046 1343 4-Hydroxy-2-nonenoic acid lactone 1.012 1414 Vanillin 0.631 All concentrations are the average of three replicates

Microwave 1.018 0.042 0.699 0.007 0.232 0.000 0.630

Table 2 Relative Concentration of Pyrazines1 ng/gm Oat Flakes Porridge Oat Flakes Stove Top Microwave Pyrazine 14.94 IC 17.20 10.43 18.87 2C 26.46 12.46 3C 10.26 12.47 12.57 4C 8.86 9.65 11.66 All concentrations are the average of three replicates Specific pyrazines isomers were not identified using NPD

These results indicate that preparation conditions do influence the composition of volatile compounds in oatmeal porridge. Observation that higher molecular weight pyrazines increase in concentration during porridge preparation is consistent with the earlier work of Heydanek and McGorrin (7). The differences in volatile concentrations resulting from the two preparation

420

conditions can not be explained on the basis of this work. However, there are four possibly interrelated factors that may have lead to the differences: [1] the point during preparation that oatmeal combined with the water, [2] different cooking times, [3] changes in the viscosity of the porridge due to beta-glucan release and starch gelatinization, and [4] the temperature profile during cooking. 3.2. Results based on the weight of porridge A second way to evaluate differences resulting from the two preparation methods is on the basis of the porridge weight. It is important to evaluate these differences as different amounts of water were lost during the two preparations: stove top 19% and microwave 5%. Relative concentrations of oxidation products and nitrogen heterocycles, based on porridge weight, are given in Table 3 and Table 4. As seen in Table 3, with the exception of hexanal the concentration of oxidation products is higher in the stove top than in the microwave porridge. Table 4 shows that relative concentrations of methyl and dimethypyrazines are greater in the microwave porridge; but, the concentration of C3 pyrazines is greater for the stove top porridge. It may be significant that the C3 pyrazine has a greater relative concentration in the stove top porridge. In Fors summary (20), flavor thresholds for the C3 pyrazines are approximately one to two orders of magnitude lower than those for methyl and dimethylpyrazines. However, it should be noted that all pyrazines concentrations are below their literature flavor thresholds.

Table 3 Relative Concentration of Oxidation Products ug/gm Porridge Porridge Stove Top Microwave

Hexanal
0.158 0.017 0.163 0.006 0.079 0.004 0.212

All concentrations are the average of three replicates

0.174 0.007 0.119 0.001 0.039 n.d. 0.102

421

Table 4 Relative Concentration of Pyrazines ng/gm Porridge Porridge Microwave Stove Top Pyrazine 2.56 2.18 IC 3.21 2.61 2C 2.13 2.61 3C 1.97 2.03 4C AQ concentrations are the average of three replicates Specific pyrazines isomers were not identified using NPD

It is interesting to note that differences do exist between the volatile compounds isolated fi:om porridge prepared in different ways. However, these results in the absence of corresponding sensory data are insufficient to make an assessment of how these changes influence the flavor quality and strength of oatmeal porridge. 3.3. S F E procedure Although not discussed, extractions using 2, 3, & 4 ceU volumes of supercritical carbon dioxide were evaluated before determining 3 ceU volumes provided the greatest overall recoveries of the desired analytes. It should also be noted that palmitic and stearic acids were tentatively identified as the substances recovered in greatest quantities under these extraction conditions. This is not surprising as the SFE conditions chosen fall within the within the "fat band" described by Gere et. al (21). The alumina added to the extraction cells helped to reduce recovery of these fatty acids and chromatography conditions were selected to disregard these compounds. Finally, silicon based artifacts from the Hydromatrix were recovered with the volatiles of interest. Future work should include comparisons of other desicants. In spite of these Hmitations, supercritical carbon dioxide extraction provided mild thermal conditions, the isolates could be analyzed by both GC/MS and GC-NPD, and the time from porridge preparation to chromatography was ca 5 hrs for 4 samples (8 cells) or 1.25 hr/sample. Therefore, SFE is a viable procedure for isolation of volatile compounds from cereal products.

4. CONCLUSIONS Results from this investigation demonstrate that the preparation procedure affects volatile compounds in oatmeal porridge. Further research is needed to determine how these compounds correlate to sensory attributes and how to manipulate the starting oat flakes in order to provide optimum flavor in porridge.

422

5. REFERENCES 1. W. Grosch and P. Schieberle, Cereal Chemistry, 74 (1997) 91. 2. W. GanBmann and K. Vorwerk, in The Oat Crop, R.W. Welch (ed.), Chapman & Hall, London (1995) 369. 3. M.R. Sahasrabudhe, J. Am. OU Chem. Soc, 56 (1979) 80. 4. V.L. Youngs, in Oats: Chemistry and Technology, F.H. Webster (ed.), American Association of Cereal Chemists, Inc.; St. Paul, MN (1986) 205. 5. M.G. Heydanek, and R.J. McGorrin, J. Agric. Fd. Chem., 29 (1981) 1903. 6. M.G. Heydanek, and R.J. McGorrin, J. Agric. Fd. Chem., 29 (1981) 950. 7. M.G. Heydanek, and R.J. McGorrin, in Oats: Chemistry and Technology, F.H. Webster (ed.), American Association of Cereal Chemists, Inc.; St. Paul, MN (1986) 335. 8. S.M. Fors, and P. SchHch, in Thermal Generation of Aromas, T.H. Parliment, R.J. McGorrin, and C-T. Ho (eds.), ACS Symposium Series 409, American Chemical Society, Washington,DC (1989) 121. 9. H. Guth, and W. Grosch, Z. Lebensm Unters Forsch. 196 (1993) 22. 10. E. L. Molteberg, E.M. Mangus, J.M. Bjorge, and A. Nilsson, Cereal Chem., 73 (1996) 579. ll.E.L. Molteberg, R. Solheim, L.H. Dimberg, and W. Frolich, J. Cre. Sci., 24 (1996) 273. 12.U. Biermann and W. Grosch, Z. Lebensm Unters Forsch, 169 (1979) 22. 13. J. HrdHcka and G. Janicek, Nature, 201 (1964) 1223. 14. J. Hrdlicka, and G. Janicek, Nature, 204 (1964) 1201. 15.M.J. MoreUo, in Thermally Generated Flavors: Maillard, Microwave, and Extrusion Processes, T.H. ParHment, M.J. MoreUo, and R.J. McGorrin (eds.), ACS Symposium Sereis 543, American Chemical Society: Washington, DC (1994) 95. 16.L.T. Taylor, Supercritical Fluid Extraction John Wiley & Sons, Inc.,New York, NY 1996 17.K. Kerrola, Food Rev. Int., 11 (1995) 547. 18.N.L. Porter, A.F. Rynaski, E.R. CampbeU, M. Saunders, B.E. Richter, J.T. Swanson, R.B. Nielson, and B.J. Murphy, J. Chromatogr. Sci., 30 (1992) 367. 19.R.P. Adams, Identification of Essential Oils by Gas Chromatography/ Mass Spectroscopy, AUured Publishing Corp., Carol Stream, IL, USA 1995 20. S. Fors, in The Maillard Reaction in Foods and Nutrition, G.R. Waller, and M.S. Feather (eds.), ACS Symposium Series 215, American Chemical Society, Washington, DC (1983) 185. 21.D.R. Gere, C.R. Knipe, P. Castelli, J. Hedrick, L.G. Randall Frank, H. Schulenberg-ScheU, R. Schuster, L. Doherty, J. Orolin, and H.B. Lee, J. Chromatogr. Sci., 31 (1993) 246.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

Characterization

of f l a v o r of t e a p r o d u c e d d i f f e r e n t

Miyuki K A T O ' a n d M a s a s h i

423

tea

area

OMORl'

^Faculty of Education KAGAWA University. T a k a m a t s u KAGAWA 760 J a p a n . ^Department of Food Science Otsuma Wumen's University. 12 Sanban-cho Chiyoda-ku Tokyo 102 J a p a n Ab 8 t r a c t

India, Sri Lanka and China are major, well known tea producing a r e a s . In recent years, the production of Kenian and I n d o n e s i a n tea also have been increased. In this study, a u t h o r s were comparatively found out about flavor components of these t e a s produced different tea a r e a . (l)It was showed high contents of polyphenol in Kenian tea, high contents of catechins in A u s t r i a n and Kenian t e a . (2)The correlations among the scores (axes 1 and axes 2) obtained by the quantification system III and p e a k s in the gaschromatograms obtained by analyzing the flavor components were determined. The correlation coefficients of the Axes 1 and 2 showed t h a t possible to do group with the flavor components in each black tea s a m p l e . 1.INTRODUCTION

Well-known black tea-producing a r e a s include Darjeeling and Assam in I n d i a , K e e m u n in China, Uva,Dimbula and Nuwara Eliya in Sri L a n k a . A number of studies have been performed on the aroma components and coloring components of these black tea products. Black tea products grown in Indonesia and Kenya have been frequently brought to the m a r k e t in recent years. There h a s been an increase in the production and exportation of black tea in these a r e a s and Southeast Asia. Gianturco et al. and Y a m a n i s h i et al. identified a number of aroma components [1-3]. On the black tea while Takino et al.[4] determined the structure of theaflavin (TF) as a coloring component. Many workers have studied the structure of t h e a r u b i g i n (TR), however no r e m a r k a b l e finding has been e s t a b l i s h e d since Roberts et al. used the name "TR" to refer to a mixture of compounds[5,6]. Since information of black tea aroma compounds formation is lacking, we studied the formation mechanism of black tea aroma components by using tea species grown in J a p a n . We have reported t h a t /3 -carotene plays an i m p o r t a n t role in the formation of the a r o m a [ 7 , 8 ] . In this study, we have p r e s e n t clarify information to the flavor composition of black tea products other t h a n those obtained in the m a i n producing a r e a s . We have performed m u l t i v a r i a t e analysis(MVA) on aroma components, the MVA was able to d e m o n s t r a t e d differences in aroma components t h a t were dependent the producing a r e a s .

424 2.MATERIAL AND M E T H O D S 2.1.Samples The s a m p l e s utilized were black t e a s Bangladesh, Myanmar, Viet Nam and A u s t r a l i a .

produced

in Kenya,

Turkey,

2.2. Sensory test Sample were t e s t e d for quality using the tea evaluation method[9]. Two hundred mL of boiling w a t e r was added to 3g of each sample in a cup, and allowed to s t a n d for 3 min. The extract was filtered and then e v a l u a t e d . The sensory t e s t s were carried out and discussed by skilled p a n e l i s t s . 2 . 3 . D e t e r m i n a t i o n a n d a n a l y s i s of b l a c k t e a e x t r a c t color Black tea extracts were prepared in the same m a n n e r as follows. One hundred mL of each extract was re-extracted with the same amount of methyl isobutyl ketone (MIBK). The MIBK layer was s e p a r a t e d from the aqueous layer. The aqueous layer was further extracted with the same amount of butanol (BuOH), and the BuOH layer was s e p a r a t e d . Absorption of each extract at 380nm was recorded. The polyphenol content was determined by the method of I w a s a et al [10]. The content of theaflavin was determined by the method of Anan et al [11]. For theaflavin, 1 g of black tea leaves was extracted with 50 mL of 20% acetone containing 0.6 mg of puropurogallin for 1 h; the volume of the acetone layer was brought to 100 mL, High performance liquid chromatography (HPLC) was performed with Innersil ODS-2 column (10 /xm, Gasukuro kogyo,4.6X250mm) for a n a l y s i s of theaflavin as described below. 2 . 4 . A n a l y s i s of a m i n o a c i d s Amino acids were analyzed according to the method of T s u s h i d a et al [12]., in which 1 g of sample was extracted with 50 mL of boiling w a t e r for 60 min. The resulting m a t e r i a l was made up to 100 mL with distilled water. The extract was p a s s e d through a 0.45 /x m millipore m e m b r a n e filter, and analyzed with a Shimadzu HPLC model LC-6A amino acid analysis system, using the o-phthalaldehyde method. 2 . 5 . A n a l y s i s of c a t e c h i n s Cathechins were analyzed according to the method of Ikegaya et al [13]. In this method,100 mg of sample was extracted with about 30 mL of 20% acetone for 60 min and filtered. The filtrate was extracted with about 100 mL of ethyl a c e t a t e before condensing. The remaing m a t e r i a l was made up to 10 mL with 25% acetonitrile. The extract was p a s s e d through a 0.45 M m millipore m e m b r a n e filter and t h e n injected into the Hitachi L-6200 i n s t r u m e n t .

425 2 . 6 . A n a l y s i s of a r o m a c o m p o n e n t s Black tea s a m p l e s were analyzed in accordance with the method of Horita et ai[14]. Analysis was preformed by a s i m u l t a n e o u s distillation and extraction (SDE) method, where distilled w a t e r was employed of a level 20 times t h a t of each s a m p l e . The d i s t i l l a t i o n was continued for 60 min. Gas-chromatography (GC) was performed from 50-180°C a t a heating rate of 2V> /min. A silica capillary column (0.35 mm i.d. X 50 m) coated with PEG-20M was used. Flame ionization detection (FID) was used as a detector. The content of components was expressed relative to the area of ethyl decanoate which was used as an i n t e r n a l s t a n d a r d and identified using a Shimadzu GC-MS QP lOOOA. 2.7.

Multivariate a n a l y s i s method Multivariate a n a l y s i s was performed by determining the area r a t i o of the aroma components of each sample with the use of "Multi Tokei System" m a r k e t e d by S h a k a i Joho Service. By using the area ratio of the aroma components, the characteristic vector value of each factor was first calculated and then the contribution ratio of the factor was calculated by using the vector value. Thus, axes 1 and 2, were determined and served as the main components for analyzing the vector p a t t e r n . For each tea sample, the factor load was calculated and the vector p a t t e r n plotted by referring the ordinate and the abscissa respectively as the first and second components. 3 . R E S U L T A AND D I S C U S S I O N 3.1.

Morphological characteristic The s a m p l e s of Kenya, Bangladesh, A u s t r a l i a , Turkey, Viet Nam and Myanmar all had a round shape owing to the Crush Tear Curl(CTC) production process. 3.2.

C h a n g e s in c o l o r i n g c o m p o n e n t s of b l a c k t e a e x t r a c t s Table 1 shows the composition of black tea coloring components, the solvent fractionation method and the theaflavin contents. The black tea s a m p l e of Kenya showed the g r e a t e s t content of the coloring components followed by those of Bangladesh, A u s t r a l i a , Turkey, Viet Nam and Myanmar. The s a m p l e of Sri L a n k a (Uva), showed the middle content of coloring compounds. It was considered t h a t the coloring components should be easily eluted in the case of the Kenya black tea which is t a k e n as milk tea in general. As the result of the solvent fractionation method, the Kenya black tea showed the largest content of the MIBK layer containing theaflavin (TF), while the samples of Turkey, Myanmar and Viet Nam showed small contents. Compared with the Uva black tea sample, the Kenya black tea sample showed a large content of the BuGH

426

layer containing thearubigin (TR) while the samples of other a r e a s showed small contents thereof. All of the s a m p l e s showed small polyphenol contents, though those of Kenya, Myanmar and Australia showed relatively large polyphenol contents. When examined by HPLC, the Kenya black tea sample showed a high TF content while those of Myanmar and Viet Nam showed s m a l l ones, similar to the d a t a of the MIBK layer obtained by the solvent fractionation method. The Kenya black tea sample showed a large TF content. Table 1 Color comparison of black tea infusion (mg/lOOg dry matter) Kenya conponents Infusion* MIBK layer* BuOH layer* Polyphenol contents** Contents of theaflavins*** Theaflavin Theaflavin-3-monogallate Theaflavin-3'-monogallate Theaflavin-3-3'digallate

Turkey Bangladesh

6.40 2.47 3.00 13.0

676.8 240.3 221.0 113.8 Black tea leaf was extracted with 200ml *0D at 380nm, **%

Myanmar Viet nam Australlia

7.2

5.26 1.39 1.92 12.2

4.94 1.45 2.48 15.0

122.2 56.8 69.4 65.6

193.3 113.3 127.1 154.1

404.0 316.0 237.0 242.0

3.84 0.48 2.00 10.0

3.97 0.52 1.46

67.0 55.0 56.2 72.6 of boiling water for 3min.

4.19 0.23 1.77

5.26 1.39 1.92

5.9

9.7

372.6 207.5 213.5 257.8

301.4 231.7 182.8 267.1

Sri Lanka

3.3.

C h a n g e s in a m i n o a c i d c o n t e n t s Table 2 shows the amino acid contents of the black tea grown in various region. The samples of each area were rich in t h e a n i n e , an amino acid contained in a large amount in t e a . The samples of Bangladesh, Myanmar, Australia and Kenya showed large total amino acid contents, while those of Viet Nam and Turkey showed smaller total amino acids content. The s a m p l e s of each area was rich in a s p a r t i c acid, serine and glutamic acid which are amino acids contained in a large amount in tea Kato et al[15]. 3.4.

C h a n g e s in c a t e c h i n c o n t e n t s Table 3 shows the results of catechin content determination. Compared with the s a m p l e s of the major producing a r e a s such as Uva and Assam, the black tea s a m p l e s employed in this study contained catechins in small a m o u n t s . In particular, the samples of Turkey, Bangladesh and Viet Nam showed small catechin contents. With respect individual catechin, these s a m p l e s were characterized by being rich in (-)-epigarocatechin gallate [(-)-EGCg] and (-)-epicatechin gallate [(-)-ECg].

427 Table 2 Contents of amino acids in black tea (mg/lOOg dry matter) Kenya Turkey Bangla- Myanmar Viet nam Aust trallia desh 160.5 101.9 147.7 170.5 146.5 Asp artic acid 284.8 33.4

Theronine Theanine Glutamic acid Isoleucine

824.0 200.1 10.5 18.4 54.4

Leucine Tyrosine

26.3 622.1 228.7 17.2

26,8 1487.4 162.2

19.5 40.5

13.6 11.5

16.6

26.5 1043.2

19.9 960.4

60.4 1084.4

176.9 24.9

165.8 16.9

171.6 17.1

27.9 25.7

18.5 31.9

Sri Lanka 24.8 10.6 284.9 44.7 3.2 14.8 20.8 34.4

Phenylalnine

26.4

176.1

50.0

71.1 43.9 66.2

54.4

26.6

Histidine

trace

6.4

trace

6.7

5.1

trace

2.1

Lysine

77.5

14.1

0.2

16.8

4.9

trace

8.9

Arginine

61.0

49.5

17.2

40.9

30.0

41.9

2.6

HPLC instrument:Shimadzu LC-6A. 0 -phthalaldehyde method Table 3 Contents of catechins in black tea (g/lOOg dry matter)

(-)-EGC (-)-EC (-)-EGCg (-)-ECg Total

Kenya

Turkey Bangradesh

0.722 1.092 1.900 1.994 5.707

trace trace 0.076 0.075 0.150

Myanmar

Vietnam Australlia

Sri Lanka

0.157 0.020 0.331 0.312 0.820

0.319 trace 0.282 0.193 trace 0.062 0.139 0.348 1.118 0.451 0.852 5.123 1.199 0.444 1.135 3.342 2.636 0.957 2.408 9.006 (-)-EGC:(-)-Epigallocatechin,(-)-EC:(-)-Epicatechin,(-)-EGCg:(-)-Epigallocatechingallate (-)-ECg:(-)-Epicatechin gallate HPLC instrument:Hitachi L-6200; coliumn:Hibar Lichrosorb RP-18(5Mm), (^4.0mm X 250mm; column temp.:30°C;mobile phase;acetonitrile:acetic acid :methanol:H2O(113:5:20:862);detector:280nm;flow rate:1.0ml 3.5.

C h a n g e s in aroma

components

T a b l e 4 shows the aroma volatile composition relative to ethyl d e c a n o a t e as

internal

standard.

Data indicated

t h a t the black tea

a r e a s contained less aroma components in general.

s a m p l e s in

these

Compared with t h e s a m p l e s

of the major producing a r e a s , t h e s e black tea s a m p l e s showed lower levels of t h e t e r p e n e compounds c h a r a c t e r i s t i c to black t e a . Uva and Darjeeling black tea s a m p l e s were rich in geraniol, benzyl alcohol and 2-phenyl e t h a n o l .

However,

t h e black tea s a m p l e s employed in t h i s s t u d y contained t h e s e compounds only in

small

amounts.

These

samples

components observed in green t e a .

contained

somewhat

larger

amounts

of

428

Table 4 Composition of volatile flavor compound ' tR (min)

Compound

Kenya

Turkey

Bangla- Viet n a m

Aus-

Sri

desh

trallia

Lanka

10?7

Hexanol

L16

0.69

0.47

0^8

0.67

0.56

12.1

(Z)-3-Hexanol

1.25

0.61

0.27

0.66

0.45

2.51

15.9

Linalool oxide

0.24

0.46

0.62

0.35

0.39

0.39

0.50

0.59

0.90

0.15

0.39

1.89

(cis-furanoid) 18.4

Linalool oxide (trance-furanoid)

21.0

Linalool

4.71

10.04

1.30

2.05

0.59

3.95

27.4

Eo-terpineol

0.60

A}

trace

A}

0.10

0.97

31.2

Linalool oxide pyranoid

0.60

0.78

0.28

0.57

0.17

0.26

32.1

M e t h y l salicylate

0.31

trace

0.52

A}

0.25

2.23

32.8

1-phenyl e t h a n o l

0.21

0.32

trace

0.14

0.03

0.45

37.1

Geraniol

1.26

1.26

0.70

0.36

0.53

2.36

38.4

Benzyl alcohol

0.13

1.77

0.87

0.41

0.16

3.34

42.0

2-phenyl e t h a n o l

18.63

0.06

5.60

1.73

6.78

4.06

49.0

Nerolidol

trace

1.12

trace

0.02

trace

0.95

* Numbers refer volatile composition relative to the internal standard (ethyl decanoate).

3.6.

R e s u l t s of m u l t i v a r i a t e a n a l y s i s on a r o m a c o m p o n e n t s Figure 1 shows the principal component loading r e s u l t s of the m u l t i v a r i a t e a n a l y s i s of black tea aroma components. Figure 2 shows the results of the m u l t i v a r i a t e analysis of the aroma components. Each sample approache green tea with an increase in the second major component (i.e., positive value) axis 2 and the characteristics of black tea became evident as the second major component became negative. The samples from Viet Nam and Turkey were somewhat different in aroma from the Uva and Assam t e a s .

429

1 0.9 O.S

5«

0.7 0.6 ^ 0.5 m

S 0..4 0.3-| 0.2-]

12*

0.1-1 0- 0 .

-0.6

-0.4

-0.2

0.2

0.4

axis 2

Figure 1. Scattergram of tea on axis 1 and axis 2 in principal components analysis l,2,3:green tea, 4,5,6:oolong tea, 7: vie tnam black tea,8:turkey black tea, 9:darjiling tea,10:keemun black tea, ll:assam black tea, 12:sri lanka uva black tea.

axx 5-1

3

2H

^'

11

• • — J —

-1.5

-1

-0.5

0.5

— I

1-5

axis 2

-1-*

Figure 2. Scattergram of flavor componets on axis 1 and axis 2 in principal components analysis

430 4. R E F E R E N C E S l.M.A.Gianturco, R.E.Diggers and B.H.Ridling,J.Agric.Food Chem.,22(1974) 758 2.G.W.Sanderson,J.Food Sci.,36(1971)231 3.T.Yamanishi,Y.Kita, K. W a t a n a b e and Y.Nakatani,Agric. Biol. Chem.,36 (1972)1153 4.Y.Takino, A.Ferretti, V . F l a n a g a n , M.Gianturco and M.Vogei,Tetrahedron Lett.,45(1965)4019 5.E.A.H. R o b e r t s , J . Sci. Food Agric,9(1958)212 6.E.A.H. Roberts and M.Meyers,J. Sci. Food Agric, 10(1959)167 7.Y.Obata,M.Omori,S.Yabuuchi,M.Kato,T.Takeo and R.Saijo,Bulletin of Faculty of Domestic Science, Otsuma Women's University,No. 12(1976)1 8.M.Omori,M.Kato,Y.Obata,R.Saijo and T.Takeo,J.Home Econom. J a p a n , 3 2 (1981)712 9.Association on Sensory t e s t of tea, National Tea Research I n s t i t u t e . : S t u d y of Tea, No.41,(1971)50 lO.K.Iwasa and S.Torii,Study of Tea,No.26(1962)87 l l . H . A n a n , H . T a k a y a n a g i and K.Ikegaya,Nippon Shokuhin Kogyo G a k k a i s h i , 35(1988)487 12.T.Tsushida,T.Murai,M.Omori and J.Okamoto,Nippon Nogeikagaku kaishi,61(1987)817 13.K.Ikegaya and H . T a k a y a n a g i , S t u d y of Tea,No.70(1989)121 14.H.Horita and T . K a r a , S t u d y of Tea,No.66(1984)41 15.M.Kato, T.Yano, M.Komatsu, M.Omori and Y.Hara,Nippon Shokuhin Kogyo Gakkaishi,40(1993)133

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

431

Studies on the formation of special aroma compounds of Pouchung tea made from different varieties Y.S.Chen^ H.R.Tasy*' and T.H.Yu'' * Department of Food Nutrition, Hongkuang Institute of Technology, Salu, Taichung, Taiwan, R O C . ^ Department of Food Engineering, Da-Yeh University, 112, Shanjeu Road, Da-Tsuen, Chang-Hwa, Taiwan, R O C .

Abstract Changes in the volatile and the nonvolatile compounds of Chin-sin Oolong and TTES12 tea leaves in different stages of tea processing were studied. The amino acid contents were found to first decrease and then increase during tea processing. Four tea catechins, (-)epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC), were found in the tea leaves; EGCG and EGC were the two dominant ones. Palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were detected in the leaves of Pouchung tea. Linoleic acid and linolenic acid were found to be the two dominant free fatty acids in the tea leaves. The fatty acid contents decrease during processing, and decrease at a higher rate in TTES-12 than in the Chin-sin Oolong. Furthermore, the sugar content was found to decrease during tea manufacturing. The analytical results indicated that there are no significant differences in the changes of volatile compounds of Pouchung tea made from different varieties during tea manufacturing. Most of the volatile components increased during fermentation of the raw leaves, but decreased during high temperature treatment. In the rolled and dried stage, volatile compounds are believed to be generated from the thermal degradation or thermal interactions of nonvolatile flavor precursors existing in the tea leaves. Chin-sin Oolong contains more volatile compounds which are generated from soluble sugars whereas TTES-12 has a higher quantity of volatile compounds which are generated from terpene alcohols.

1.

INTRODUCTION

During processing different varieties of teas commonly produce special aromas [1]. Pouchung tea is one of the partially fermented teas, possessing a unique floral flavor with a pleasant taste. The formation of special aromas strongly affects the sensory quality and consumption of Pouchung tea. Many researchers have investigated the correlation between flavor and the chemical compounds of processed tea [2-5]. Chen and Tsai reported that the total amino acids, and the total nitrogen and soluble solids of Pouchung tea made relatively high contributions to the first principal component (PC) of the taste quality of Pouchung tea; sucrose, alkaloids and

432

EGC to the second PC and total catechins, alkaloids and carbohydrates to the third [2]. Yamanishi et.al. studied the effects of processing conditions on the flavor quality of Indonesian black tea [3]. The results showed that there was a very high positive correlation between tea quality and the ratio of theaflavins (TF)/total color (TC). Takeo [4] reported that the ratios of linalool and its oxides contents to the total monoterpene alcohol content could be used to determine the clonal specificity of the tea plant. Yamanishi [5] reported that the ratio of linalool concentration to (£)-2-hexenal concentration correlated positively with sensory evaluation and the market price of the tea. There have been many studies done on the formation of tea flavor; the formation of black tea aroma [6], the withering effect on the aroma formation during Oolong tea manufacturing [7], the thermal generation of aroma compounds from tea and tea constituents [8], the flavor constituents of Pouchung tea and a comparison of the aroma pattern with Jasmine tea [9], and the thermal generation of aroma compounds from Oolong tea by model reactions [10]. However, there are few studies on the formation of special flavors from different varieties during tea processing and their correlation to the changes in chemical compounds. The purpose of this study is to investigate the changes in chemical compounds and special aroma of Pouchung tea during processing and the correlation between chemical compounds and the special aroma formation of Pouchung tea made from different varieties.

2. MATERIALS AND METHODS 2.1. Materials Pouchung tea was freshly produced by tea farmers during the fall season in Lukung Valley Nantou country from Chin-sin Oolong and TTES-12 varieties. The samples were divided into the nine stages during tea processing: raw, solar withered, indoor withered (I), indoor withered (II), fermented, pan fired, rolled, dried and the final tea product. The samples were quick frozen and stored at -40°C for analyses of the chemical compounds and aroma. 2.2.1. Chemical components analyses The free amino acids were determined by an automatic amino acid analyzer (AAA). The fatty acids were determined by gas chromatograph (GC). Catechins, glucose, fructose and sucrose were determined with a high performance liquid chromatograph (HPLC). 2.2.2. Aroma analyses GC analyses was carried out using an HP5890 series II gas chromatograph equipped with a flame ionization detector (FID). The column was J&W Scientific DB-Wax 50 m x 0.32 mm. i.d. The column temperature was programmed from 40**C to 220**C at a rate of 2*'C/min. The injector and detector temperatures were 250''C and 270°C, respectively. The nitrogen carrier gas flow rate was 1.2 ml/min. Peak identification was determined on the basis of the GC-MS results and coincidence of retention time (tR) and Retention Index (RI) [11].

433 3. RESULTS AND DISCUSSION 3.1. Changes in non-volatile compounds of Pouchung tea with produced by different varieties during processing As indicated in Table 1, the fatty acids in Pouchung tea include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:l), linoleic acid (C18:2) and linolenic acid (C18:3). Linoleic acid and linolenic acid were found to be the two dominant free fatty acids in the tea leaves. The fatty acid content in the Chin-sin Oolong was higher than in the TTES-12. Table 1 Changes in the content of fatty acids in Pouchung teas during tea preparation. Content (% , based Chin-sin Oolong variety 18:1 18:2 16:0 18:0 18:3 2.06 0.58 2.13 0.69 8.26 Raw 1.84 2.06 0.61 7.38 0.59 Solar withered 1.52 0.57 0.53 Indoor withered (1) 1.44 6.15 1.48 0.48 0.62 Indoor withered (2) 1.41 5.13 0.57 1.28 4.91 1.29 0.45 Fermented 1.16 0.68 0.41 Pan fired 1.19 4.59 1.18 1.06 0.43 0.53 RoUed 4.36 1.13 0.38 1.05 4.16 0.51 Dried 0.44 0.89 . 4.13 0.36 1.03 Made tea

on dry weight) 16:0 1.61 1.62 1.49 1.38 1.25 1.09 0.86 0.63 0.54

18:0 0.69 0.61 0.57 0.56 0.52 0.43 0.35 0.31 0.25

rrES-12 variety 18:1 18:2 18:3 0.85 6.19 1.63 6.17 0.87 1.53 0.71 1.23 5.29 4.53 0.65 1.16 0.61 3.94 0.94 3.51 0.73 0.47 0.46 2.95 0.61 2.83 0.53 0.35 0.31 2.81 0.46

The results show that fatty acids decrease during processing. The rate of decrease is higher in the TTES-12 than in the Chin-sin Oolong. The total fatty acids were reduced approximately 6 1 % and 50 % for TTES -12 and Chin-sin Oolong. Two reactions caused the decrease in fatty acids during tea processing. One was the oxidation of fatty acids and their degradation to lower molecular weight compounds; the other was the transfer of fatty acids during the formation of the volatile compounds [17]. The changes in catechins at different processing stages are shown in Table 2. These results indicate that catechins decrease during processing. EGCG and EGC were the two dominant catechins and they obviously decrease during the raw materials to fermented stage, but then remain stable from the post fermented stage to the final tea product. They were oxidized and condensed to form a yellow-orange polymer which interacts with the oral mucoprotein to induce astringency [12]. Another pathway may be the combination of chemical compounds to form volatile compounds [13,14]. Table 3 shows the changes in sugars of Pouchung tea during manufacturing. These results indicate that the three dominant sugars were sucrose, glucose and fructose. The sucrose content was higher than the others and reduced quickly during processing, particularly from the fermentation stage to the final product. This is probably associated with the carbonization of the tea which is fried at high temperature, and the Maillard reactions of free amino acids and glucose [15]. Addtionally, the sucrose degrades to glucose and fiiictose during

434 3. RESULTS AND DISCUSSION 3.1. Changes in non-volatile compounds of Pouchung tea with produced by different varieties during processing As indicated in Table 1, the fatty acids in Pouchung tea include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:l), linoleic acid (C18:2) and linolenic acid (C18:3). Linoleic acid and linolenic acid were found to be the two dominant free fatty acids in the tea leaves. The fatty acid content in the Chin-sin Oolong was higher than in the TTES-12. Table 1 Changes in the content of fatty acids in Pouchung teas during tea preparation. Content (% , based on dry weight) Chin-sin Oolong variety 16:0 18:0 18:0 16:0 18:1 18:2 18:3 2.06 0.69 0.58 2.13 0.69 1.61 8.26 Raw 0.61 1.84 2.06 0.61 1.62 7.38. 0.59 Solar withered 0.57 1.52 0.57 Indoor withered (1) 1.44 1.49 6.15 0.53 0.56 1.38 1.48 0.62 0.48 Indoor withered (2) 1.41 5.13 0.57 0.52 1.25 1.28 1.29 0.45 Fermented 4.91 0.43 1.16 0.68 Pan fired 1.09 0.41 1.19 4.59 0.35 0.86 1.18 0.53 1.06 Rolled 0.43 4.36 0.31 0.63 1.13 0.38 1.05 4.16 0.51 Dried 0.25 0.54 0.89 0.36 0.44 1.03 4.13 Made tea

rrES-12 variety 18:1 18:2 18:3 1.63 0.85 6.19 6.17 1.53 0.87 1.23 0.71 5.29 4.53 1.16 0.65 0.61 3.94 0.94 0.73 3.51 0.47 0.61 2.95 0.46 0.53 2.83 0.35 0.46 2.81 0.31

The results show that fatty acids decrease during processing. The rate of decrease is higher in the TTES-12 than in the Chin-sin Oolong. The total fatty acids were reduced approximately 6 1 % and 50 % for TTES -12 and Chin-sin Oolong. Two reactions caused the decrease in fatty acids during tea processing. One was the oxidation of fatty acids and their degradation to lower molecular weight compounds; the other was the transfer of fatty acids during the formation of the volatile compounds [17]. The changes in catechins at different processing stages are shown in Table 2. These results indicate that catechins decrease during processing. EGCG and EGC were the two dominant catechins and they obviously decrease during the raw materials to fermented stage, but then remain stable from the post fermented stage to the final tea product. They were oxidized and condensed to form a yellow-orange polymer which interacts with the oral mucoprotein to induce astringency [12]. Another pathway may be the combination of chemical compounds to form volatile compounds [13,14]. Table 3 shows the changes in sugars of Pouchung tea during manufacturing. These results indicate that the three dominant sugars were sucrose, glucose and fructose. The sucrose content was higher than the others and reduced quickly during processing, particularly from the fermentation stage to the final product. This is probably associated with the carbonization of the tea which is fried at high temperature, and the Maillard reactions of free amino acids and glucose [15]. Addtionally, the sucrose degrades to glucose and finctose

435 Table 2 Chan ges in the content of catechins in Pouchung teas during tea preparation. Content (% , based on dry weight) Chin- sin Oolong varietyr TTES-12 variety

Raw Solar withered Indoor withered (1) Indoor withered (2) Fermented Pan fired Rolled Dried Made tea

EC

ECG

EGC

1.31 1.32 1.28 1.07 1.27 1.03 1.17 1.16 1.14

1.62 1.51 1.48 1.46 1.45 1.43 1.42 1.36 1.34

4.62 4.58 4.51 4.42 4.41 3.92 3.82 3.76 3.73

EGCG 8.73 8.62 8.55 8.52 8.47 8.16 8.13 8.04 7.96

EC

ECG

EGC

1.41

2.16 2.15 2.13 2.14 2.11 2.06 1.97 1.79 1.83

4.81 4.71 4.65 4.61 4.55 4,03 3.89 3.78 3.47

1.4 1.39 1.39 1.38 1.05 1.12 1.06 1.02

EGCG 9.61 9.64 9.48 9.38 9.29 9.26 8.92 8.82 8.59

Table 3 Changes in the content of sugars in

Raw Solar withered Indoor withered (1) Indoor withered (2) Fermented Pan fired RoUed

Dried Made tea

Pouchung teas during tea preparation.

Content (% based on dry weight) j TTES-12 tea variety Chin-siEI Oolong variety Sucrose Glucose Fructose Sucrose Glucose Fructose 1.77 0.91 0.68 2.36 0.67 0.54 0.83 1.85 0.71 2.34 0.64 0.52 1.75 0.56 0.65 2.21 0.63 0.54 0.61 1.73 0.55 2.25 0.61 0.51 1.68 0.34 0.59 2.18 0.59 0.49 1.03 0.53 0.31 1.24 0.37 0.45 0.86 0.17 0.36 0.78 0.33 0.24 0.66 0.23 0.06 0.63 0.31 0.13 0.58 0.24 0.04 0.61 0.30 0.15

during processing. The difference in the rate that the sugar decreased between the Chin-sin Oolong and the TTES-12 during tea preparation was not significant. Changes in free amino acids of Chin-sin Oolong and TTES-12 at different processing stages are shown in Figures 1 and 2, respectively. These results indicate that theanine, glutamic acid and aspartic acid are the three dominant free amino acids in the tea leaves, and that the theanine content was approximately 50-60%. At first the amino acid content decreased; however, during tea processing they increased [18].

436

•S

600^-

*

•S" 3od-

s

M*9 D

alBI HlH fl H» I

1 4 II!

i" 4" J

2oa-

c§

H'

ill R9I SlPrS ifS Indoor withered (2) Indoor withered (1) Solar wiihocd Raw

Figure 1. Changes in the content of free amino acids in the Pouchung tea made from Chin-sin Oolong during tea preparation

Figure 2. Changes in the content of free amino acids in the Pouchung tea made from TIES-12 variety during tea preparation

3.2. Aroma divided by correlated precursors Changes in the concentration of volatile compounds in the Pouchung tea made from the Chin-sin Oolong and TIES-12 varieties during tea manufacturing processes are shown in Tables 4 and 5. The results indicate that there are 81 and 78 volatile compounds identified by GC-MS for Chin-sin Oolong and TTES-12, respectively. This study divided the volatiles according to their possible precursors into six groups including lipids, amino acids, carotenoids, sugars, terpenes and terpene alcohols. Compounds generated from lipids include methylbenzene, l-penten-3-ol, (Z)-3-hexenyl butyrate, c/5'-6-nonenol, 4-methyl-3-penten-2-one, c/5-3-hexenyl hexanoate in Chin-sin Oolong, and hexanal, 3-hexyne, 1-nonanol, d5-3-hexenyl 2- methylbutyrate, decoanoic acid, c/5-3-hexenyl benzoate in TTES -12. These compounds contribute the special "Grassy" aroma. The volatile compound content generated from lipids was higher in TTES-12 than in Chin sin Oolong. Compounds generated from terpenes and terpene alcohols contribute the special "Fruity" and "Floral" aroma. These compounds are the quality index of Pouchung tea made from different varieties. These compounds are ocimene, famesene, /ra«5-a-bergamotene, Pmyrcene, linalool, nerolidol, hotrienol, /raw^-geraniol and linalool oxide. And the content of the compound generated from terpenes in Chin-sin Oolong was larger than that in TTES-12. Compounds generated from amino acids contribute the special "jasmine" aroma. Particularly, IH-indole was the important volatile compound to the Pouchung tea. And other contributions were "Seaweed" and "Milk". Compounds generated from carotenoids and sugars contribute the special aroma "slightly floral" and "fire", respectively. These volatile compounds were generated by enzyme reaction and thermal degradation of carotenoids and sugars during tea manufacturing.

437

Table 4 Changes in the concentration of volatile compounds in the Pouchung tea made from Chin-sin Oolong variety during tea preparation. Yield ( ppb, based on dry weight) Compound

RL*

Compounds probably generated from Lipids methyl benzene 338.34 949.26 hexanol 13.03 3-penten-2-one 55.5 4-pentent-l-yl acetate 4-methyI-3-penten-2-one 75.77 402.26 l-pentcn-3-ol 21.55 (E)-2-hexenal 97.94 octanal (Z)-3-hexen-l-ol acetate 264.38 2-methyl-l-pentanol 8.72 277.23 1-hexanol 7.54 (E)-3-hexen-l-ol 1938.56 (Z>3-hexen.l-ol nonaxul trace 192.04 (E)-2-hexen-l-ol hexyl ester butanoic acid trace hexyl -2-methyl butyrate trace cthvl nonanoatc trace 1-heptanoI 73.17 (Z)-3-hexenyI butyrale 40.17 cis-3-hexeny 3-niethylbutyrate 28.23 decanal trace benzaldehyde 8.57 1-octanol 36 hexyl hexanoate trace cis-3-hexenyl hexanoate 33.4 cis-6-nonenol 48.4 hexyl bcnzoate 103.05 cis-3-hexenyl benzoate 89.45 33.19 cis-3-hexenyl phenylacetate sub-total 5135.75 Compounds probably generated from Terpene ocimenc (isomer 1) 1481.35 ocimene (isomer 2) 28.72 camphene 7.32 trans-alpha-bergamotene 29.97 famesene 24.09 delta-cadinene 130.17 famesene trace 1701.62 sub-total

SW*

IWl*

IW2*

254.18 160.96 296.12 53.66 77.73 282.23 16.16 19.66 45.81 779.22 148.13 783.49 35.8 34.84 66.09 103.22 91.6 211.13 186.08 567.17 17.5 82.96 109.68 59.14 197.65 36.61 377.99 17.1 77.62 76.82 395.57 99.14 167.62 7.36 19.44 7.86 2623.97 3299.72 4594.23 10.44 trace 45.54 56.98 103.2 405.91 trace trace 5.89 trace trace trace .12.6 trace 45.73 493.36 582.49 245.82 36.47 96.03 232.53 29.54 19.12 20.51 11.07 13.87 trace 7.96 14.64 16.62 43.21 83.1 86.52 12.14 trace 52.83 315.81 111.64 406.57 45.92 130.96 198.42 79.97 48.79 105.23 96.09 251.75. 381.36 33.09 21.93 49.76 4547.78 6851.69 10143.2

FL* 311.13 26.96 41.51 1342.06 36.3 74.85 441.15 59.78 26.23 63.67 306.92 17.78 2504.37 71.76 256.8 8.57 9.17 37.89 414.15 316.62 62.4 48.79 trace 60.99 92.08 455.6 191.23 57.04 359.91 21.28 7716.99

PF* 393.86 12.44 10.99 35.42 132.04 5.58 61.5 14.34 trace trace trace trace 12.24 49.84 trace 13.53 18.4 26.8 45.22 119.33 19.92 trace trace 65.03 109.02 473.67 119.49 57.79 444.98 60.94 2302.37

RL* 846.03 25.27 32.89 83.78 276.74 16.74 121.95 15.06 trace trace trace trace 16.03 142.99 trace 18.97 32.09 38.96 49.4 172.45 71.5 trace trace 70.79 189.62 795.8 216.87 87.61 756.49 109.36 4187.39

PL*

MT*

897.28 43.27 trace 54.05 230.88 9.03 63.21 10.52 trace trace trace trace trace70.92 trace trace

—

—

30.41 trace 151.73 150.35 trace trace 39.57 196.1 715.55 229.53 119.97 851.45 24.3 3888.12

44.85 trace 188.37 237.57 trace trace 47.03 56.67 232.41 294.38 76.11 328.09 69.3 4311.26 81 717.07

72.53 68.7 8.54 34.54 52.14 102.99 trace 339.44

150.97 916.11 87.9 2458.79 11412.41 12893.11 10.55 12.37 11.45 71.07 51.06 63.29 586.52 3736.34 3815.07 253.4 54.87 275.58 20.97 16.6 27.68 3552.27 16443.78 16930.06

17.31 32.89 57.21 1268.52 2694.31 2120.2 10.56 trace 590.21 1177.94 1039.84 3818.16 6679.79 9079.81 31.44 54.15 66.45 43.08 87.34 70.45 5779.28 10726.42 12433.96

Compounds probably generated from Terpene Alcohols linalool oxide (isomer 1) 518.28 467.36 996.04 linalool oxide (isomer 2) 1118.92 5629.64 6684.06 linalool 19.12 hotrienol 19.72 27.25 (Z)-citraI 25.81 24.54 a -tcrpineol 28.68 17.77 16.31 epoxylinaiool geranyl acetate 38.63 59.7 gamma-isogeraniol 39.45 35.08 61.41 ncrol 64.74 7958.4 trans-geranoid 8047.66 geranyl acetone 14.29 trace cis-jasmone 91.32 40.9 nerolidol (isomer 1) 17.55 17.05 nerolidol (isomer 2) 228.47 402.21 gamma-ylangene 26.14 35.88 t-murrolol 34.67 33.89 famesol 14.43 trace nerolidol (isomer 3) 24.24 40.29

787.92 1240.64 1261.06 1072.9 1658.19 1653.22 15294.4 14728.2 13652.13 66.85 216.5 230.87 52.44 54.55 50.92 108.13 279.63 223.29 35.87 47.84 27.8 94.23 64.41 152.13 69.49 63.93 52.95 110.61 149.61 159.68 14241.2 13092.28 9682.66 25.01 64.59 41.69 582.99 472.21 252.46 25.41 48.55 38.03 2941.46 3133.45 2993.05 37.8 35.71 73.84 138.94 19.87 134.44 124.54 133.05 222.42 10.41 31.49 51.36

163.81 66.77 2533.28 313.36 trace trace 22.15 82.67 33.23 109.84 969.13 57.52 235.67 14.47 3460.66 58.24 74.94 209.46 17.86

—

282.11 86.6 3417.82 373.6 trace trace 44.51 169.72 59.08 159.11 1627.4 101.35 489.34 47.24 6036.75 95.8 173.02 451.96 41.25

829.06 57.63 trace 212.16 747.24 268.41 105.33 148.82 30.5 74.61 37.31 35.41 trace 190 trace trace

210.88 42.49 1884.11 184.54 trace trace trace 205.57 85.29 120.59 1322.38 95.94 481.65 55.41 7880.43 87.64 219.22 512.89 45.2

—

306.89 3189.1 trace trace 4294.11 158.61 92.69 2813.81 505.45

— —

trace 140.67 trace 103.16 975.7 92.28 130.47 trace 3427.13 40.27 136.59 176 trace ,

438

(Table 4 continued ) Yield ( ppb, based on dry weight)

RL*

Compound sub-total

15961

IWl* FL* IW2* 16958.88 36426.97 35841.26 30041.07

SW*

Compounds probably generated from Amino Acids 6.55 pyridine 5.92 192.52 173.7 isoamyl alcohol acelophenonc 31.47 27.61 1790.1 1963.66 methyl 2-hydroxybcnzoate 214.2 265.88 benzyl alcohol 290.4 369.96 phcncthyl alcohol 47.9 39.43 bcnzcneacclonilrile 26.24 trace 2-mcthyl-2-phcnylethylpropanoic acid 16.22 17.51 2-methoxy-4-{ 1 -propenyl)-phcnol 40.06 45.21 2-methoxy-4-<2-propneyl>phenol 74.08 52.21 IH-indole 3009.2 2681.63 sub-total Compounds probably generation from Carotenoids 1.5.5.6-tetramethyl-1.3-cyclohcxadiene trace*** trace cyclopentenc 38.18 1.3-dimethyl-bcnzene 1. l-dimethyl-3-methylidene-215.8 vinylcyclohexane trace bcta-ionone beta-demascenonc trace 53.98 sub-total

PF*

RL*

PL*

8792.83 183.21 195.44 61.16 93.78 257.69 57.35 143.54 168.26 124.39 164.29 512.38 1961.49

10.21 300.38 90.68 3369.9 789.32 903.16 632.91 trace 55.93 59.72 1418.66 7630.16

75.36 313.22 158.49 3326.81 740.77 641.3 1027.56 17.39 53.85 167.23 690.91 7212.89

79.8 405.22 147.89 2246.91 131.86 148.57 619.86 100.98 30.57 68.16 483.4 4463.22

40.02 34.97 97.61 65.47 73.06 57.66 244.07 38.07 6.21 115.67 1025.36 1798.17

94.74 65.55 179.23 106.1 154.62 103.55 488.46 82.42 12.24 226.12 2164.73 3677.76

140.84 160.17 168.81 61.65 185.68 126.84 448.64 53.89 94.18 372.81 2615.91 4429.42

4.92 7.86 37.9

7.43 12.85 55.15

17.9 19.54 79.82

43.73 40.51 186.11

21.08 9.75 15.63

33.46 15.54 .23.42

trace trace 10.81

29.14 13.01 16.78 109.61

524.94 7.93 19.37 627.67

589.41 12.19 15.08 733.94

443.42 13.25 16.41 743.43

134.29 22.87 32.45 236.07

278.28 28.88 60.89 440.47

254.94 15.44 43.21 324.4

Compounds probably generated from Sugars 38.39 26.37 30.81 48.53 acctaldehyde 52.81 107.1 153.43 266.28 1232.58 863.09 2-propanone 13.18 68.97 9.72 24.19 49.5 2-pentanone 12.27 6.21 4.32 17.48 4-mclhyl-2-pentanonc 5.61 160.96 52.9 3.83 26.98 4.83 2-butanol 197.84 137.87 149.92 38.01 30.65 1-cyclopropyl-2-propanonc 2,3-dihydro-6-methyl-2-propyl-4H-157.21 47.78 54.23 20.97 23.93 bcnzopyran-4-one 2H-1 -benzopyran-2-one 62.57 52.55 315.29 10.9 40.11 479.89 471.91 1007.5 1044.4 1516.47 sub-total Toul 27378 25681.74 56096.26 70846.98 60374.66

• RL : Raw leaves SW : Solar withered IWl : Indoor withered (1) PF: Pan fired RL: Rolled DL : Dried MT: Made tea ** — : not detected *•• trace: <0.05

MT*

8423.06 13656.66 13434.23

114.49 1533.42 14.8 8.51 6.63 30.19

-—** 149.09

— —

149.09

241.26 197.48 408.13 2887.44 18277.62 14424.89 62.78 255.38 77.48 10.21 19.37 100.16 10.43 58.3 15.45 68.81 61.23 287.04

128.41 144.58 94.39 145.15 88.23 57.88 219.63 341.58 1860.31 3513.74 19329.27 15688.03 2G399J 36202.44 53839.4 35196.81

IW2 : Indoor withered (2)

FL : Fermented

439 Table 5 Changes in the concentration of volatile compounds in the Pouchung tea made from TTES-12 variety during tea preparation. Yield (ppb, based on dry weiight) PF* IWl* ][W2* RL* FL* Compound RL* Compounds probably generated from Lipids 38.54 427.44 10189 56.04 33.6 16.34 34.81 hexanal 30.64 5144 39.21 50.26 50.45 heptanal 16.19 35.35 57.37 59.4 51.5 15.78 90.79 36225 cis-3-hexenal 89.57 trace 31.18 trace 1-pentanol 37.71 8.53 19.5 51.69 95.14 319.93 124.16 26.57 15169 254.53 cis-famesol 266.03 13.18 trace trace trace 8.01 27.12 3.27 (z)-2-penten-l-ol 46.16 trace trace 54.69 208.19 3-raethyl-2-buten-l-ol 53.44 17.01 33.18 21.44 50.36 13.35 trace 18.23 6-methyl-5-hepten-2-one 14.08 74.67 262.9 48.49 83.25 147.42 557.95 1-hexanol 139.94 1499.8 94.41 73.32 cis-3-hexenol 611.91 1199.67 2468.85 1214.9 18.31 9.25 trace 55.26 16.21 21.33 hexyl butyrate 14.61 360.74 174.63 9.91 trace (E)-2-hexen-l-ol 86.09 17.49 33.65 trace trace trace 379.39 597.16 cis-3-hexenyl butyrate 73.66 125.5 63.34 87.21 95.41 49.52 1-heptanol 178.39 85.56 78.92 135.9 157.42 193.22 229.71 93.83 228.09 ds-3-hexenyl 2-methylbutyr.ite 56.39 trace 53.45 trace 44.54 41.21 2-cyclohcxen-l-one 27 35.22 8171 100.91 119.39 88.67 100.45 10163 1-ocianol 129.68 22.92 28.62 28.78 trace 6.53 24.76 hexylhexanoate trace 16121 78.56 168.78 25.36 30.69 81.91 5.21 3-hexyne 79.44 208.41 111.53 117.12 141.38 21.08 583.81 cis-3-hexcnyl hexanoatc •97.27 3237.06 5117.75 74.4 234.85 48.06 38.93 1-nonanol 36.79 trace 12.44 14.78 trace 12.02 trans-2-chlcro-cyclohexanol trace 39.08 trace trace 34.61 25.19 14.26 19.63 1-decanol 4152 29.14 16.63 57.63 39.96 26.35 octanoic acid 10.56 74.77 48.57 84.3 118.1 1626 44.42 14.44 cis-3-hexenyi benzoate 15.23 25.12 15.77 1136 trace 19.62 trace nonanoic acid 33.54 18.14 17.01 20.93 29.51 17.89 methyl hexadecanoate 17.13 52.31 93.04 71.85 19.55 38.11 28.85 6.34 etiiyl hexadecanoate 371.38 480.87 245.69 40.42 69.43 96.61 decoanoic acid 7.56 54.44 33.47 28.7 trace 21.2 23il 37.94 methyl (z^)-9.12-octadecadienoate 2107 27.65 trace 10.67 19.48 19.65 dodecanoic acid trace 192.09 4132 51.22 62.67 54.59 109.33 methyl (z.z,z)-9,l2,15-octadecatrienoate 37.07 27.38 69.64 9.63 68.49 10.22 12.33 tetradecanoic acid 12.03 55.34 87.23 276.34 hexadecanoic acid 32.81 35.19 10.92 44.95 28.6 74.06 67.74 137.16 33.96 22.56 butyl 2-methylpropyl ester 1,222.15 benzenedicarboxylic acid sub-total 3387.22 6957.26 4431.04 559181 6881.39 3315.61 1651^8 Compounds probably generated from Terpenea 125.47 526.43 682.37 67.81 41.05 39.12 ^ -rayrcene 29.64 181.64 62.47 36.54 50.21 44.3 171.9 ocimene (isomerl) 30.13 ocimenc (isoraer2) 35.61 32.93 454.42 2005.54 476.97 3381.4 448.94 famesene 21.39 637.41 2226.91 2390.76 16.25 34152 1176.36 r -cardinene trace trace trace trace 12.33 44.03 35.21 sub-total 966.71 3497.22 6328.71 3042.04 129.98 111.63 1751.77 Canpound probably generated from Tcrpene Alcohols cis-3-hexenyl acetate 3.29 165J2 162.48 148.42 10.05 trace trace linalool oxide (isomerl) 552.71 304.63 541.32 650 1328.3 143.08 109.29 linalool oxide (isoraer2) 1112.5 684.69 1355.61 1120.63 1778.8 66.91 65.44 linalool 2905.85 2475.02 4217.64 5745.97 5455.21 1307J 1022.71 Z-citral trace trace trace trace 181.23 268J7 130.51 geranyl formate 31J4 21.41 53.92 114.68 trace trace trace a -tcrpineol 16.81 49.85 33.46 41.75 44.9 97.08 58.88 E-citral 38.45 90.12 71.61 86.56 216.65 7639 19.68 epoxylinalol 43.46 157.05 190.17 55.03 66.56 26.61 16.95 a -isogeraniol 24.11 3171 14.28 30.87 21.49 trace trace isogeraniol 42.69 52.27 60.43 47.34 77.16 28.28 5147 trans-geraniol 9676.89 7154.77 15014.6 9652.56 14140.8 950J 797.72

sw*

MT*

PL* 44.77 54.94 73.25 trace 111.54 trace

— —

53.54 58.77 trace trace trace 96.5 235.06 trace 106.11 29.8 197.54 146.84 5024.3 trace

—

159.42 48.54 147.43 trace 56.65 trace

— —

42.47 60.45 trace trace 123.39 105.42 248

—

91.24 33.59 247.39 104.66 317.47

— —

124.77 258.39 43.11 35.3 114.33 455.08 trace 37.6 64.68 83.34 73.39 211.39

trace 39.74 70.91 171.1 258.62

7734.3

3224.31

46.72 44.51

131.05 42.33

348.83 2858.9 24.93 3323.9

61.2 15011 32.61 1769.29

—

128.74 80.77 14814 306.08

—

13334 73.18 25.76 trace 46.93 1313.4

60.37 187.78 93.21 45.21 131.74 379.51

—

—

115.23 95.18 2025.94 194.68

—

192.48 81.98 33.67

—

99J8 1881.54

440

(Table 5 continued ) Yield ( p p b , basccI on dry weight) Compound cis-jasraone ncrolidol

RL*

57.32 29.95 10022.1 sub-total Compounds probably scnerated &om Amino Acids 93.45 3-methyl-l-butanol acctophcnonc 10.04 471.61 2-hydroxymcthyIbcnzoatc 2-hydroxy-1 -mclhylcthylbcnzoate 1377.02 benzyl alcohol 72.18 57.04 phencthyl alcohol benzene acctonitrile 33.89 trace 2-phcnylcthyl benzoatc 17.28 8-quinolinol trace 2-methoxy-4 -(1 -propeny l)-phenoI IH-indolc 67.73 2200.24 sub-total QsnpouDds probably generated from Carotenoids 2-cyclohexen-l-ol 135.17 39.77 9-hydroxytheaspiran A 4-methylcyclohept-2-cnone 22.02 10.15 3.7-dinielhyl methyl 2.6-octadienoatc 14.02 (2-methylcnebutyl) cyclopropane 14.24 /9-damascenone yS-ionone 24.59 trace 3-(2-propcnyl)cyclopct_emc_ trace methyl jasmonate 405.82 sub-total Compounds probably generated from sugars 2274.13 2-propanone 3.77 2-pentanone 13.63 2-butanal 9.05 2.3-dihydrobenzofuran 2300.58 sub-total

SW«

IWl*

DL*

FL*

PF*

RL*

267.99 3415.09 18383.7

194.11 2850.6 4223.2

94.18 1939.6 2979.48

189.94 3878 5660.7

154.41 3047.29 5490.95

36.01 47.16 325i7 trace 46.43 1132 66.85 63.15

41.48 40.35 8113 trace 72.2 26.24 40.65 4109 73.11 44.37 675.67 1867.66

77.13 151.23 7570.36

111.15 566.71 16136i

IW2* 92.02 1136.55 11259J1

16.48 18.28 383.5 946J2 65.24 48.11 45.26 trace 22.15 trace 44.2 1589.54

60.22 44.28 766.9 1706.8 143.18 136.65 195.36 28.27 25.65 24.59 341.83 3473.73

228.33 65.81 5812 1378il 55.69 53.32 156J8 30.63 23.16 14.99 225i4 2814.76

17534 154.78 644.48 142151 197.18 163.72 638.93 97.91 49.61 7439 1283.96 490181

35.71 50.38 599.41 trace 70.42 25.41 100.47 46.01 88.49 53.95 1177i 2247.8

5232 841i7 158192

36.14 53.75 704.76 trace 56.47 15.84 63.5 21.45 91.07 64.13 1186.4 2293.6

91M

MT*

66J3 17.68 28.54 13.59 15.94 15.98 29.58 trace trace 187.84

105.07 38.89 33.62 19.76 17.09 15.22 45.65. 18.95 20.44 314.69

14739 45J2 31.96 26.28 37.62 28.11 39J8 7.18 21.15 384J9

148.46 48.65 48.07 33.62 trace 35.01 45.79 20.02 40.48 420.1

190.19 26.01 trace trace trace 27.68 30.69 trace 77.21 351.78

204.2 23i8 trace trace trace 36.87 61.13 trace 261i4' 587.42

213.06 2107 trace trace trace 35.51 79.81 trace "89.89 509.48

200.17 18.99 trace trace trace 89.49 146.35 trace 71.59 828.26

834.69 9.35 118.59 5.25 967.88

131.49 11.31 36.61 29.97 209.38

477.17 31.64 310.96 12.97 832.74

24138 17.48 50.04 47.15 356.05

1634.8 53.86 324.74 42.66 2056.1

5067 75.46 401.55 22.03 5566.04

4553 81.05 169.96 40.24 48443

5963 85.7 55.29 51.34 6155.33

• RL : Raw leaves SW : Solar withered IWl: Indoor withered (1) PFi Pan fired RL : Rolled DL : Dried MT: Made tea ** — : not detected • • • trace: <0.05

IW2 : Indoor withered (2)

FL : Fermented

3.3. Changes of volatile content of Pouchung tea generated from precursors during processing. r u i +1 Changes in volatile content according to the possible generation sources of the volatile compounds in the Pouchung tea made from Chin-sin Oolong and TTES-12 are shown m Figures 3 and 4. The results indicate that there are no significant differences in the changes of volatile compounds of Pouchung tea made from different varieties during tea processing. Chin-sin Oolong contains more volatile compounds generated from soluble sugars, whereas TTES-12 has a higher quantity of volatile compounds that are generated from terpene compounds.

441

Figure 3. Comparisons of the flavor composition of the Pouchung tea made from Chin-sin Oolong variety during tea preparation (according to the possible generation sources of the volatile compounds).

Figure 4. Comparisons of the contration of volatile compounds in the ouchung tea made from TIES-12 variety during tea preparation (according to the possible generation sources of the volatile compounds).

4. ACKNOWLEDGEMENTS The authors thank the National Science Council of the Republic of China for financial support (NSC 85-2321-B-241-001).

5. REFERENCES 1. A.O. Chen, R.C. Chang, and Y.S. Tsai, In Proceedings of the International Symposium on Tea Science, The Organizing Committee of ISTS (1991) 67-71. 2. A.O. Chen, Y.S. Tsai, In Proceedings of the International Symposium on Recent Development in Tea Production, Taiwan Tea Experiment Station, Taoyuan, Taiwan (1988)249-271. 3. T. Yamanishi, Y. Musalam and A. Kobayashi, In Proceedings of the International Symposium on Recent Development in Tea Production, Taiwan Tea Experiment Station, Taoyuan, Taiwan (1988) 229-236. 4. T. Takeo, Nippon Nogeikagaku Kaishi, 56 (1982) 1982. 5. T. Yamanishi, Report to AARD-IADS from RITC Indonesia, (1985). 6. G.W. Sanderson and H.N. Graham, J. Agric. Food Chem. 21 (1973) 576-585. 7. Takeo, Agric. Biol. Chem. 48 (1984) 1083-1085. 8. T. Yamanishi, M. Kawakami, A. Kobayashi, T. Hamada and Y. Masalam, In Thermal Generation of Aroma, T.H. Parliament, R.J. McGorrin and C.-T. Ho (eds.), American Chemical Society, Washington, DC (1989) 310. 9. T. Yamanishi, N. Kosuge and Y. Yokitimo, Agric. Biol. Chem. 44 (1980) 2139-2142. 10. M.J. Yang, Master Thesis, Department of Food Engineering, Da-Yeh Institute of Technology, 1996.

442

11. T. Yamanishi, L. Shaojun, G. Wenfei and A. Kobayashi, In Frontiers of Flavor, G. Charalambous (Ed), Elsevier Science, Amsterdam, (1988) 181-190. 12. I.M. Juan, Proceedings of the International Symposium on Tea Science, The Organizing Committee of ISTS,1991. 13. Y. Musalam, T. Suhartika and T. Yamanishi, In Proceedings of the International Symposium on Tea Science, The Organizing Committee of ISTS (1991) 47-51. 14. S. Luo, W. Guo and H. Fu, In Proceedings of the International Symposium on Tea Science, The Organizing Committee of ISTS (1991) 57-61. 15. D. Ferreia, J.P. Steynberg, B.C.D. Bezuidenhoudt, A. Cronje, S.L. Bonner and L.J. van Lelyveld, In Proceedings of the International Symposium on Tea Science, The Organizing Committee of ISTS (1991) 72-75. 16. A. Gulati and S.D.Ravindranath, J. Sci. Food. Agric. 71 (1996) 231-236. 17. P.O. Owuor and M.Obanda, J. Sci. Food. Agric. 70 (1996) 288-292. 18. M. Kawakami, S.N. Ganguly, J. Banerjee and A. Kobayashi, J. Agric. Food Chem. 43 (1995) 200-207.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

443

EGYPTIAN ONION OIL Nadim A. Shaath and Frederick B. Flores Kato/Nickstadt-Moeller, 1169 Edge water Ave., Ridgefield, NJ 07657 ABSTRACT The common onion (AlUum cepa) belonging to the Lily family, is among the oldest known cultivated plants, and was cultivated in Egypt, China and India before the beginning of recorded history. Onion contains a volatile oil, fixed oil, protein, cellulose, sugars and minerals. The volatile oil content of onion depends on the variety of onion used and the distillation processes employed. The average yield is 0.015 percent oil. The chief constituents of the oil are di-n-propyl disulfide and dimethyl-n-propyl disulfide. Generally, one pound of the oil has the flavoring strength of 5,000 pounds of fresh onions or about 500 pounds of dehydrated onions. This steam distilled oil contains numerous volatile components, mostly alkyl and allyl di- and tri-sulfides. We identified 32 of these chemicals using dual capillary column Gas Chromatography in combination with Gas Chromatography/Mass Spectrometry. The commercial significance of the oil and its full chemical analysis is discussed.

INTRODUCTION Onions^ are believed to be native to Southwestern Asia. They are known to be among the oldest known cultivated plants. Egyptian priests placed onions and garlic as offerings on the altars of their Gods. Onion has an ancient reputation as a curative agent, highly valued by the schools of Galen and Hippocrates. It is high in vitamins A, B and C and shares many of the properties of garlic, to which it is closely related. Raw onion helps to keep colds and infections at bay, promotes strong bones and a good blood supply to all the tissues. It acts as an effective blood cleanser which, along with the sulfur it contains, helps to keep the skin clear and in good condition. It has a sound reputation for correcting glandular imbalance and weight problems [1]. The common onion, an herbaceous biennial plant, has a single large bulb from which arise one or more leafless stalks that may grow to about 2 ¥2 feet to 6 ^ Onions (Allium cepa in Latin) are: Basal (Arabic), Yang Tsung (Chinese), Ui (Dutch), Sibuyas (Filipino), Oignon (French), Kremethi (Greek), Cipolla (Itallian), Luk (Russian) and Cebolla (Spanish).

444

feet high (depending on the variety and growing conditions), with a terminal cluster of many small greenish-white flowers. These are characterized by their penetrating pungent and long-lasting aroma, remarkably characteristic of fresh onions. Although the onion is hardy and may be grown under a wide range of conditions, it is a temperate climate crop and prefers relatively cool, moist soil rich in organic matter but moderately low in soil acidity. The onion may be grown from its small black seed, from small bulbs called "sets", or from transplants; commercially, most onions are grown from seed. The bulb itself is used as a vegetable or as a seasoning, and the green parts of the plant may be mixed in a fresh salad. The seed is planted in rows 12 to 16 inches apart or less, depending on conditions. In well-cultivated land, with effective weeding, herbicidal control, adequate irrigation, and fertilizer application, yields of 4 to 12 tons or more of fresh onion bulbs per acre may be obtained, depending on the weather, the fertility of the soil, and the variety of onion. In the United States, storage and spring/summer. outer skin and a firm inside. color skin and is more fragile

onions are basically divided into two categories -The Storage Onion has a thicker, darker, paper-like The spring/summer onion has a thinner and lighter than the storage onion.

The Storage Onion is more pungent than the spring/summer onion because it has a lower water and sugar content. They also do not bruise as easily. Their season begins in August and goes through March. Typical storage onions are available in red, yellow and white varieties. Spring/Summer Onions are sweeter and milder than storage onions and are sometimes called, "designer onions". Their season begins in April and ends in August. Many of the varieties are well-known to onion lovers: Texas Sweet, Sweet Imperials, Carzalia, Maui, Vidalias, and Walla-Walla being chief among them. Turkey is among the largest onion producing countries in the world, and ranks third largest in production of all vegetables after tomatoes and potatoes. Onions in Turkey are classified as follows: Short and Medium-Day Onions are usually white skinned, have high crop yield, high water content and a relatively sweet taste. They are non-storage onions and have a low solid content (5% - 8%). These onions are good for fresh consumption only. Long-Day Onions are usually domestic varieties, have purple/violet inner skins, high solid content (8% -11%) and are stronger in taste. Under suitable conditions they can be stored throughout the year.

445

There is no remarkable relationship between the onion type and cultivating regions. There are nine major cultivation regions in Turkey with different climatic conditions. Within these regions both storage and non-storage onions are grown. In Egypt, terminology such as short, medium and long-day onions does not exist. Its equivalent is: Winter crop where production holds for 4 months during November, December, January and February. At that time, the crop is whiter in color, contains a higher percentage of water and, consequently, onions collected during that period are sold for dehydration or fresh consumption. Summer crop (long-day onions) can, as the name implies be stored for a long time. Long-day onions can be shipped fresh to Europe and are the onions that are produced from the second half of April through June/July. Some of the "giant producing" areas of the world are the Peoples Republic of China - 3, 980, India - 3, 400, the former U.S.S.R. - 2, 200, U.S.A. - 2, 200, and Turkey - 1, 600. (All quantities expressed in thousand of tons.) It should be noted that the Egyptian, Dutch and Italian production of onions are about the same levels of 500 thousand tons each.

ONION OIL Onion oil is obtained through the distillation of crushed fresh onions which have been allowed to stand for a few hours before distillation. As onion constituents are partially water-soluble, a special recovery technique is necessary to achieve a high quality oil in a yield of about 0.01 to 0.03 percent, depending on the type of onion, ripeness of the bulb, and season. Onion contains a volatile oil, fixed oil, protein, cellulose, sugars, minerals, etc. The volatile oil content of onion depends on the variety of onion used and the distillation processes employed. The extractives are, to some extent, watersoluble. The average yield is 0.015 percent oil which has a tendency to crystallize upon standing. The chief constituents of the oil are di-n-propyl disulfide and methyl-n-propyl disulfide. Generally, 453.5 g (1 lb) has the flavoring strength of two tons of fresh onions or about 227 kg (500 lb) of dehydrated onions. Basic research is continuously being conducted for: i. ii. iii.

yield improvement; reducing the relative GC ratio compounds at the head and tail ends; increasing relative GC ratio compounds of the middle section or "body" of the oil.

446

Some of the main procedural parameters that directly influence oil production are: Parameters

Influence

Maceration

extraction time temperature

yield quality

Distillation

extraction time steam pressure condensation temp

yield equilibrium in GC (head, body, tail end)

Whereas the dehydration industry uses onions with 18 to 22 percent solids, onions used for distillation of onion oil requires low solids content. There is a direct correlation between solids and oil content. Egyptian onions have a total solid content of 8.0 to 9.3 percent depending on crop time. Although the chemical composition of onion oil has been the subject of numerous studies [2-5], until recently nobody had looked at the physiological distribution of the characteristic flavor components. In 1975, Freeman reported that there was a consistent flavor distribution throughout onion, garlic, and leek [6]. This information should prove useful to the onion oil distiller as there is a belief held by many distillers that there are localized portions of these alliaceous plants which contain higher proportions of flavor compounds [7]. In another paper on onion oil, Galetto and Bednarczyk used the technique of multiple regression analysis to correlate GC data with sensory evaluation data. They found that there were six major compounds responsible for the aroma of onion oil. Three of these compounds were methyl propyl disulfide, methyl propyl trisulfide and di propyl trisulfide, while the other three were unknown [8]. Onion oil is used exclusively in flavors, rarely in fragrances, although such application is very possible (violet, hyacinth, rose, etc. can benefit from minute traces of onion oil for a distinct part of the top note). The oil has found an increasing outlet in the canning industry in flavors for soups, meat, table sauces, dressings, etc. There are wide limits for a suggested usage-level concentration, according to the acidity and overall type of finished product in which the oil will be incorporated [7]. The Food Chemical Codex [9] describes onion oil as follows: A volatile oil obtained by steam distillation of the bulbs of allium cepa L. (Liliaceae Family). It is a clear, amber yellow to amber orange liquid having a strong pungent odor and taste characteristic of onion. It is soluble in most fixed oils, in mineral oil, and in alcohol. It is insoluble in glycerin and in propylene glycol. Onion oil is purchased

447

mainly on the basis of its odor and flavor, which render definite specifications of little value. Identification. The infrared absorption spectrum of the sample exhibits a characteristic pattern. Heavy Metals (as Pb) Refractive Index Specific Gravity

Passes test. Between 1.549 and 1.570 at 20^C Between 1.050 and 1.135

Packaging and Storage. Store in full, tight, preferably glass or aluminum containers in a cool place protected from light. Functional Use in Foods. Flavoring agent. The EOA [10] lists the following specifications for onion oil: An amberyellow to deep-amber oil; some samples may show a crystalline deposit; specific gravity (25°C) 1.0500 to 1.1350; refractive index (20°C) 1.5495 to 1.5695. The EOA specification points out that the sensory quality of this oil is of more importance than physical characteristics. Onion oil, garlic oil, leek oil and mustard oil all possess significant concentration of sulfur compounds. Sulfides occur frequently in plants due to glucoside degradations [11]. Twenty volatile sulfur-containing compounds were identified. Onions have little smell until the tissues are cut or bruised. Once this takes place the enzymatic reaction results in a complex mixture of sulfides that can be recovered as oil of onion by distillation.

FLAVORING FORMS Dehydrated Onion. The dehydration of onions to produce onion powder and pieces of various sizes is a major activity in Egypt. The onions are first flame peeled, washed to remove the burnt outer skin, and then mechanically sliced into a perforated belt. The drying is carried out in a tunnel drier, the hot air circulating through the holes in the conveyor belt. Onions enter the system with a moisture content of about 80 percent and are dehydrated to about 4 percent. The dehydrated product may be sold as such, kibbled to various mesh sizes, or milled to a moderately fine powder [11]. Several methods have been proposed for the assessment of the flavoring strength and pungency of both fresh and dehydrated onions. There is a good correlation between the enzymatically produced pyruvic acid, odor strength and pungency [12]. The relative flavor strength of fresh and dehydrated onion is difficult to determine, as the two profiles are quite different. However, it is

448

generally accepted that onion powder is about ten times stronger than fresh onion [11]. Toasted Dehydrated Onion. Onions contain reducing sugars whose quantity depends on the variety and/or conditions of storage. These sugars may undergo Maillard reaction during the latter stages of dehydration. High temperatures result in a marked toasted note. There is a demand for toasted onion products that have the odor and flavor associated with sauteed onions. These are available in both powder and kibbled form in qualities ranging from lightly to heavily toasted [11]. Onion Salt. This is a mixture of onion powder and salt, often with an anticaking agent such as starch, tricalcium phosphate or silicon oxide, to maintain dry free-flowing properties. Standardized dispersions of onion oil, with or without onion extract, are available for use in blended seasonings [11]. Encapsulated Onion Flavors. Onion oil encapsulated by spray drying in gum acacia or a modified starch is available as a flavoring ingredient. The strength of these products depends on the manufacturer and may range from equal to ten times stronger than onion powder [11]. Onion Juice/Extract (Oleoresin). Onion juice carries all of the available flavor, actual and potential, and is a good basis for the preparation of a watermiscible onion flavoring. The juice is obtained by hydraulic pressing of the washed onions [11]. Oil of Onion. This is obtained by the distillation of crushed fresh onions that have been allowed to stand for some hours before distillation. As onion oil constituents are partially water-soluble, a special recovery technique is necessary to achieve a high-quality oil in a yield of about 0.01 to 0.03 percent, depending on the type of onion and the season.

TECHNICAL DATA ON EGYPTIAN ONION OIL The herbaceous plant of Onion is of extreme popularity throughout the world as a vegetable and a spice used in many food preparations and as a flavoring ingredient [13,14]. The plant contains a volatile oil which has been the subject of abundant research. Studies of the chemical constituents of this oil have dated back to as early as 1892 by Semmler when he noted the empirical formula of the major component to be C6H12S2 [15]. In 1964, Sikher et al were the first to use gas chromatography to quantitatively analyze onion oil [16]. Now we know that the major components of this volatile oil are organic alkyl and allyl sulfides of the empirical formulas of C6H14S2 and C6H12S2 [17]. Methyl propyl disulfide, methyl propyl trisulfide, and dipropyl trisulfide are three of six chemicals

449 responsible for this characteristic odor [18]. Others who have studied the chemical analysis of onion are Carson (1967), Schwimmer (1968, 1971), Galetto and Hoffman (1976), and Albrand et al (1980) [6,12,18-24].

The physicochemical properties of the steam distilled Egyptian Onion oil are listed below: Appearance Odor Refractive Index (@ 20°C) Specific Gravity (@ 25°C) Flash Point Solubility

Yellow to amber clear liquid. Strong pungent odor, characteristic of fresh onion. 1.5300 - 1.5500 1.0310 - 1.1010 115°F±10°F Insoluble in water, propylene glycol, glycerine; soluble in most fixed oils, mineral oil and alcohol.

Several parameters directly influence the distilled oil product. For example, the time, temperature and agitation of the maceration process influence the yield and quality of the essential oil. Time, steam pressure and condensation temperature of the distillation process has direct bearing on the yield and balance of volatile components in the oil. We have found that different geographical locations within Egypt also have an effect on the product oil. This steam distilled oil contains hundreds of volatile components mostly alkyl and allyl di- and tri-sulfides. We identified thirty-two of these chemicals using dual capillary column Gas Chromatography in combination with Gas Chromatography/Mass Spectrometry. The instruments used for the identification were the Perkin-Elmer Autosystem GC II and the Ion Trap Detector. The capillary columns used were non-polar (Ultra 1) and polar (CBXS).

CHEMICAL ANALYSIS Figure 1 shows a gas chromatogram of one region of Egyptian Onion Oil run on a Ultra 1 column at 4°C/minute. Thirty-two of the most prominent peaks of onion are numbered and identified with their respective levels in all the data reported.

450

Figure 1: Tj^ical GC profile of Egyptian Onion Oil

Table I is a summary sheet of the min/max levels found in three regions of Egyptian production sites. TABLE I: Summary m/n/max data of Egypf/an Onion production regions I, II, and III.

\REGION lA

REGION II

REGION IB

REGION II THEORETICAL

CHEMICi«lNAME

T

MIN

MAX

-

AVERAGE

MIN

-

MAX

AVERAGE

MIN

MAX

-

0.31

1.75

AVERAGE

MIN

MAX

AVERAGE

AyERAGE

1.66

0.93

Allyltiiiol

0.13

-

0.72

0.32

0.13

0.23

0.62

0.82

0.92

2.52

2

Mffthylallyi(ulfid«

0.02

-

0.09

0.03

0.01

-

0.02

0.02

0.00

0.34

0.08

0.02

0.12

0.07

0.06

3

Dimethyl Disulfide

0.06

-

0.59

0.22

0.02

-

0.03

0.09

0.04

0.35

0.20

0.12

0.55

0.26

0.23

4

Hexanol

0.02

0.24

0.15

0.03

-

0.17

0.11

0.01

0.12

0.08

0.02

0.10

0.06

0.10

5

trara-2-Hexanol

0.04

0.61

0.24

0.02

-

0.03

0.41

0.09

1.25

0.68

0.74

3.78

2.18

1.03

6

Meihyt ethyl disulfide

0.11

0.61

0.20

0.11

-

0.20

0.21

0.18

0.61

0.30

0.18

0.58

0.40

0.30

7

Allyl Propyl Sulfide

0.06

0.29

0.11

0.14

0.27

0.15

0.05

0.10

0.10

0.06

0.11

0.08

0.10

8

2,4-DimethylThJophene

0.04

0.11

0.07

0.06

0.09

0.10

0.08

0.34

0.14

0.15

0.29

0.20

0.14

3,4-DimethytThiophefw

0.42

0.72

0.52

0.31

0.45

0.55

0.62

2.46

0.95

0.91

1.88

1.24

0.90

10 cis-MethyM-Propenyi Disulfide

0.09

0.21

0.15

0.02

0.06

0.06

0.03

0.13

0.09

0.05

0.13

0.09

0.11

11 Methyl propyl disulfide

2.80

6.24

3.99

2.09

2.94

2.92

2.81

6.00

3.73

2.29

5.54

4.03

3.92

12 trans-Methyi-1-Propenyl Disulfide

0.77

1.41

1.09

0.27

0.57

0.55

0.98

1.70

1.24

1.21

3.94

2.41

1.58

13 Dimethyl Trisulfide

0.70

1.75

1.00

0.11

0.40

0.29

0.55

1.51

0.89

0.54

2.01

1.02

0.97

14 cis-Propyl Propenyl Disulfide

0.68

1.03

0.83

0.71

1.07

0.82

0.44

0.62

0.59

0.34

0.47

0.40

0.61

9

15 Nonanal

0.27

0.70

0.59

0.28

0.49

0.34

0.19

0.24

0.29

0.15

0.23

0.19

0.36

16 Dipropyl disulfide

13.89

20.92

17.46

33.03

41.64

33.34

15.65

34.33

21.15

10.13

21.10

15.58

18.07

4.40

5.86

5.01

4.94

7.94

5.30

4.50

6.78

5.49

5.11

17 trans-Propyl-Propenyl Disulfide

2.96

5.61

4.54

16 Methyl Propyl Trisulfide

7.05

6.60

7.83

3.48

6.75

5.00

8.04

13.37

7.87

5.68

9.21

6.85

7.52

19 cis-Methyl Propenyl TrisuHide

0.41

1.07

0.58

0.08

0.32

0.20

0.32

0.90

0.53

0.58

1.48

0.97

0.69

20 trans-Methyl Propenyl Trisulfide

0.44

1.00

0.66

0.10

0.35

0.24

0.00

0.99

0.61

0.75

1.63

1.21

0.83

21 Dimethyl TetrasuHide

0.26

0.50

0.33

0.00

0.18

0.14

0.27

0.47

0.35

0.32

0.67

0.49

0.39

22 Decanal

0.02

-

0.42

0.18

0.10

0.17

0.29

0.02

0.16

0.23

0.31

0.77

0.50

0.30

23 Methyl-1-(Methyl Thjo) Propyl Disulfide

0.06

-

0.29

0.17

0.04

0.06

0.09

0.04

0.56

0.28

0.53

1.60

0.99

0.48

24 Methyl Nonyl Ketone

0.21

-

0.40

0.35

0.49

3.18

2.60

0.35

-

0.54

0.86

0.23

0.49

0.43

0.77

0.58

0.12

0.39

0.32

0.57

28.19

15.31

11.97

13.99

13.79

13.37

0.55

25 cis-Propyl Propenyl Trisulfide

0.48

•

0.91

0.82

0.54

0.90

0.66

0.43

-

26 Dipropyl trisulfide

8.66

-

12.47

11.00

15.88

16.95

16.93

15.70

-

27 trans-Propyl Propenyl Trisulfide

0.17

-

2.58

1.25

1.09

3.24

2.88

1.09

-

7.67

3.02

5.13

5.44

5.38

3.22

28 Allyl propyl trisulfide

2.21

-

6.27

3.12

0.00

2.58

0.93

0.00

-

6.32

2.57

4.40

5.26

6.06

3.92

14.40

-

26.24

17.60

2.87

4.12

3.82

0.57

-

0.83

4.85

2.44

3.00

2.73

8.39

30 Methyl Undecyi Ketone

0.76

-

1.74

1.31

0.74

3.00

2.15

0.17

•

0.57

0.94

0.18

0.28

0.25

0.84

31 Dipropyl Tetrasulfide

0.62

-

1.35

1.12

1.10

1.24

1.22

0.33

-

1.22

0.96

0.94

1.70

1.34

1.14

32

4.82

-

6.14

5.67

1.81

3.06

2.11

0.42

-

0.65

1.83

0.35

0.97

0.70

2.73

29

2-n4<exyl-5-Methyl-2,3-Dihydrofuran^-one

2-n-Octyl-5-Methyl-2.3-Oihydrofura^3-one

-

451 A prominent percentage of dipropyl disulfide (DPDS) is found in onion oil. The increase and decrease of this level is not due to any procedural variations, however it varies because of the onion itself. The time of storage from the crop to the distillation is directly proportional to the level of DPDS; the longer the storage the greater the level.

98

100X

39

111

INT

182

\\\\ III, l,[ll 40

60

I

• ' • I ' « • I ' ! ' I • I ' I '

88

lee

120

148

« • I '

160

180

Figure 2: Mass Spectrum of 2-n-Hexyl-5-methyl-2,3-dihydrofuran-3-one C6H14S2O2 MW=182

98

imt

111 INT

39

55

69

85

210 • I ' I ' I ' { ' '"I""' ' ' I ' 180 120 140 48 60 160 180 200 220 Figure 3: Mass Spectrum of 2-n-Octyl-5-methyl-2,3-dihydrofuran-3-one C8H18S2O2 MW=210 •

^

^

^

The 3,4-dimethyl thiophene is believed to be formed from the decomposition of methyl propenyl disulfide when exposed to heat and sun. Methyl undecyl ketone is a degradation product of myristic acid. The 2-n-hexyl-5-

452

methyl-2,3-dihydrofuran-3-one (see mass spectrum, Figure 2) is believed to be "formed from a fatty acid precursor via the C-hydroxy diketone" [2]. Boelens in this pubHcation gave mass spectral data for hexyl dihydrofuranone, and we have found an unknown (pk# 32 on Figure 1) which has a molecular weight 28 mass units higher, which we have identified as 2-n-octyl-5-methyl-2,3-dihydrofuran-3one (Figure 3). The volatiles in the extracted oil are not present in the intact tissue of onion but are produced only when the tissue is injured or cut. The odiferous components are produced by the enzymatic cleavage of alliins. Alliins immediately react with the enzyme alliinase to form alliicin, which then readily form the mixture of sulfides that are found in the distilled essential oil [24]. Thiolsulfinates, pyruvic acid and ammonia are released by the interaction of the enzyme alliinase and the precursors, the S-alk(en)yl-L-cysteine sulphoxides [25]. The thiosulfinate is unstable and decomposes to a thiosulfonate and a disulfide. This explains the presence of alkyl di- and tri-sulfides. The total amount of pyruvic acid released depends on the pungency of the onion [26]. Other sulfides with lower molecular weights than methyl propyl disulfides in are unlikely to be found in large percentages in the oil because of their higher solubility in the water phase of the distillation and their greater volatility. Therefore the presence of such chemicals as allyl disulfides and alkyl sulfides in large percentages would indicate tampering with the natural oil. Garlic oil is abundant in allyl disulfides; therefore any adulteration of onion with garlic will significantly alter the concentrations of methyl allyl disulfide and 1-propyl-allyl disulfide. Another indication of adulteration would be the synthetic addition of allyl disulfides. Synthetic allyl disulfides contain only one of the isomers, while natural allyl disulfides occur as both the cis- and trans - isomer and at a characteristic ratio [4].

ACKNOWLEDGMENT The authors would like to thank Gideon Andemicael, Nehla Azzo and Ben Benveniste from Kato Worldwide Ltd and Eng. Mohamed Abed Al Al from Kato Aromatic for their assistance and data used in this paper.

REFERENCES 1 2 3

J. Lawless, The Encyclopedia of Essential Oils, Barnes and Noble (1995) 142. M. Boelens, P. J. De Valois, J. Wobben, and A. van der Gen, Volatile Flavor Compounds from Onion, J. Agr. Food Chem., 19, 5 (1971) 984. M.H. Brodnitz, C. L. Pollock, and P.P. Vallon, Flavor Components on Onion Oil, J. Agr. Food Chem., 17, 4 (1969) 760.

453 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

M.H. Brodnitz and C.L. Pollock, Gas Chromatographic Analysis of Distilled Onion Oil, Food Technology, 24 (1970) 78. A. Sass-Kiss, M. Petro-Turza, J. Sparfoeldi-Szalma, and J. Pino Investigation on the volatility of Mako onion. Nahiung 33, 5 (1989) 413. G. Freeman, J. Sci Food Agric, 26 (1975) 471. B.M. Lawrence, Essential Oils 1976-1977, Allured PubUshing Corporation, (1979) 14. W.G. Galetto and A.A. Bednarczyk, Relative flavor contribution of individual volatile components of the oil of onion. J. Food Science, 40 (1975) 1165. Food Chemical Codex, 3rd edition. National Academy Press (1981) 208. EGA No. 183, ECC III (1981). G. Reineccius, Source Book of Flavors, 2nd edition. Chapman & Hall (1994) 85-86. J.F. Carson, Chemistry and Physiology of Flavors, Avi (1967) 390. S. Arctander, Perfume and Flavor Materials of Natural Origin, D. Van Nostrand Company, Inc. (1960) 468. K.T. Farell, Spices, Condiments, and Seasonings, 2nd Ed., Van Nostrand Reinhold (1990) 146-153. F.W. Semmler, Arch. Pharm., 230 (1982) 443. A. R. Saghir, L. K. Mann, R. A. Bernhard, and J. V. Jacobsen, Proc. Amer. Soc. Hort. Sci., 84 (1975) 386. M. H. Brodnitz and C. L. Pollock, Food Technology, 24 (1970) 78. W.G. Galetto and P.G. Hoffman, J. Agric. Food Chem., 24 (1976) 852. S. Schimmer, Phytochemistry, 7 (1968) 401. S. Schimmer, J. Agric. Food Chem., 19 (1971) 980. R. A. Bernhard, J. Food Sci., 33 (1968) 298. G. Freeman and R. Whenham, J. Sci. Food Agric, 25 (1974) 517. M. Albrand, P. Dubois, P. Etievant, R. Gelin, and B. Tokarsha, J. Agric. Food Chem., 28 (1980) 1037. C. Bandyopadhyay, A. N. Srirandarajan and A. Sreenivasan, J. Chrom., 47 (1970) 400. E. Block, Sci. Amer., 252 (1985) 94. A. A. Gbolade, J. Essent. Oil Res., 4 (1992) 381.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

455

Melanoidins in the Maillard reaction T. Obretenov^ and G. Verninb*

^Higher Institute of Food and Flavor Industries, Department of Organic Chemistry, 26 Maritza Blvd, BG-4000 Plovdiv, Bulgaria

t>Laboratoire de Chimie des Aromes-Oenologie (CNRS, ESA 6009) Faculte des Sciences et Techniques de St-Jerome, Case 561, Avenue Escadrille Normandie-Niemen F 13397 Marseille Cedex 20, France

Abstract This paper is a review of the studies on melanoidins, the end heavy products of the Maillard reaction. It deals with the description of melanoidins in foods and model systems as well as their physical and chemical methods of analysis, their chemical properties and their biological and antioxidative activities in vivo and In nature. Some reconstructed mass spectra using our SPECMA 2000 data bank are also reported. The paper includes research prospects in a field which has been almost unexplored until now. 1.

INTRODUCTION

A great number of chemical reactions occur during the thermal and technological treatment of food. The main reactants are reducing sugars and aamino acids. Their reaction yields specific aromas and brown pigments known as melanoidins. French chemist, L-C. Maillard from Lorraine, was the first to describe this reaction in 1912, during a lecture at the French Academy of Sciences [1]. He was both a doctor and a biologist. He thought of replacing glycerol with glucose during the synthesis of peptides from amino acids. A large amount of carbone dioxide was given off, and brown pigments formed which he named melanoidins. This work was abstracted in Chemical Abstracts in 1950. The studies were continued by Hodge in 1953 [2]. * Address for correspondence

456

The reaction was then called the "non-enzymatic browning reaction". Maillard aroma is a well studied topic with many books and six International Symposia devoted to them. The situation is quite different as far as the brown pigments are concerned. They can be compared in some ways to humic substances. This paper reviews the literature on melanoidins 2.

MAIN PHASES OF THE MAILLARD REACTION

According to Hodge [2] the outstanding features of the Maillard reaction can be summarized in three steps: 2.1. Primary steps * Condensation between a reducing sugar and an a-amino acid; formation of a Schiff base, then of a glucosyl amine; formation of Amadori (1-amino-1-deoxy-2ketose) and Heyns (1-aldose-2-amino-2-deoxy) intermediates. * Rearrangement of the previous intermediates to reductones and dehydroreductones. This step does not give rise to an absorption in the visible or in the near UV. 2.2. Intermediate steps * Dehydration of the glucidic moiety. * Ose fragmentation. * Amine degradation which shows a strong absorption in the near UV. * Strecker reaction giving rise to a-amino ketones and aldehydes from aamino acids and a-dicarbonyl compounds. 2.3. Final steps * Polymerization between aldehydes and amines leading to the formation of melanoidins giving a strong absorption in the visible range. * Formation of volatile aroma compounds, especially heterocycles Including furans, pyrazines, pyrroles, oxazoles, thiazoles, etc. This scheme has been updated by Vernin etal [53 ](See Scheme 1). The food industry and consumers are directly affected by the formation of melanoidins because they result in a loss of nutritional value. They are not only found in every day consumer foods (coffee, caramel, baked bread, cereals, dairy products, cooked meat etc.), but also in biological systems. Hence, they are very interesting as far as nutrition, physiology and the environment are concerned.

457

n

REDUCING SUGARS + a-AMINO ACIDS MAILLARD

-[ Cetosylamines and aldosylamines

)

AMADORI AND HEYNS INTERMEDIATES

1 -Amino-I -deoxy-2-ketoses + 2-Am ino-2-deoxy-1 -aldoses

t

2H2O

t Reductones

^ .^

STRECKER

Aldehydes and a-Amino ketones

^

-3H20

Dehydroreductones

I

Furfural, 5-Hydroxymethyl furfural 5-Hydroxymethyl N-alkyl-2-formyl pyrrole

Retroaldolization

a-Dicarbonyl-, and a-hydroxylated carbonyl compounds

H2S NH3

Aldolization

Aldols Aldimines Cetimines Polymerization

t VOLATILE FLAVORING COMPOUNDS Aliphatics, alicyclics, heterocycles (more than 3,000 compounds)

"MELANOIDINS" Brown pigments (M.W.: 10,000-100,000)

Scheme 1. The various steps of the Maillard reaction according to Hodge [2], up-dated by Vernin etal [53].

458 3.

MELANOIDINS IN FOOD AND MODEL SYSTEMS

The main work on melanoidins is summarized in Table 1. Amino compounds (a-amino acids, peptides, proteins), carbonyl compounds (reducing sugars), vitamins and minerals (oligo elements) are found in all foods. When food is subjected to thermal or technological processes (whether prepared, canned, pasteurized, frozen etc.) it undergoes changes in color, texture, odor and flavor as well as in biological activity [3,4]. Such changes depend on the water activity, the pH and the temperature as well as the presence of catalytic metal ions. Numerous studies were devoted to the changes in color due to the formation of melanoidins which can be measured by their UV absorption between 420 and 700 nm. The first melanoidins studied were those in soy sauce [5]. Soy sauce is of outstanding importance as far as Asiatic food (especially Japanese) is concerned. The studies were extended to other melanoidins such as those found in cane syrup (molasses) from the alkaline degradation of sugars [7], malt [8,9], beer [10], roasted coffee [11], cooked meat [12], pasteurized dairy products [13], roasted barley [14] etc. Those of grapefruit juice were separated by HPLC. The influence of storage time and temperature were also studied [15]. Melanoidins found in malt [16], beer [17], meat [18-20], bread [21-24], and coffee [11,25,26] have been isolated and characterized. The melanoidins formed in model systems containing amino and carbonyl compounds were also studied [28-30]. The reaction is a first order reaction as far as the reducing sugar is concerned and a second order reaction as far as the two reactives are concerned. The reactions can follow different kinetic patterns as described by various equations [33-35]. Twenty years after Maillard, Enders [36] synthesized melanoidins (called standard melanoidins) from a glucose/glycine model system. Other models were studied (See Table 2). These studies proved that the elemental composition of melanoidins is strongly influenced by the nature of the starting materials, i.e. sugars (ribose, glucose, fructose, maltose, sucrose) and a-amino acids, especially glycine, as well as their molar ratio. Some experiments were carried out without a solvent. In aqueous medium, the elemental analysis of melanoidins obtained from the reaction of glucose, fructose and 5-hydroxymethylfurfural with glycine (or methionine) are very close. The amount of nitrogen is about 6%. For glycine, the polymer obtained results from the combination of one mole of the amino acid and one mole of the carbonyl compound. The reaction leads to the loss of three water molecules and does not show any maximum absorption in the UV [38]. The melanoidins obtained by the action of D-glucose on para-chloroaniline are less stable, but permethylation increases their stability. The molecular weight is about 1000. Oxidation, reduction and thermolysis reactions generate free radicals observed by ERR, as well as aromatic compounds.

CO

c o CO

0 •D

CO

C/)

c

CD

"o c JO

E c o 0 Q. CO

Q.

CO

E E CO


s o

<

•g

o c JO 0

0) Q. O •D C CO CO CD C

c

o c:

I

o

;r^ T— s-^ ^

Q> CO « CM > Tt ® ^

CO _ ~

CD C CO

"cB -g ^

c ^-c

r:.-£^

® o CO

"3 ;:^ "kS

C

CO ••«. "T^

oo" "co . ^ t-" ;r; 2 h*

^ o >,! a> S S S"co 2 ? ^-g 2 2 2

irT "co 03

CO" T—

0)

"co

1 CO

(D

^

C CD O ^ -tt •« -K o D .£ "o u J3 -Q -Q Js: O (D

i

CD "O 0 CO

^

- i CO

(D

"fe ^ "^

TS ^ CO ^

CD

G , CO

O

J2 ^ c C ^

(0

2^5 to"

8« 5 ^

c 'o c CD OC m O CE O Q

8^ "co "S $81832-5

:t= CD

II I

i^CQI-OCOOOSDCS

(D

si o o CO 2

CO CD

459

I 2

I 03 «

gill i f ^1 >- .J5 .J2 O )

O (^ cS CC

460

•o

0 '••-»

c c o O

cd

n -S .a c

mm "nj * ^

mill 0) D

"5 S X3 •g (0 (0 0)

g. 2a (/>

.ti

c .o

8. E o O

nil

-I O

- ^ ^ " X : "te 76 f^ ^ "^

LL ^

oi 0

"5 V

i

"co

.>

CM . > CVJ

T - CM

t=

0%

im

en

^ o "ft ^

00

co^

S fT o> e

»-"«;£

J's-ia i| ^^

. 0)

•§*5: S3>^ ^mg i^ScS h^ " - ^ ^ •cB^-S ^ "ft To

CO

LL 0 0

co" G>^

"cc 0)

^

CO ^ "fe- 0)

8S z --

U . LU

£ D

0)

.c

"co

S S •S

c c

1

CO 0)

CM

IE

(D

1 iS

1

i

8 g

z (0

g

^

§:||

4

S5.1E

>^^-^

lili if 1^ lilt t|!

IIJI

.^ .>

1 s

^o CQ

s<

"•d-

.o

CO

CO

c LU

CO

95

J

I

§

CD

(3

CO

o 0 > CD

0) I 0) .•S «

5 CO

-S

CO

2 N

i'i il

^-v

CD

^

CO

^ ^ .

-PR E

^

I X DC

"-^^ • F CO £ N

CO

0

c

^^ a

CO

"2

(0

c

(D

E

CO

CO

o 0 .>

t ^

Is

.1

o c i5

^

•B I -^

I ^

5 .2 ?.2 S, Is "(H ^ l ) "S •5 J "o 'co

F ^ ^ O

3 D « o o ?>

-c Z

461

462

•g CO

o

g CO Q. CO CO

0 O)

c o c

•D

CO 0 (O O

o O) E o **c

CO

"D

o c i5

0

E "o •D

C >^

o E E o CO *^ o CO cvi'S ^ £ JQ CO

<

I

g

I

E

CJ> 3

V)

< 1 o> c

cr

8 X

c

o

i (A

0

S

(A

"CO

i

11 1 s

t $ CO

cvi

CO

M

1 %s

< d

CM

12 O

1

(D

^-^

Ii

5

O

o

(0

I1

8.0

8,1

LU

II I 0)

1

o

I $ 10 10

SI

s

T-

I 9,

CD

I 2 OI 2,(5 ^

463

Reaction between D-glucose and glycine [40] or lysine [41] when done in the presence of hydrogen sulfide or sulfur (IV), are slower, showing a weaker absorption at 450 nm. Sulfur is also incorporated. Sulfur incorporation in organic sediments during the first steps of diagenesis is an important factor to control because it determines the formation of geopolymers of a melanoidin kind in sediments. This geochemical aspect was discussed by Ikan et al [30]. Metal ions (Ca, Cu, Zn, Mn), the formation of free radicals by thermolysis, and the modification of a-amino acids are important factors which can influence the chemical nature of melanoidins arising from model systems. 4. PHYSICO-CHEMICAL METHODS OF MELANOIDIN ANALYSIS Elemental analysis (C,H,0,N) of melanoidins is achieved using microanalyzers on a 1.5 mg scale. The quantity of sugar per mole of amino acid included in the melanoidin molecule can be calculated [37,40]:

C(ia + Pb - x)

H(ma + qb)

0(na + rb - 2x - y)

Nb

in which

a is the number of aldose molecules which contain I, m, n atoms of C, H, O. b is the number of amino acid molecules containing p, q, r, atoms. X and y are CO2 and H2O molecules released during the reaction. This formula allows one to draw Important conclusions on the composition of melanoidins. For instance, the ratio y / a defines the level of dehydration. Most of the time it is equal to 3. The level of decarboxylation (x) is also interesting, but is more prone to variation. Wedzicha et al [40] have set the following equation on the basis of the glucose/glycine model system:

1.25C6H1206 + C2H5NO2

— > - C9.3Hi2.5NO5 + 3.73 H2O + 17 0 0 2

This equation could be applied to other models. It was noticed that the melanoidins whose molar mass is between 12,000 and 100,000 have very similar stoichiometric ratios. This means that nondialysable melanoidins are very homogeneous and that polymerization reactions are not very specific and regularly take place [40].

464 Dialysis, high performance liquid chromatography (HPLC) and electrophoresis are widely used to purify and separate model melanoidins [33,34,37,46,47]. As far as food melanoidins are concerned, the process is more complex and requires a combination of several techniques such as extraction, dialysis, gel permeation, HPLC, ion exchange chromatography, lyophilization etc.[12,16,17,48]. In all cases HPLC remains the most frequently used method [10,15,24,37]. As melanoidins are not volatile compounds, gas chromatography cannot be used. Among spectroscopic techniques, UV is not the best because melanoidins do not show accurate maxima owing to the numerous chromophores they contain [12,16,17,27,38]. Melanoidins strongly absorb in the visible region, but do not give clear maxima. This region is used to monitor the Maillard reaction in food and model systems. "•H- and ''^C-NMR give characteristic signals for the following compounds: olefins, aromatics, carbonyl (See below):

Groups

1HNMR chemical shifts (ppm)

- CH2" and CH3 (aliphatics) - CH2 (alicyclics) CH3 (aromatics)

1.05 -2.5 1.25 -2.0 2.0 - 2.2

- CH2NC. CH3NC (amines) - CH-0, - CH2O -, CH3O - (ethers)

2.2 - 2.9 3.25 -3.6

HC = C C (ethylenics) I Aromatic (ArH) and heterocyclic (Het-H) protons

4.5- 7.5 7.0- 7.25

Analyses performed using 13c- and ISfsj-NMR show the incorporation of a carboxylic function (COOH) and a carbon atom a to the amino acid as well as indolic, pyrrolic and amido fragments [29,30,38]. Feather and Nelson [38] recorded "I^C-NMR spectra of melanoidins from the following model systems: D-glucose/glycine, D-glucose and D-fructose with the glycine labeled "^^C on carbons in the 1 and 2 positions. The "^^C NMR spectrum of D-glucose/glycine 1-''3C-labeled reveals a broad signal between 155 and 166 ppm which is characteristic of a carbonyl function.

465 It seems that part of the carbon in position 1 is eliminated as CO2, while part is incorporated in the polymer. With the glycine labeled on the carbon in position 2, a signal is observed at 60 ppm which is characteristic of substituted methyl groups. It seems that the two carbon atoms of glycine are incorporated in the polymer. Dialysable portions of polymers obtained from the glycine labeled on the carbon in position 1 and the fructose give signals at 175.6, 173.6 and 172.1 ppm, while that obtained from the glycine labeled on the carbon in position 2 gives signals at 43.4, 42.7 and 42.2 ppm. The spectra suggest structural differences between polymers obtained from glucose and fructose. "•^C-NMR spectra of polymers obtained from glucose/glycine are very similar to those of the corresponding Amadori intermediates. EPR analysis shows the formation of radicals but does not give any information about molecular structure. Several IR studies were done on food melanoidins and model systems [17,25,29,30,33-35,37]. Different functions were characterized: -OH, :rNH at3240cm-1, ^ C - H at 1930cm'^, ::c = 0 at 1710cm""", ;::C = N-, Z:C = C C at1630cm-^ ' ^ C - O - , and and

^C-NC

at 1200cm""",

— 9-C-at C - C - at 1020 cm"'

New techniques such as combination pyrolysis by Curie points, GC, NMR or "•^C-CP-Mass NMR have confirmed Maillard's hypothesis about a similarity between humic substances and melanoidins [29,30,50]. Melanoidins show a main peak in the aromatic region centered at 135-136 ppm. "•^N-CP-Mass NMR have been applied to analyse xylose/glycine ("^^N) melanoidins [102]. Observed signals in the region of 60-150 ppm are suggested to be due to secondary amide, pyrrole and indole-Iike nitrogens. In the case of glucose/glycine ("^^N) the "•^N.cp-Mass NMR spectra show a broad peak at 0-70 ppm, a large peak at 70-120 ppm corresponding to conjugated enamines and partly to amides, and a shoulder peak at 120-170 ppm estimated to be mainly due to - C = N--K . The corresponding graphs have been reported by

Hayase[129]. From these analytical data, one can formulate hypotheses on the structure of melanoidins [28-30,37,39]. Some authors suggest furanones and pyranones as a basic unit. Others tend to show a structure based on pyrazines and aromatics. Kato and Tsuchida [109] claim that the furan ring is the most abundant.

466 With the extreme variety of starting materials as well as the various experimental conditions involved in the formation of melanoidins, whether food or model systems, one cannot assert a single structure. There are different structures even if they show the same patterns. As an example, aldolization between aldimines and aldehydes (See Scheme 2) as well as polymerization between 5-hydroxymethyl furfural (or the corresponding pyrrole derivative) and 1-(2-oxo-hydroxyethyl) furan are well known. In this case, N-alkyl-2-formyl-5-hydroxymethyl pyrroles also can polymerize as their furan homologs. A basic unit for brown pigments from Heyns intermediates has been suggested by Kato and Tsuchida (See Scheme 3).

5. CHEMICAL

PROPERTIES

Melanoidins are brown amorphous substances both hygroscopic and photosensitive. Their solubility in water depends on their molar mass and polarity. Those which have a low molar mass are soluble in ethanol and chloroform, and partially soluble in organic solvents of low polarity. They are not soluble in nonpolar solvents such as aliphatic and aromatic hydrocarbons. They are quite stable in anhydrous solvents and in a dark environment. The polarity of melanoidins mainly depends on the presence of carboxylic groups. Those obtained from sugars and amino acids are more polar than melanoidins formed from lipids. In this case, they are strong lipophiles [12,16,18-20]. In aqueous medium their structures depend on the pH, which involves a change in the absorption maximum and its intensity. In practice one substitutes mobile hydrogens by methyl groups. The methylation is done using methyl iodide in DMSO under a dry nitrogen atmosphere, at room temperature (20°C). The methylated product is extracted with methylene chloride, then washed with distilled water and dried over anhydrous sodium sulfate [43]. Melanoldin hydrolysis is done at high temperature and in acidic (HCI, H2SO4) or basic (KOH) medium. Products thus obtained are extracted with diethyl ether [39,50]. The action of oxidizing agents (KMn04, H2O2, K2Cr07, O3, meta- chloroperbenzoic acid) gives information on the polymeric structure of melanoidins. Hydrogenation by Raney nickel in THF (or by LiAIH4) was also used [39]. Melanoidins give metallic complexes with different salts. They also trap hydroxy free radicals and hydrogen peroxides and superoxides [129]. Both properties are responsible for their in vivo antioxidative activities [52]. Above 200°C and under an inert atmosphere, melanoidins can depolymerise especially those obtained from glucose and para-chloroaniline [39]. Products thus obtained are benzene, naphthalene and quinoline derivatives.

467

CHa I CH Ml

CH2

I CHa I C=0

I HC — I NH I

Ri

Polymers

I CH I C=0 I

Scheme 2. Polymerization of aldimines with carbonyl compounds

Polymers

CH=0 1 C = N-R I CH2 I HC-OH I HC-OH 1 R'

t HOHgC

N I R

X—CH HC I CH II C-OH I •CH I R'

CHO

R I N —

CH II

CI CH C-OH I HC-OH I R'

R I N

CH2 R I I C — N I

r 3

1

CH I I — r.H R'

CH=0 I C-NH-R II CH I HC-OH I HC-OH I R'

CH2 R I I C - N II I CH I C-OH II C-OH I R'

Scheme 3. Basic unit for brown pigments from Heyns intermediates according to Kato and Tsuciiida [109] (R' = H or CH2OH).

468 The thermal degradation of beer melanoidins releases furan derivatives [17]. During the thermal degradation of apple juice melanoidins at 300°C under a nitrogen atmosphere, furans, benzofurans, volatile phenols, aromatic and naphthalenic hydrocarbons were identified [131]. Some mass spectra reproduced from our SPECMA 2000 data bank are given in Figures 1-4. On the whole, the chemical properties of melanoidins have been little studied. 6. BIOLOGICAL AND ANTIOXIDATIVE ACTIVITY 6.1. Biological and physiological activities Melanoidins have interesting biological and physiological activities. Their desmutagenic effects have been discussed by Hayase [105,106,132]. According to the Ames test [101], melanoidins from the glucose/glycine model system have strong desmutagenicity against their heterocyclic amines such as 3-amino 1methyl-, and 1,4-dimethyl, -H-pyrido [4,3b] indoles (Trp-P-1) and 2-amino-6methyl-dipyrido [1,2a:3',2'd] imidazole which are mutagenic and carcinogenic [129,130]. They are formed by heating sugars and amino acids (or protein pyrolysates) at high temperature [133]. Melanoidins from the ribose/lysine model system (whose molar mass is about 12,000) could be desmutagenic and antibacterial [46]. Besides heterocyclic amines melanoidins also showed a desmutagenic activity of 25-75% against mutagenic aromatic or heterocyclic compounds such as aflatoxin B i , benzo [a] pyrene, 2-aminofluorene, 4-aminobiphenyl and 2-aminonaphthalene. They have no mutagenicity without metabolic activation by cytochrome P-450 in hepatocyte. Desmutagenicity of melanoidins is due to the action against hydroxylamine from the heterocyclic amines. Melanoidins are supposed to react directly with NHOH group of the amines or scavenge the active oxygen species. It also was suggested that they show the desmutagenic activity in vivo in digestive organs against Trp-P-1 because part of them were absorbed through the gastrointestinal tract of rats [134]. Melanoidins showed no desmutagenic activity against mutagenic and carcinogenic nitrosamines, which are formed by the nitrosation between nitriles and secondary amines in digestive organs and in processed foods. However, owing to their strong reducing ability (as well as ascorbic acid) they inhibited their formation by the reduction of nitrite. Hydroxyl radicals liberated by oxidation of hydroxylamines may damage DNA molecules. ESR studies have shown that melanoidins at a concentration of 0.3% scavenged 86% of these radicals.Scavenging activity of melanoidins on hydroxyl radicals was much higher than that of known scavengers such as fructose, mannitol and bovin serum albumin. It may be due to the unique partial structures in their molecules such as: reductones, enamines or pyrrole-like structures. On the other hand, melanoidins give rise to related stable free radicals which are supposed to scavenge hydroxyl radicals.

469

Chemical name:

2-ETHYL-5-METHYLFURAN

Origin & ref:

WINES;REF.MS :APPLE JUICE MELANOIDINS;VERNIN & OBRETENOV, 1996.

Molecular formul C7 HIO Ol IKA:

770

IKP:

FEMA: 0

(110)

1025

COE 0

R.N (CAS): 1703 52 2 DIK

255

CA: 107 38191 d

Family: Heterocycles Furans

lOFI 2

Occurence:

Maillard/Model Systems

Descriptor odor/Flavor ETHEREAL /

0

20

40

60

80

100

120

140

160

200

180

220

240

260

280

300

320

El: 43(100) 95(62) 39(28) 110(20) 96(12) 58(10) 42(10) 53(6) 51(5) 65(3)

^.^•-BIMETHYL^qH) FUBANOND Chemical name:

2,5-DIMETHYL-3(2H) FURANONE

Origm & ref:

APPLE JUICE (MELANOIDINS);VERNIN & OBRETENOV, 1996.

Molecular formul C6 H8 0 2 (112) IKA:

915

FEMA: 0

R.N (CAS): 14400 67 0

IKP: 0

DIK

0

COE 0

lOFI 2

CA:

Family: Heterocycles Furans Occurence:

Maillard/Model Systems

Descriptor odor/Flavor

100

20-1

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I'

100

120

140

160

180

200

220

240

260

280

El: 43(100)97(18)112(3)

Figure 1. Mass spectra of 2-ethyl-5-methylfuran and 2,5-dimethyl-3(2H) furanone

300

320

470

N° 105

Chemical name:

FURFURAL

Origin & ref:

APPLE JUICE,MELANOIDINS,VERNIN & OBRETENOV ,1993,N,2933.

Molecular formul C 5 H 4 0 2 (96) IKA:

820

IKP:

FEMA:2489

1450

R.N(CAS): 98 01 1 DIK

COE 2014

630

lOFI 2

CA:0:0

Family: Heterocycles Furans Occurence:

Essential oils & Maillard

Descriptor odor/Flavor SWEET,WOODY,ALMOND,FRAGRANT,BAKED BREAD / IDEM 1 0 0 - -EI

39 96 95

80-

//

1\

^J

.H

\

60-

0 40-

20-

29

0 -

'^li r^"T"'f*pi'

0

20

40

|67

60

1 ""'"''"''""T'i'T" 1 1 111 1 1 11 1 1 1 1 1 1 1 111 11 1 1 1 1 i"'i'i"f^

80

100

120

140

160

180

200

220

240

1 .1 .

260

M 1 M M 1 1 1 1 r |l

280

300

320

El: 39(100) 96(94) 95(87) 29(22) 67(8) 51(4)

WimWimJh ALCOHOL Chemical name:

FURAN,2-HYDROXYMETHYL

Origm & ref:

WINES;REF.MS:APPLE JUICE( MELANOIDINS);VERNIN & OBRETENOV, 1996.

Molecular formul C5 H6 0 2 (98) IKA:

850

IKP:

FEMA: 2491

1635

COE 2023

R.N (CAS): 98 00 0 DIK 785 lOFI 2

CA: 0

Family: Heterocycles Furans Occurence: Maillard

Descriptor odor/Flavor LOW;COOKED SUGAR,CHARACTERISTIC, M1LD,MUSTY HAY,BURNT / BITTER,WARM,CREAMY,WOODY IN BEER AND SUGAR CANE;TV:5 ppm/Water. 1 0 0 - -EI

98

80-

a^»

60-

40-

42

53 81

20-

1:9 1 31

0-

'1 p Mil

1 1 1 11 1 11 1 i

0

20

40

97

60

80

1 1 1 II 1 1 II 1 1 II 1 1 11 1II 1II1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 100

120

140

160

180

200

El: 98(100) 53(45) 42(42) 97(36) 81 (35) 70(18) 39(17) 51 (16) 69(14) 31 (10)

Figure 2. Mass spectra of furfural and furfuryl alcohol

220

240

260

280

300

320

471

N° 103

Chemical name:

PHENOL

Origin & ref:

APPLE JUICE(MELANOIDINS), VERNIN & OBRETENOV, 1996.

Molecular formul C6 H6 Ol IKA:

955

IKP:

FEMA: 3223

(94)

1930

R.N (CAS): 108 95 2 DIK

COE 0

975

CA: 0:0

Family: Aromatics Phenols

lOFI 2

Occurence:

Maillard/Model Systems

Descriptor odor/Flavor

100

0

20

40

60

80

100

120

140

160

180

200

El: 94(100) 39(50) 66(35) 65(30) 40(26) 55(14) 50(10) 51(9) 95(7) 63(7) 27(4)

:2,|KDIMETHYLFimM0L <^,$-XYIJBNOL) N° 444

Chemical name:

PHENOL,2,5-DIMETHYLL

Origin & ref:

APPLE JUICE(MELANOIDINS),VERNIN & OBRETENOV, 1996..

Molecular formul C8 HIO Ol IKA:

(122)

R.N (CAS): 95 87 4

1150

IKP: 0

DIK

0

FEMA: 3595

COE 0

lOFI 2

CA: 0:0

Family: Aromatics Phenols Occurence:

Essential oils & Maillard

Descriptor odor/Flavor MEDICINAL,PHENOLIC. /

00- -EI

107 OH

80 -

1

M

122

60-

40 -

1^43

20-

41

121

77 |53 |65

^

0- 1 1 1 1 1 1 1 M l| 0

20

40

60

91

|79 80

100

f ^ 'l 1 1 1 1 1 1 M 1 1 1 11M 1 11 111 11 11 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 120

140

160

180

200

220

240

260

El: 107(100) 122(66) 121 (50) 39(48) 43(44) 77(26) 41 (22) 91 (22) 79(14) 65(12) 53(12) 63(7) 93(5)

Figure 3. Mass spectra of phenol and 2,5-dimethylphenol

280

300

320

472

g-MHTHYI^wETHYLBENZ^FURAN N° 2006

Chemical name:

BENZOFURAN,2-METHYL-3-ETHYL

Origin & ref:

APPLE JUICE( MELANOIDINS), THERMAL DEGRADATION,VERNIN & OBRETENOV, 1996.

Molecular formul CI 1H12 0 1 (160) IKA:

1280

FEMA: 0

IKP: 0

DIK 0

COE 0

lOFI 2

R.N (CAS): 40484 94 4

CA:0:0

Family: Fused heterocyclic compounds Benzofurans Occurence:

Maillard/Model Systems

Descriptor odor/Flavor

100

El: 145(100) 160(58) 39(53) 43(41) 51 (41) 124(41) 110(31) 159(28) 123(25) 78(25) 53(23) 41 (20) 77(20) 55(19) 91 (19) 63(13) 65(12)

%MMTWm-'J-WmYLBENm¥VMAN Chemical name:

BENZOFURAN,2-METHYL-7-ETHYL

Origm & ref:

APPLE JUICE(MELANOIDINS),THERMAL DEGRADATION,VERNIN & OBRETENOV, 1996.

Molecular formul CI 1H12 0 1 (160) IKA: 1288 FEMA: 0

IKP: 0 COE 0

DIK 0 lOFl 0

N° 2007

R.N (CAS): 0-0-0

CA:0:0

Family: Fused heterocyclic compounds Benzofurans Occurence: Maillard/Model Systems

Descriptor odor/Flavor 100

43

•EI

160 ^9 159

5 121

20 4

TTTT-

20

180

200

220

240

260

280

300

El: 43(100) 145(94) 160(89) 39(83) 159(72) 51(66) 41(63) 55(48) 115(34) 53(33) 78(32) 91(30) 121(30) 50(30) 77(28) 57(26) 110(24) 52(23) 124(20) 135(20) 105(19) 65(17) 63(16) 79(13)

Figure 4. Mass spectra of 2-methyl-3-ethylbenzofuran and 2-methyl 7-ethylbenzofuran

320

473

6.2. Antioxidative effects of melanoidins Initiation of the oxidation of either carbohydrates or lipids stimulates the oxidation of the other, and these autooxidation reactions generate reactive oxygen species including oxygen, and hydroxyl radicals, peroxides and metal-oxo complexes. Reactive dicarbonyl intermediates and aldehydes are also formed in these reactions leading to browning and crosslinking Maillard reactions. It is now evident that non-enzymatic oxidation reaction known as "oxidative stress" modulates the impact of the Maillard reaction in vivo. Baynes [135] claims that reactive oxygen species are formed in metabollic reactions particularly as side products of mitochondrial electron transport, and also in non-enzymatic autooxidation reactions involving oxidizable substrates, metal ions and molecular oxygen. On the other hand, the nonoxidative adduction of sugars to proteins or nucleic acids yields Schiff bases and Amadori products. Consquently, DNA and protein may be damaged directly in these oxidation reactions during natural aging and in diseases such as diabetes and artherosclerosis. Modification of proteins by carbohydrates is accelerated during hyperglycemia in diabetes and is implicated in the development of the long-term complications of these diseases including retinopathy, nephropathy and generalized vascular diseases. These reactions are greatly accelerated in the presence of oxygen and transition metal. Ascorbate a strong reducing agent which is often used as a sacrificial antioxidant in foods, inhibits browning reactions in reducing catalytic metal ions and reactive carbohydrate intermediates in oxidation reactions. The browning of protein by ascorbate first requires the oxidation of ascorbate to the dicarbonyl sugar, dehydroascorbate. Then, browning of proteins takes place under anaerobic conditions as well as deoxyosones. The antioxidative activity of melanoidins discovered by Franzke et al [62] was tested on products of the Maillard reaction. They were obtained from amino acids, protein hydrolysates and sugars [63-67,112]. It was also tested on melanoidin fractions [68,69]. Melanoidins are supposed to scavenge hydroxyl radicals, peroxides and superoxides depending on their concentrations and molar masses. Glycated proteins resulting from the condensation of reducing sugars with proteins, possess antioxidative activity and are expected to have scavenging activity against these active oxygen species. 7. DECOLORiZATION OF MELANOIDINS BY MICROORGANISMS Melanoidin decolorization by microorganisms is an up-to-date topic, particularly in the food industry (cane, molasses, soy sauce, "miso", cooked meat products.).

474

One of the first reports on enzyme decolorization of melanoidins by various Coriolius species was made by Watanabe etal [58]. Thus, Coriolius species No 20 decolorized a melanoidin solution with a decrease of A/ 80% in darkness under optimal conditions. The decolorization occurred with an intracellular enzyme prepared from an extract of integrated mycelia , and required aeration and various sugars, particularly, glucose and sorbose. The fraction with melanoidin decolorizing activity (MDA) was collected and purified by DEAE-cellulose and sephadex G-200 column chromatography. The optimal pH and temperature were pH 4.5 and 35°C, respectively. The molar mass was ^ 200,000 by SDS-gel electrophoresis. The purified enzyme was identified as sorbose oxidase, and decolorization proceeded in the presence of oxygen and sugars as maltose, sucrose, lactose, galactose and xylose, besides glucose and sorbose. Glucose in the reaction mixture was converted to gluconic acid. Melanoidin was suggested to be decolorized by active oxygen formed by the reaction. The distribution of molasses pigment melanoidin decolorizing activity (MDA) was investigated in various Basidiomycetes for possible application to biological treatment of molasses fermentation wastewater. MDA was only found in some genera of the white-rot fungi group of which Coriolius versicolor Ps4a showed high activity and a decolorization yield of A^ 80% under the optimal conditions. MDA by C. versicolor was almost completely coincident with the growth of mycelia . The main MDA was due to intracellular enzymes and induced by the molasses pigment. The induced enzyme consisted of two types, namely a sugardependent enzyme, and a sugar independent enzyme. The decolorization by C. versicolor was due to the decomposition of the molasses pigment [136]. The same authors extracted melanoidin decolorizing enzymes (MDE) from mycelia of C. versicolor Ps4a and putrified them by DEAE-Sephadex, DEAE-Sephacel, and Sephadex G-200 column chromatography. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction. The P-fraction was composed of ^ 5 enzymes. P-lll and P-IV in the P-fraction were picked as typical enzymes of this strain and their enzymatic properties were investigated. P-lll had a molecular weight of 48,400-50,000, an optimum pH of 5.5, and an optimum temperature of 30-35°C. It required glucose and oxygen for activity and was inhibited by p chloromercuribenzoate (p -CMB), N -bromosuccinylimide (N -BSI), Ag+, and o phenanthroline. On the other hand, P-IV had a molecular weight of 43,800-45,000, an optimum pH of 4.0-4.5, and an optimum temperature of 30-35°C. P-IV could decolorize melanoidin in the absence of glucose and oxygen, and was inhibited weakly by Ag+, p-CMB, and A/-BSI. P-IV attacks the melanoidin directly, in contrast to P-lll which attacks melanoidin indirectly as in the such-reaction of sugar oxidase [137].

475

Among a total of 228 strains belonging mainly to the class Deuteromycetes, of these, 9 showed decolorization yields of > 50%. They required different culture conditions, such as the glucose concentration, medium pH and nitrogen sources for a high decolorization yield. The isolated strain, D-90 showed the highest decolorization yield (/v^QSy©) when it was cultivated at 30° for 8 days in a molasses pigment solution containing glucose 2.5%, yeast extract 0.2%, KH2PO4 0.1% and MgS04, 7H2O 0.05%, the pH being adjusted to 6.0. This potent strain was identified as being of the order Mycelia sterilia [138,139]. Autoclaved mycelium of Aspergillus orysae Y-2 32 adsorbed melanoidin, especially lower molecular weight fractions, and the degree of the adsorption was influenced by the kind of sugars utilized for growth. The melanoidin-adsorbing ability of mycelia was repressed by a high concentration of salt. Furthermore, it decreased to half the initial level on washing with a 0.01%Tween 80 solution and was entirely lost on washing with a 0.1% SDS solution [140]. Decolorization of molasses melanoidin by bacteria was carried out by strain C-82 Immobilized in calcium alginate wastewater from a bakers' yeast factory that has been treated by activated sludge. Decolorization reached 19% by free cellules and was A^ 2-4-fold greater by immobilized cellules [141]. Melanoidins are decolorized without loss of taste and flavor by treating them containing materials (e.g. soy sauce, molasses, amino acids containing condiments) with lactic acid bacteria. Lactobacillus brevis JMC 1059 was anaerobically cultured in a medium containing glucose, peptone, yeast extract and salts at 30°C for three days, and the bacteria was separated by centrifugation. Soy sauce treated by the bacteria for 3 hours at 37°C was 22.1% decolorized [141]. The antioxydative activity of decolorized melanoidin was reviewed by Yamaguchi [142]. Decolorization was achieved by hydrogenation, oxidation by ozone, and by a microorganism C. versicolor , as previously described. Actomycete strain Streptomyces werraensis TT14 isolated from soil decolorized the model melanoidin prepared from glucose and glycine, the decolorization rate being 64% in the optimal medium (starch 2.0%, yeast 1.0%, NaCI 0.3% and CaCOa 0.3%, pH 5.5) and 45% in a synthetic medium. Lower molecular weight compounds increased in the decolorized melanoidin [143]. Rhizoctonia species D-90 decolorizes molasses melanoidin and synthetic melanoidin media of 87.5% and 84.5%, respectively, under optimal experimental growing conditions. The color of mycelium grown in melanoidin solutions turned dark-brown. However the melanoidin (dark brown color) can be eluted from the mycelium by washing in sodium hydroxide solution. The maximum elution yield of melanoidin from mycelium by 5.0 N sodium hydroxide solution was 96.1%. The melanoidin decolorization mechanism of Rhizoctonia sp. D-90 was such that the melanoidin pigment was absorbed into the cellules as a macromolecule and was accumulated intracellularly as a melanoidin complex in cytoplasm and around the membrane which then could be gradually decomposed by intracellular enzymes [144,145].

476

Aspergillus fumigatus was also found to be useful for decolorization of lignin, dyes, humic substances and melanoidins [146]. Terasawa et al [147] investigated the decolorization of model pigments and browned foods by microorganisms such as Coriolus versicolor IFO 30340, Paecilomyces canadensis NC-1 and Streptomyces werraensis TT 14 cultured at 27°C and 37°C, respectively. The decolorization rates differed by model brown pigments and foods. P. canadensis NC-1 mainly decolorized phenol-type model brown pigments, coffee, and black tea. C. versicolor IFO 30340, mainly decolorized model melanoidins and amino-carbonyl reaction type, pigments. S. werraensis TT 14 decolorized xylose-glycine and glucose-lysine model melanoidins and some caramel-type pigments. 8. MELANOIDINS

IN VIVO AND IN

NATURE

The discovery of the Maillard reaction in living organisms including the human, opens new fields to research. The attention of research scientists [28,29,77] has been focused on nonenzymatic glycation. Proteins in the eye, crystalline, collagen and many other proteins slowly react (over many years) with reducing sugars in the organism to induce cataracts, artherosclerosis, and a decreasing elasticity of the muscles [78-83]. The formation of AGE (Advanced Glycosylation End Products) as well as Amadori intermediates, pyrazinic structures and yellow-brown products which have very specific spectral characteristics have been suggested [84-87]. The amount of glycosylated products in diabetics is higher than in normal individuals [78-80,88]. The Maillard reaction is certainly the oldest in nature. Research has been devoted to the participation of melanoidins as a matrix in the synthesis of proteins in the prebiotic period. Their role in the formation of humic substances as well as that of natural hydrocarbons in rocks has also been studied [28,30,89,90]. According to Ikan et al [30] brown acidic polymers known as humic substances account for much of the organic material that occur in soils, natural waters and sediments. It has been suggested that these substances may be not only formed from lignin/proteins system but also by condensation reactions between sugars with amino acids, peptides and proteins [91-93]. Melanoidins also called synthetic humic acids possess isotopic analysis and spectroscopic properties similar to the natural geopolymers arising from the marine humic acids [30,50,94,95].

Research Prospects Melanoidins have remained insufficiently known because of their extreme complexity. Numerous studies need to be conducted in order to elucidate their structures including the chromophores present and their molar mass.

477

More efficient extraction and analytical methods need to be devised. We also need to extend our knowledge of their biological and antioxidative activity in vivo and in nature as well as their influence on food In general. The melanoidins obtained from foods cooked in microwave ovens has not been yet studied. Acknowledgements: The authors wish to thank I. Vernin-Rainaldi for the translation of the paper and H. Arzoumanian for his interest with the manuscript. Thanks are also due to G.M.F. Vernin and R.M. Zamkotsian for their collaboration.

9. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

L-C. Maillard, C.R. Acad. Sci., Paris, 154 (1912) 66; idem., Ann. Chim. Paris, 9 (5) (1916) 258. J.E. Hodge, J. Agric. Food Chem., 1 (1953) 928; idem., Symposium on Food Chemistry and Physiology of Flavours, The Avi Pub. Co, Inc, Westport, Connecticut (1967) 465. R.F. Hurrel, The Maillard Reaction in Food Processing, Human Nutrition and Physiology, P. Finot, H. Aeschbacher, R.F. Hurrel, R. Liardon (eds.), Birkhauser Verlag, Boston (1990) 245. G. Rizzi, Maillard Reaction in Chemistry, Food and Health, T. Labuza (eds.). The Royal Society of Chemistry, London (1994) 11. S. Kawamura, Dev. Food Sciences, 13 (1986) 3. S. Homma, Denpun Kagaku, 38 (1) (1991) 73. J. Keramat, H. Nursten, Food Chem., 51 (4) (1994) 417. T. Tolstoludskaya, N. Gretchko, N. Emelyanova, V. Ovod, Izvest. vyssh. Ucheb. Zaved. TecknoL, 6 (1990) 69. T. Tolstoludskaya, N. Gretchko, N. Emelyanova, Izvest. vyssh. Ucheb. Zaved. TecknoL, (1-3) (1991) 63. L Lusk, H. Goldstein, D. Ryder, J. Amer. Soc. Brew. Chem., 53 (3) (1995) 93. T. Obretenov, R. Maneva, M. Kuntcheva, G. Somov, J. Agric. Food Chem., 34(2) (1987)243. T. Obretenov, S. Ivanova, M. Kuntcheva, A. Burzev, C. Krachanov, N.T. of HIFFI, Plovdiv, 34 (2) (1993) 653. S. Meshcheryakova, K. Agienko, Dostizh. v Obi. Khim. Gigieny Obespech. Povysh. Kach. Moloch. Prod., Moskva, (1987) 95. B. Milic, B. Grujic-lnjac, M. Piletic, S. Lajsic, L Kolarov, J. Agric. Food Chem.,23(5)(1975)960. R. Rouseff, J. Fisher, S. Nagy, J. Agric. Food Chem., 37 (1989) 765. T. Obretenov, M. Kuntcheva, S. Mantchev, G. Valkova, J. Food Biochem., 15(4) (1991)279. M. Kuntcheva, T. Obretenov, Z. Lebensm.-Unters. Forsch., 202 (1996)238. T. Obretenov, M. Kuntcheva, S. Ivanova, Nahrung, 27 (10) (1983) 889. M. Kuntcheva, S. Ivanova, T. Obretenov, Nahrung, 27 (10) (1983) 897.

478

20 G. Somov, S. Ivanova, M. Kuntcheva, T. Obretenov, Nahrung, 27 (10) (1983)901. 21 T. Obretenov, M. Kuntcheva, A. Vangelov, M. Karadjova, E. Stamenova, NT. of HIFFI, Plovdiv, 2 (1984) 137. 22 T. Obretenov, E. Stamenova, M. Kuntcheva, A. Vangelov, N.T. of HIFFI, Plovdiv, 2(1984) 145. 23 A. Vangelov, E. Stamenova, T. Obretenov, G. Jatov, M. Kuntcheva, M. Karajova, 2(184) 173. 24 H. Steinhart, A. Moller, H. Kletschkus, Colloq. Sci. Intern. Cafe, Montpellier (1989) 197. 25 T. Obretenov, R. Maneva, M. Kuntcheva, C. Krachanov, N.T. of HIFFI of Plovdiv, 34(2)(1987) 255. 26 H. Steinhart, A. Packert, Colloq. Sci. Intern. Cafe, Montpellier, 15(2)(1993) 593. 27 W. Binkley, Intern. Sugar J., 62 (1960) 36. 28 A. Pissarnitzki, I. Egorov, Priklad. Biokhim, 25(5)(1989) 579. 29 F. Ledl, E. Schleicher, Angew. Chem., 29(6)(1990) 565. 30 R. Ikan, Y. Rubinbsztain, A. Nissembaum, I. Kaplan, The Maillard Reaction. Consequences for the Chemical and Life Sciences, R. IKan (ed.), Wiley & Sons Ltd, New York (1996) 1. 31 W. Baisier, T. Labuza, J. Agric. Food Chem., 40(5)(1992) 707. 32 T. Labuza, Maillard Reaction in Chemistry, Food and Health, T. Labuza et al (eds.). The Royal Society of Chemistry, London, England (1994) 176. 33 T. Obretenov, M. Kuntcheva, I. Panchev, J. Food Process.Preserv, 14(4) (1990) 309. 34 M. Kuntcheva, I. Panchev, T. Obretenov, J. Food Process. Preserv, 18(1 )(1990) 9. 35 S. Rogacheva, I. Panchev, T. Obretenov, Z. Lebensm.-Unters. Foesch., 200(1) (1995)52. 36 C. Enders, K. Theis, Brenstoff Chemie, 19(1938) 439. 37 B. Cammerer, L Kroh, Food Chem., 53 (1995) 55. 38 M. Faether, D. Nelson, J. Agric. Food Chem., 32(1984) 1428. 39 V. Lessig, W. Baltes, Z. Lebensm.-Unters Forsch., 173 (1981) 435. 40 B. Wedwicha, M. Kaputo, Food Chem., 43(1992) 359. 41 N. Suzuki, R. Philp, Org. Geochem.,15(4)(1990) 361. 42 S. Wang, F. Bobblo, P. Bobbio, Anales Acad. Brasil. Chem.,59(1)(1978) 55. 43 V. Migo, M. Matsumura, E. Rosario, H. Kataoka, J. Ferment.Bioeng., 76(1) (1993)29. 44 V. Lessig, W. Baltes, Z. Lebensm..-Unters. Forsch., 175(1982) 13. 45 J. Rafalska, M. Engel, W. Lanier, Geochim. Cosmochim. Acta, 55(1991) 3669. 46 M. Daglia, G. Stoppini, M. Cuzzoni, C. Dacarro, F. Zani, P. Mazza, Riv.ltal. Sci. Aliment. 21(1)(1992) 65. 47 A. Tomlinson, J. MIotkiewicz, Lewis I., Food Chem., 49(1994) 219. 48 M. Lee, H. Kim, J. Park, D. Kim, J. Korean Soc. Food Nutr., 21(6)(1992) 686. 49 L-C. Maillard, Ann. Chim. Paris, 7(9)(1917) 113.

479

50 51 52 53

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

L Benzing-Purdie, J.A. Ripmeester, Soil Sci Soc. America,47(1)(1983) 56. V. Lessig, W. Baltes, Z. Lebensm.-Unters. Forsch., 172(1981) 466. H.S. Cheigh, C. Lee, J. Korean Soc. Food Nutr.,22(2)(1993) 246. G. Vernin, J. Metzger, T. Obretenov, Actualite Chimique, 3 (1983) 7. G. Vernin and C. Parkanyi, The Chemistry of Heterocyclic Flavouring and Aromas Compounds, G. Vernin (ed.), E. Horwood Publ. Chichester, England (1982) 151. S. Kim, F. Hayase, H. Kato, Amino-Carbonyl Reaction in Food and Biological Systems, M. Fujimaki, M. Namiki, H. Kato (eds.), Elsevier, Amsterdam, The Netherlands (1986) 383. S. Ivanova, N. Popov, T. Obretenov, Nahrung, 33(10)(1989) 912. S. Sirianuntapiboon, P. Somchai, S. Ohmono, P. Atthasampunna, Agric. Biol. Chem., 52(1988)387. S. Ohmono, M. Kainuma, K. Kamimura, S. Sirianuntapiboon, I. Aoshima, P. Attasampunna, Agric. Biol. Chem.,52(1988) 381. Y. Watanabe, R. Sugi, Y. Tanaka, S. Hayashlda, Agric. Biol. Chem.,46(1982) 1623. I. Aoshima, Y. Tozawa, S. Ohmomo, K. Ueda, Agric. Biol. Chem., 49(1985) 2041. S. Ohmomo, I. Aoshima, Y. Tozawa, N. Sakurada, K. Ueda, Agric. Biol. Chem., 49(1985)2047. M. Murata, N. Teresawa, S. Homma, Biosci. Biotech. Biochem., 56(1992) 1182. C. Franzke, H. Iwainsky, Deut. Lebensm.-Rundschau, 50(1954) 251. N. Yamaguchi, Y. Okada, Nippon Shokuhin Kogyo Gakkai-shi, 15(5)(1968) 187. S. Lee, C. Rhee, D. Kim, Hanguk Sikp urn Kwahakhoe Chi, 7(1)(1975) 37. H. Lingnert, C. Eriksson, J. Process. Preserv., 4(3)(1980) 173. H. Lingnert, C. Eriksson, G. Waller, Maillard Reaction in Food and Nutrition, ACS Symp. Ser.,215(1983) 335. T. Obretenov, S. Ivanov, D. Peeva, Amino-Carbonyl Reaction in Food and Biological Systems, Kodansha, Tokyo, Japan (1986) 281. J. Won, D. Kim, Hanguk Sikp um Kwahakoe Chi, 12(4)(1980) 235. N. Yamaguchi, Y. Koyama, M. Fujimaki, Prog. Food Nutr. Sci., 5(1981) 429. N. Kirigaya, H. Kato, M. Fujimaki, J. Agric. Chem. Soc. Japan, 43(7)(1969) 484. H. Itoh, K. Kawachima, I. Chibata, Agric. Biol. Chem., 39(1 )(1975) 283. K. Kawashima, H. Itoh, I. Chibata, J. Agric. Food Chem., 25(1)(1977) 202. H. Lingnert, C. Eriksson, J. Food Process. Preserv., 4(3)(1980) 161. H. Lingnert, J. Food Process. Preserv., 4(4)(1980) 219. H. Lingnert, B. Lundgren, J. Food Process. Preserv., 4(4)(1980) 235. R. Koening, A. Cerami, Proc. Nat. Acad. Sci., USA, 72(1975) 3687. A. Cerami, Maillard reaction in Chemistry, Food and Health, T. Labuza (eds.). The Royal Society of Chemistry, London (1994) 1. R. Shapiro, M. McManus, C. Zulit, H. Bunn, J. Biol. Chem., 255(1980) 3120. V. Monnier, A. Cerami, Science, 211 (1981) 491.

480

80 I. Waite, M. Tanzer, Handbook of Biochemistry in Aging, R. Florini (ed.), CRC Press (1981) 346. 81 D. Fujimoto, Biochem. Int., 5(1982) 743. 82 S. Shnider, R. Kohn, Exp. Gerontol., 17(1982) 185. 83 A. Eble, S. Thorpe, J. Baynes, J. Biol. Chem., 258(1983) 9406. 84 W. Brownlee, H. Vlassara, A. Cerami, Ann. Int. Med., 101(1984) 527. 85 R. Bucala, P. Model, A. Cerami, Proc. Nat. Acad. Sci., USA, 81(1984) 105. 86 V. Monnier, R. Kohn, A. Cerami, Proc. Nat. Acad. Sci., USA, 81(1984) 583. 87 S. Pingor, R. Ulrish, F. Bebesath, A. Cerami,, Proc. Nat. Acad. Sci., USA,81 (1984)2684. 88 V. Monnier, Amino-Carbonyl Reactions in Food and Biological Systems, M. Fujimaki, M. Namiki, H. Kato (eds.), Kodansha, Tokyo, Japan (1986) 459. 89 F. Stevenson, Humus Chemistry: Genesis, Compositions, Reactions, J. Wiley & Sons, New York, 1982. 90 T. Telegina, Z. Masinovskii, G. Mokhnatina, T. Pavlovskaya, Zhur Evol. Biokhim. i. Fiziol., 25(5)(1989) 561. 91 W. Flaig, Sci. Proc. Royal Dublin Soc.,4(1969) 49. 92 A. Nissenbaum, J. Kaplan, Limnol. Oceanogr., 17(1972) 570. 93 J. Hedges, P. Parker, Geochim. Cosmochim. Acta, 40(1976) 1019. 94 J. Ertel, J. Hedges, Aquatic and terrestrial Humic Materials, R. Christman, E. Gjessing (eds.), Ann. Arbor. Science, Michigan, (1983) 143. 95 Y. Rubinsztain, P. loselis, R. Ikan, Z. Aizenshtat, Org. Geochem., 6(1984) 791. 96 W. Ruckdeschel, Z. Gesamte Brauwes, 37(1914) 430. 97 K. Kurono, H. Katsume, Nippon Nogei Kagaku Kaishi, 3(1927) 594. 98 K. Korono, Japanese Pat N° 2199 (1927) 99 K. Kurono, T. Fukai, Nippon Nogei Kagaku Kaishi, 3(1927) 1292. 100 T. Fukai, Jozogaku Zasshi, 5(1928)451. 101 J. Ames, L Bates, D. MacDougall, Maillard Reaction in Chemistry, Food, and Health, T. Labuza (eds.), The Royal Society of Chemistry, London (1994) 129. 102 LM. Benzing-Purdie, C.I. Ratcliffe, Dev. Food Science., 13 (1986) 193: L M. Benzing-Purdie, M.V. Cheshire, B.L Williams, G.P. Sparling, C.I. Ratcliffe and J.A. Ripmeester, J. Agric. Food Chem., 34 (1986) 170. 103 M. Feather, R. Huang, J. Carbohydrate Chem., 4(1985) 363. 104 T. Gomyo, H. Kato, K. Udaka, M. Horikoshi, M. Fujimaki, Agric. Biol. Chem., 36(1972) 125. 105 F. Hayase, S. Kim, H. Kato, Agric. Biol. Chem., 48(1984) 2711. 106 F. Hayase, S. Hiroshima, G. Okamoto, H. Kato, Agric. Biol. Chem., 53(12) (1989)3383. 107 R. Ikan, P.loselis, Y. Rubinsztain, Z. Aizenshtat, M. Frenkel, K. Peters, J. Therm. Anal., 42(1 )(1994) 31. 108 H. Kato, G. Noguchi, M. Fujimaki, Agric. Biol. Chem., 32(1968) 916. 109 H. Kato, H. Tsuchida, Prog. Food Nutr. Sci., 5(1981) 147.

481

110 H. Kato, S. Kim, F. Hayase, Amino-Carbonyl Reactions in Food and Biological Systems, M. Fujimaki, M. Namiki, H. Kato (eds.), Elsevier, Amsterdam, (1986) 215. 111 H. Kato, I. Lee, N. Chuyen, S. Kim, F. Hayase, Agric. Biol. Chem., 51 (1987) 1333. 112 S. Kim, F. Hayase, H. Kato, Agric. Biol. Chem., 49(1985) 785. 113 S. Kim, Y. Park, Bull. Korean Fish Soc, 19(1986) 36. 114 H. Motai, S. Inoue, Agric. Biol. Chem., 38(1974) 233. 115 H. Motai, Agric. Biol. Chem., 38(1974) 2299. 116 S. Nam, M. Kim, Korean J. Food Sci. Technol., 16(1984) 218. 117 H. Nursten, R. O'Reilly, Food Chem., 20(1986) 45. 118 Y. Naohiko, New Food Ind., 33(1 )(1991) 76. 119 T. Obretenov, M. Kuntcheva, I. Panchev, Food Process. Preserv., 10 (1986)251. 120 C. Pakaew, S. Ohmono, H. Kataoka, Microb. Util. Renewable Resour., 6 (1989)271. 121 M. Miura, T. Gomyo, Agric. Biol. Chem., 52(10)(1988) 2403. 122 M. Miura, T. Gomyo, Nippon Eiyo, Shokuryo Gakkaishi, 46(4)(1993) 309. 123 M. Hirano, M. Miura, T. Gomyo, Biosci. Biotechnol. Biochem., 58(5)(1994) 940. 124 M. Fujimaki, S. Homma, N. Arakawa, C. Inagaki, Agric. Biol. Chem., 43 (1979)497. 125 H. Takeuchi, Y. Nishioka, M. Fujishiro, K. Muramatsu, Agric. Biol. Chem., 51(1987)969. 126 N. Yamaguchi, Aichi-ken Shomuhin Kogyo Shikensho Nenpo, 28(1987) 88. 127 K. Taguchi, Y. Samoei, Org. Geochem., 10(1986) 1081. 128 B.L Wedzicha, M.T. Kaputo, Food Chem., 43(1992) 359. 129 R. Ikan Ed., The Maillard Reaction: Consequences for the Chemical and Life Sciences, J. Wiley and Sons, New York (1996). 130 I.E.Lee, N.V. Chuyen, F. Hayase and H. Kato, Biosci. Biotechnol. Biochem., 58(1994) 18. 131 G.Vernin, T. Obretenov, S. Rogacheva, to be published. 132 F. Hayase, in ref. 129, (1996) 89. 133 J.W. Wong and T. Shibamoto, in ref. 129, (1996) 129. 134 I.E. Lee, N.V. Chuyen, F. Hayase, H. Kato, Biosci. Biotechnol. Biochem., 56(1992)21. 135 J.W. Baynes, in ref. 129, (1996) 55. 136 I. Aoshima, Y. Tozawa, S.Ohmomo, K. Ueda, Agric. Biol. Chem.,49(7) (1985)2041. 137 S. Ohmono, I. Aoshima, Y. Tozawa, N. Sakurada, K. Ueda, Agric. Biol. Chem., 49(7)(1985) 2047.

482

138 S. Sirianuntapiboon, P. Somcha'i, S. Ohmomo, P. Atthasampunna, Agric. Biol. Chem., 52(2)(1988)387. 139 S. Sirianuntapiboon, P. Sihanonth, P. SomchaT, P. Atthasampuna, S. Hayashi, Microb. Util. Renewable Resour., 6(1989)261. 140 I. Aoshima, P. Atthasampunna, Agric. Biol. Chem., 52(2)(1988) 381. 141 C. Pakaev, S. Ohmono, H. Kataoka, Microb. Util. Renewable Resour.,6(1989) 271. 142 N. Yamaguchi, New Food Ind., 33(1 )(1991) 76. 143 M. Murata, N. Terasawa, S. Homma, Biosci. Biotechnol. Biochem., 56(8) (1992)1182. 144 S. Sirianuntapiboon, P. Sihanonth, S. Hayashi, Microb. Util. Renewable Resour., 8(1993)505. 145 S. Sirianuntapiboon, P. Sihanonth, P. SanchaT, P. Atthasampunna, S. Hayashida, Biosci. Biotechnol. Biochem., 59(7)(1995) 1185. 146 T. Nakamura, Y. Sakamoto, T. Ootani, Jpn Kokai Tokkyo Koho, JP 06,153,913 (1994).

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

483

Formation of volatile sulfur compounds in reaction mixtures containing cysteine and three different ribose compounds Donald S. Mottram and Ian C.C. Nobrega The University of Reading, Department of Food Science and Technology, Whiteknights, Reading RG6 6AP, United Kingdom Abstract The Maillard reaction between cysteine and ribose is an important route to the characteristic flavors of cooked meat. The main sources of ribose in meat are inosine monophosphate (IMP) and smaller quantities of free ribose and ribose 5phosphate. This paper reports on a comparison of the volatiles produced in reactions of these compounds with cysteine in heated model systems. Complex mixtures of aroma compounds were formed, which included thiophenes, thiophenones, mercaptoketones, thiophenethiols, furanthiols and a large number of disulfides. The largest quantities of volatiles were obtained from the reactions containing ribose 5-phosphate and the smallest from the IMP reactions, where the concentrations of many components were up to 50-fold lower. The mechanisms for the formation of the major sulfur-containing components are discussed. 1. INTRODUCTION Meat flavor is generated, during cooking, by a complex series of reactions involving non-volatile water-soluble precursors and/or lipids. The main watersoluble precursors in raw meat, which participate in such reactions, are amino acids, peptides, thiamine and carbohydrates. Sulfur-containing volatile compounds are considered to make a particularly important contribution to the characteristic aromas of cooked meat [1]. An important route to these compounds is the Maillard reaction between reducing sugars and sulfur-containing amino acids, such as cysteine or methionine. Meat contains significant quantities of ribose, which is a pentose sugar, and its reaction with cysteine, in model systems, has been shown to give meat-like aromas [2,3]. The reaction is widely used in the preparation of reaction-product flavorings with meat-like characteristics. The main sources of ribose in meat are inosine 5'-monophosphate (IMP) and smaller quantities of ribose 5-phosphate and free ribose. IMP is formed in muscle post-slaughter from the enzymic dephosphorylation and deamination of

484

adenosine triphosphate (ATP), the ribonucleotide which is essential to muscle function in the live animal [4]. Further enzymic breakdown of IMP may lead to hypoxanthine, ribose and ribose 5-phosphate (Figure 1), although most of the ribose in meat remains bound within IMP. IMP is well recognized as a flavor potentiator and is associated with the taste sensation known as "umami" [5,6]. However, IMP may also provide a source of ribose for Maillard reactions occurring during the cooking of meat. The

HoN OH OH OH

I

I

H9C—O—P

N

0

P—P—OH

0

I!

0

II

adenosine-5'-triphosphate (ATP)

H

^O. 'H

OH I H2C-0-P-OH

^'

HO OH

hypoxanthine

o

H OH

OH

inosine-5'-monophosphate (IMP) (inosinic acid)

II

H OH

ribose-5-phosphate

l^Pi H

^0...^^^?^20H

HO

H HO

OH

OH

inosine

ribose

hypoxanthine

" OH

OH

Figure 1. Sources of ribose in meat.

OH

H

H.

OH

OH OH

485 N-glycoside link between ribose and the base, hypoxanthine, involves the reducing group of the sugar and, therefore, Maillard-type reactions will not occur until this link is hydrolyzed. Although IMP appears to have relatively high thermal stability, some hydrolysis does occur on heating in aqueous solution and this is enhanced under acidic conditions [7]. In a recent investigation of the volatiles produced from model systems containing cysteine and IMP, many sulfur compounds were formed, including thiols, disulfides and mercaptoketones arising from the reaction of the pentose sugar and cysteine [8]. Although the potential for ribose and cysteine to generate meat-like flavors has been widely studied in model systems, the relative contributions of IMP, ribose 5-phosphate and free ribose to flavor-forming reactions have not been investigated. Since ribose may be present in any of these forms in meat, such a comparison could improve the understanding of flavor formation in cooked meat. This paper reports on a comparison of the volatiles produced in the reactions of these ribose compounds with cysteine in heated aqueous systems. 2. EXPERIMENTAL PROCEDURES 2.1. Preparation of reaction mixtures For each reaction, solutions of cysteine (0.1 M) and the ribose-containing compound (0.1 M) were prepared. Before making up to volume, the pH was adjusted to 5.6, using dilute sodium hydroxide or hydrochloric acid, if necessary. Equal portions of the two solutions were mixed and aliquots (6.0 mL) of the mixture were transferred to round-bottomed thick-walled Pyrex glass ampoules of 10 mL volume (S. Murray and Co Ltd, Old Woking, Surrey, UK), which were then flamed-sealed. Ampoules, containing the reaction mixtures, were heated in an autoclave for 30 min at 140 °C under a pressure of 0.28 MPa (2.7 bar). The reactions were carried out in triplicate. 2.2. Collection of headspace volatiles The reaction mixtures were transferred to 250 mL conical flasks, diluted with 20 mL distilled water, and headspace volatiles were purged onto Tenax-TA traps for 1.5 h at 60 °C using 40 mL/min nitrogen, as described previously [9]. One microliter of a internal standard (1,2-dichlorobenzene in hexane, 130 |ag/mL) was added to the front end of the trap before GC-MS analysis. 2.3. Gas Chromatography-Mass Spectrometry. All analyzes were performed on a Hewlett Packard HP5890 Series II gas chromatograph fitted with a 5972 mass selective detector. A CHIS injection port (Scientific Glass Engineering Pty Ltd, Ringwood, Australia), held at 250 °C, was used to thermally desorb the volatiles from the Tenax trap onto the front of a BPX5 fused silica capillary column (50 m x 0.32 mm i.d., 0.5 |am film thickness; Scientific Glass Engineering). During the desorption period of 5 min, the oven was held at 0 °C. After desorption, the oven was heated to 50 °C, over 1 min, and held for 2 min before heating at 4 °C/min to 250 °C. Helium at 8 psi was used as

486 the carrier gas, resulting in a flow of 1.75 mL/min at 40 °C. A series of ^-alkanes (Ce - C22) was analyzed, under the same conditions, to obtain linear retention indices (LRI). The mass spectrometer was operated in the electron impact mode with an electron energy of 70 eV, an emission current of 50 [lA and a scan rate of 1.9 scans/s over the mass range m/z 29 to m/z 400. Components were identified by comparison of their mass spectra and LRI with those from authentic compounds analyzed in our laboratory, or by comparison with spectra contained in the NIST/EPA/NIH Mass Spectral Database or in the literature. The approximate quantities of the volatile components were calculated by comparison of the peak areas, from the GC-MS chromatograms, with those of the dichlorobenzene internal standard.

0

0

SH

SH

SH

^X^^

^

^

R R

R

^

R

^O

f' ^\' V> Q^'-'-^ c l '"^ X

^

^

X

"

O

o

^Sv . s - s . JL

\\ r '^^

TR ^ \j

.S—S. >/

yK ^^-K J\

S

"o^

S—S.

ir ^^-^ j3

^ ^^

X

'^\

//

R = HorCH3

R = CH3orC2H5

X = OorS

Figure 2. Some thiols and their symmetrical and unsymmetrical disulfides found in the headspace volatiles from heated systems containing cysteine and ribose.

487 RESULTS AND DISCUSSION More than 70 sulfur-containing volatiles were identified in the headspaces above the reaction mixtures and they accounted between 50 and 88% of the total mass of the headspace volatiles (Table 1). Most compounds were present in all the reaction mixtures, the main exceptions being systems containing IMP which contained considerably fewer volatile compounds. Quantitatively, the major volatiles were mercaptoketones, furanthiols and thiophenethiols (Figure 2). More than 30 disulfides were also found, but in smaller quantities than the mercaptoketones and thiols. These were symmetrical and unsymmetrical disulfides derived from the mercaptoketones and thiols and comprised most of the possible combinations of these SH-compounds (Figure 2). Such compounds have been previously reported in cysteine - ribose model systems and several, have been found in meat where they are believed to contribute to desirable meaty aromas. Other compounds included thiophenones, dithiolanones and dithianones, acylthiophenes, alkylthiophenes, some polysulfur heterocyclics as well a number of bicyclic compounds, including thienothiophenes, some dihydrothienothiophenes and kahweofuran (Figure 3). The volatiles were dominated by the sulfur compounds, but the major non-sulfur volatiles were 2-furfural and 2,4pentanedione. 3.1. S y s t e m s containing IMP Comparison of the number and quantities of volatiles from the IMP system

O

CHO

O

^Tf\,H

n

O

R = H,CH3orC2H5

^ i CJ O

R = CH3orC2H5

LXI

^O^

Figure 3. Some heterocyclic sulfur compounds found in the headspace volatiles of heated reaction mixtures containing cysteine and ribose.

488 Table 1. A p p r o x i m a t e quantities^ (ng/0.3 mmole ribose) of some sulfur compounds and selected non-sulfur compounds identified in t h e h e a d s p a c e volatiles of cysteine model s y s t e m s containing ribose 5-phosphate, ribose or IMP. Compound

Ribose-P

Ribose

IMP

3-mercapto-2-butanone 3-mercapto-2-pentanone 2-mercapto-3-pentanone Total m e r c a p t o k e t o n e s

830 (66) 2767 (128) 561 (30) 4158

8(3) 225 (53) 32(6)

3(0) 1(1) 2(0)

265

6

2-methyl-3-furanthiol 2-methyl-3-thiophenethiol 2-furanmethanethiol 2-thiophenemethanethiol 2-thiophenethiol Total furan and thiophene thiols

883 (89) 210 (29) 994 (83) 31(6) 720 (109) 2838

225 (22) 25 (15) 784 (181) 5(2) 21(15) 1060

29(2) 6(3) 5(2)

bis(l-methyl-2-oxopropyl) disulfide l-methyl-2-oxopropyl l-ethyl-2-oxopropyl disulfide bis(l-ethyl-2-oxopropyl) disulfide l-methyl-2-oxobutyl l-ethyl-2-oxopropyl disulfide Total oxoalkyl disulfides

6(1) 64(13) 100 (52) 33 (18)

Tr 7(1) 1(0)

203

12

2-methyl-3-furyl l-methyl-2-oxopropyl disulfide 2-methyl-3-furyl l-ethyl-2-oxopropyl disulfide 2-methyl-3-furyl l-methyl-2-oxobutyl disulfide 2-furylmethyl l-methyl-2-oxopropyl disulfide 2-furylmethyl l-ethyl-2-oxopropyl disulfide 2-furylmethyl l-methyl-2-oxobutyl disulfide bis(2-methyl-3-furyl) disulfide bis(2-furylmethyl) disulfide 2-methyl-3-furyl 2-furylmethyl disulfide Total furyl d i s u M d e s

21(9) 84 (44) 15(7) 10(4) 64(6) + 23(5) 13(1) 14(2)

2(1) 12(5) 2(1) 1(0) 19(3)

2-thienyl l-methyl-2-oxopropyl disulfide 2-thienyl l-methyl-2-oxobutyl disulfide 2-furylmethyl 2-thienyl disulfide 2-methyl-3-furyl 2-thienyl disulfide 2-methyl-3-thienyl 2-thienyl disulfide bis(2-methyl-3-thienyl) disulfide 2-methyl-3-furyl 2-methyl-3-thienyl disulfide bis(2-thienyl) disulfide Total thienyl disulHdes 4,5 - dihy dro-3 (2Jfi/) - thiophenone 4,5-dihydro-2-methyl-3(2//)-thiophenone 4,5-dihydro-5-methyl-3(2//)-thiophenone dihydro-2,4-dimethyl-3(2iiZ)-thiophenone dihydro-2,5-dimethyl-3(2ii/)-thiophenone Total t h i o p h e n o n e s

+

16(9) 24(1) 14 (10)

15(6)

55 Tr Tr -

2(0)

2 _ -

13(8)

96 _ Tr

+

2(1)

24(5) 8(6) 3(2) 15(9) 15(9)

Tr Tr Tr

3(3) 1(1)

3(2)

1(1) 3(3)

252 +

78

Tr 5

9(1) 73(2) 35(3)

7(1) 5(0)

Tr Tr 117

12

Tr

Tr 8

1(0) 3(1)

Tr 4

489 Table 1 (cont/..) Compound

Ribose-P

Ribose

IMP

3,5-(iimethyl-l,2-dithiolan-4-one 3-ethyl-l,2-dithiolan-4-one 3-methyl-l,2-dithian-4-one Total d i t h i a n o n e s and dithiolanones

284 (46) 7(1) 57 (14) 348

5(2)

14(2)

2-formylthiophene 3-methyl-2-formylthiophene 5-methyl-2-formylthiophene 3-ethyl-2-formylthiophene 2-acetyl-3-methylthiophene 2-propanoylthiophene dimethylformylthiophene 2-methylthiophene 2,3- dime thyIthiophe ne Total t h i o p h e n e s

44(3) 57 (13) 12(8) 18(4) 13(2) 5(3) 87 (22) 367 (58) 18(3) 621

3,5-dimethyl-l,2,4-trithiolane 3-methyl-l,2,4-trithiane 1,2,4,5-tetrathiane Total thiolanes and thianes

-

-

-

2(1) 7

1(0) 15

20(5) 2(1) 1(0) 2(1)

Tr 2(0) 1(0) Tr

-

-

6(3) 90 (43) Tr 121

21(3) Tr 24

-

3(1) 2(1) 19(1) 24

2(1) Tr Tr 1(0) 1(0) Tr 5

3(1)

4(2)

8(1) 4(1)

-

2,3-dihydro-6-methylthiothieno[2,3c]furan thieno[3,2b or 2,3b]thiophene dihydrothienothiophene methyldihydrothienothiophene methyldihydrothienothiophene methyldihydrothienothiophene dimethyldihydrothienothiophene Total bicyclic compounds

Tr 89 (22) 21(1) 2(0) 22(1) 46(0) 9(0) 189

2-pentanone 3-pentanone 2,3-pentanedione 3-hydroxy-2-butanone 2,4-pentanedione 2-furfural Total selected non-sulfur compounds

38(9) 8(3) + Tr 761 (31) 438 (41) 1245

-

-

3(1) 1499 (132) 1506

1(0) Tr 13

4.2

3.9

5.6

Final pH (initial pH = 5.6)

+

3

1 Approximate quantities obtained by comparing GC/MS peak areas with the area of 130 ng dichlorobenzene added to Tenax trap as internal stardard; amounts are expressed in terms of means (triplicate) and standard deviations (in brackets); Tr, trace (< 0.5 ng); -, not detected; -i- present in small amounts and quantitation confounded by adjacent peak

490 with those containing ribose or ribose 5-phosphate, showed that the IMP system was much less reactive (Table 1). Although all the mercaptoketones, furanthiols and thiophenethiols, discussed above, were detected in this reaction mixture, they were only present in very low concentrations. No bis(oxyalkyl) disulfides were found and only trace quantities of furanyl and thienyl disulfides. Most of the other sulfur compounds found in the ribose and ribose 5phosphate systems were detected, but only in small amounts in the IMP system. The only exceptions were the polysulfur heterocyclics, 3,5-dimethyltrithiolane, 3methyl-l,2,4-trithiane and 1,2,4,5-tetrathiane, which were not found at all in the reactions involving ribose or ribose 5-phosphate, but were present in the IMP system. These thiolanes and thianes are believed to be formed by the thermal degradation of cysteine in aqueous solution [10,11]. They do not require the presence of ribose and the associated Maillard reactions. Their absence from the ribose and ribose 5-phosphate systems is probably due to competing reactions for intermediates of cysteine breakdown preventing their availability for thiolane and trithiane formation. These results demonstrate that IMP is relatively stable in aqueous solution, at a pH typical of that found in meat, and that very little reaction occurs with cysteine. In contrast, ribose and ribose 5-phosphate appear to undergo reactions with cysteine which give typical Maillard reaction products. At lower pH more hydrolysis of IMP may occur giving higher concentrations of such products [8]. 3.2. Systems containing ribose and ribose 5-phosphate Interesting differences were found between the ribose and ribose 5phosphate systems. The ribose 5-phosphate appeared to be more reactive, producing much larger quantities of most volatile compounds. This was particularly noticeable with the major class of volatiles, the mercaptoke tones and the corresponding disulfides. The difference in the total quantities of furanthiols, as shown in Table 1, is less. However, relatively large quantities of 2-furanmethanethiol in the ribose system are largely responsible for the class total in this system. The dominant volatile in the ribose system was 2-furfural, which was present at a level approximately 4 times higher than in the ribose 5phosphate reaction. Reaction of this compound with hydrogen sulfide (from cysteine degradation) is the probable route to 2-furanmethanethiol. A possible explanation for the increased reactivity of ribose 5-phosphate may be that different mechanisms for its breakdown and reaction with cysteine occur (Figure 4). It has been reported that, in aqueous solution, ribose 5-phosphate is relatively easily dephosphorylated and dehydrated, via 1-deoxypentosone, to yield 4-hydroxy-5-methyl-3(2ii/)-furanone [12]. This compound can readily form thiol-substituted furans and thiophenes by reaction with hydrogen sulfide, produced in the degradation of cysteine [13,14]. Diacetyl can be formed via dehydration and fragmentation of the 1-deoxypentosone intermediate, while reteroaldolization of this intermediate will give, among other products, hydroxyacetone [15]. 2,3-Pentanedione could result from its aldol condensation

491 with acetaldehyde. Reaction of these diones with hydrogen sulfide will yield the mercaptoketones, which are the dominant products of the reaction. The dephosphorylation of ribose 5-phosphate may provide an easier route the furanone and dione intermediates than the Maillard pathway, via Amadori intermediates, which is required for the free ribose system. Hence, sulfur compounds from reactions with cysteine or hydrogen sulfide were more readily produced from ribose 5-phosphate. 2-Furfural is formed via 3-deoxypentosone, which is produced from Amadori intermediates in the Maillard reaction. This is not produced by the dephosphorylation of ribose 5-phosphate and, therefore, the formation of 2-furfural will not be favored in the ribose 5-phosphate systems.

OH H2C—0-P-OH

5 HO

s

OH

4-hydroxy-5-methyl3(2//)-furanone

Reduction

I-H2O O

SH

Figure 4. Formation of 2-methyl-3-furanthiol and mercaptoketones via the dephosphorylation of ribose 5-phosphate. (RA = retroaldol)

492 4. CONCLUSION In heated aqueous solution at pH 5.6, inosine 5'-monophosphate was stable to hydrolysis and therefore did not readily undergo Maillard tj^e reactions with cysteine. Under similar conditions ribose-5-phosphate reacted readily with cysteine to give a complex mixture of volatile sulfur compounds, dominated by mercaptoketones, furanthiols, thiophenethiols and their disulfides. Although such compounds were found in a similar system containing free ribose, the quantities were much smaller. In both systems, sugar dehydration products, such as 1-deoxypentosone and hydroxymethylfuranone, are key intermediates. Such compounds are Maillard reaction intermediates, but they are formed more readily by the dephosphorylation of ribose-5-phosphate, a pathway which is not available in the free ribose system.

5. REFERENCES 1. D.S. Mottram, In Volatile Compounds in Foods and Beverages, H. Maarse (ed). Marcel Dekker: New York (1991) 107-177. 2. I.D. Morton, P. Akroyd and C.G. May, Brit. Patent 836,694 (1960). 3. L.J. Farmer, D.S. Mottram and F.B. Whitfield, J. Sci. Food Agric. 49 (1989) 347-368. 4. R.A. Lawrie, Meat Science, 5th ed, Pergamon: Oxford (1992). 5. J.A. Maga, Crit Rev. Food Sci. Nutr. 18 (1983) 231-312. 6. Y.H. Sugita, In Developments in Food Flavours, G.G. Birch, M.G. Lindley (eds), Elsevier Applied Science: London (1986) 63-79. 7. T. Matoba, M. Kuchiba, M. Kimura and K. Hasegawa, J. Food Sci. 53 (1988) 1156-1159. 8. M.S. Madruga and D.S. Mottram, J. Sci. Food Agric. 68 (1995) 305-310. 9. D.S. Mottram and F.B. Whitfield, J. Agric. Food Chem. 43 (1995) 984-988. 10. O.K. Shu, M.L. Hagedorn, B.D. Mookherjee and C.T. Ho, J. Agric. Food Chem. 33 (1985) 438-442. 11. F.B. Whitfield and D.S. Mottram, In Contribution of Low and Non-volatile Materials to the Flavor of Foods, W. Pickenhagen, C.T. Ho, A.M. Spanier (eds), Allured Publishing: Carol Stream, IL (1996) 149-182. 12. H.G. Peer and G.A.M. van den Ouweland, Reel. Trav. Chim. Pays-Bas 87 (1968) 1017-1020. 13. G.A.M. van den Ouweland and H.G. Peer, J. Agric. Food Chem. 23 (1975) 501-505. 14. D.S. Mottram and F.B. Whitfield, In Thermally Generated Flavors. Maillard, Microwave, and Extrusion Processes, T.H. Parliment, M.J. Morello, R.J. McGorrin (eds), American Chemical Society: Washington, DC (1994) 180191. 15. H. Weenen and W. Apeldoorn, In Flavour Science: Recent Developments, A.J. Taylor, D.S. Mottram (eds). Royal Society of Chemistry: Cambridge (1996) 211- 216.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

493

Flavor formation from the interactions of sugars and amino acids under microwave heating T.H., Yu*, B.R., Chen*, L.Y., Lin**, and C.-T., Ho*** *Department of Food Engineering, Da-Yeh University, 112, Shan-jeu Road, DaTsuen, Chang-Hwa, Taiwan, ROC. **Department of Food Nutrition, Hungkung Institute of Nursing and Medical Technology, Taichung, Taiwan, ROC. ***Department of Food Science, Cook College, Rutgers University, New Brunswick, NJ, USA Abstract In this study, twenty kinds of amino acids were mixed with D-glucose or D-xylose, individually in propylene glycol or glycerol. These mixtures were heated in a 650 W traditional microwave oven with full power for 2 minutes. The color, appearance, and aroma of the heated solutions were studied. The appearance of the heated Ltryptophan solutions was found to be the most intense when these amino acids were heated with D-glucose or D-xylose in propylene glycol. The appearance of the heated L-tyrosine solutions were found to be the most intense when these amino acids were heated with D-glucose or D-xylose in glycerol. The heated solutions of Lcysteine, L-methionine, L-proline, L-phenylalanine, L-glutamine, L-leucine, and Lisoleucine had very characteristic and intense flavor sensations. The flavor sensations of the heated solutions are discussed in this study.

1. INTRODUCTION The use of microwave oven in food preparation is very popular in developed countries and a number of microwave foods have also been developed in these countries. Although it has the advantages of rapid heating, reduction in the cooking time, and uniform heating, microwave heating also has several disadvantages. These include: lack of browning color, flavor loss, formation of undesired texture, and the lack of Maillard-type or caramellic flavor (1-6). Flavor researchers have made efforts to solve these problems. Several techniques or methods have been developed to improve the quality of microwave heated foods. These methods or techniques include: the modification of food formulations, the modification of flavor formulations, adding flavor precursors, using special package materials, coating flavor precursors on food surface, and the application of flavor encapsulation methods (1-12).

494 In this study, high polarity and high boiling point food grade solvents, e.g., propylene glycol and glycerol, were used as the microwave energy absorbers and solvents for the flavor compounds and Maillard reaction products. Twenty amino acids found in foods were mixed with D-glucose or D-xylose, individually, in propylene glycol or glycerol. After being stirred for 2 hrs, these mixtures were heated in a 650 W traditional rotating microwave oven with full power for 2 minutes. The color, appearance, and aroma of the heated solutions were studied to establish the potential contribution of Maillard reaction products to the fortification of the color and/or the flavor of microwaved foods.

2. MATERIALS AND METHODS 2.1. Materials: A. Amino acids: (1) L-alanine (Aldrich, 97 % purity) (2) L-arginine free base (Sigma, >98 % purity) (3) L-asparagine anhydrous (Sigma, 98 % purity) (4) L-aspartic acid (Sigma, >99 % purity) (5) L-cysteine (Aldrich, 97 % purity) (6) L-glutamine (Aldrich, 99 % purity) (7) L-glutamic acid (Aldrich, 99 % purity) (8) glycine (Sigma, 99 % purity) (9) L-histidine (Sigma, >99 % purity) (10) L-isoleucine (Sigma, 98 % purity) (11) L-leucine (TCI-GR, >98 % purity) (12) L-lysine (Sigma, >98 % purity) (13) L-methionine (Aldrich, 98 % purity) (14) L-phenylalanine (Sigma, 98 % purity) (15) L-proline (Sigma, 98 % purity) (16) L-serine (TCI-GR, >98.5 % purity) (17) L-threonine (Sigma, 98 % purity) (18) L-tryptophan (Sigma, 98 % purity) (19) L-tyrosine (Sigma, > 99 % purity) (20) L-valine (TCI-GR, > 98 % purity) B.Sugars: (1) a-D-glucose anhydrous (Aldrich, 96 % purity) (2) D-xylose (Aldrich, 99 % purity) C. Solvents: (1) propylene glycol (PG or 1,2-propanediol, Fischer, 99 % purity) (2) glycerol (TEDIA, 99.5 % purity) 2.2. Sample Preparation A. Combinations pf the mixtures of amino acids and/or sugars As shown in Figure 1, 0.01 mole of each amino acid listed in 2.1.A. was mixed with or without one of the sugars listed in 2.I.B. in 50 g of propylene glycol (PG) or glycerol in a 250 mL Erienmeyer flask. Each flask was then stirred without heating

495

on a stirrer (Thermolyne cimarec 2) for 2 hr. After that, each flask was put into a regular microwave oven with a rotating glass plate (Sunpentown Co., Model SM1201) and heated under 650 W power for two minutes. After heating, each flask was removed and cooled immediately. One hundredth of a mole of glucose or xylose was also mixed with propylene glycol or glycerol without amino adds, and then stirred and heated with the same procedure as shown above to act as control samples. 0.01 mole amino acid and/or 0.01 mole a-D-glucose or D-xylose

4^ mixed with 50 g of propylene glycol or glycerol

4^ stirred in a 250 mL Erienmeyer flask for 2 hr put into a microwave oven

4^ heat for 2 min under 650W power

4^ cool immediately

4^ color measurement and odor description Figure 1. Flow chart for the preparation of microwave heated samples of amio acids and/or sugars B. Determination of the maximum absorption wavelengtlis and Hunter "L" values of the heated samples After being diluted 200 times with PG or glycerol (depending on which is the original solvent system), the UV and visible absorption of the microwave heated solutions were measured on a Beckman DU-70 Spectrophotometer to find the maximum absorption wavelength. The absorption of the diluted microwave heated solutions at wavelength 420 nm were also measured. Hunter "L" values of the diluted samples were measured on a color analyzer (Color Mate OEM, Milton Roy Co., USA). C. Observation of some properties of the samples The solubilities and the pH values of the heated or unheated samples were also observed or measured in this study. The odors of the heated solutions were evaluated and described by one trained flavorist.

496

3. RESULTS AND DISCUSSIONS 3.1. Solubilities of sugars and/or amino acids Solubilities of the samples of sugars and/or amino acids are shown in Table 1 to Table 7. In general, heating caused solubilization of the mixtures. Exceptions are noted in the tables.

Table 1 Some properties of various sugars in propylene glycol and glycerol System D-glucose in PG* D-xylose in PG D-glucose in Glycerol D-xylose in Glycerol

Solubility

pH

Solubility

before heating SS*** SS SS SS

before heating 7.21 6.97 7.48 7.23

after heating

s*** S S S

pH

Maximum

after absorption wave heating length (nm)** 5.10 319 5.52 319 355 4.09 350 3.88

OD* value 0.0098 0.0159 0.0857 0.0300

PG: propylene glycol Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS : slightly soluble S: soluble

3.2. pH values of sugars and/or amino acids pH values of the samples of sugars and/or amino acids are shown in Table 1 to Table 7. As shown in Table 1, pHs of the sugars In PG or glycerol were approximately 7.0 before heating and dropped to 3.88-5.52 after heating. The decrease In pHs of these samples after heating probably resulted from the release of acid compounds from the degradation of sugars during microwave heating. As shown in Table 2, pHs of all the amino acids In PG except L-glutamine, L-glutamic acid, and L-lysine increased after microwave heating. The increase in the pHs of these samples probably resulted from the release of ammonia or amines from these amino acids during microwave heating. As shown in Table 3, pHs of all the amino acids in glycerol except L-arginine, L-glutamic acid, and L-lyslne increased after microwave heating. The increase in the pHs of these samples probably also resulted from the release of ammonia or amines from these amino acids during microwave heating. As shown in Table 4 to Table 7, L-arginine and L-lysine systems had pH values higher than 7 either before or after heating. pHs of proline with either Dglucose or D-xylose in PG or in glycerol were found to increase significantly after microwave heating.

497

3.3. Visible wave absorptions of sugars and/or amino acids The maximum absorption wavelengths of the various solutions are shown in Table 1 to Table 7. As shown in these tables, the maximum absorption wavelengths of these samples were found in the range of 285 to 360 nm. The OD values of these samples after 200 x dilution and measured at 420 nm (widely accepted for the

Table 2 Some properties of various amino acids in propylene glycol Solubility pH Solubility pH Amino acid*

before heating SS*** S SS SS SS SS SS SS SS SS SS S SS paste S SS SS SS SS SS

before heating 6.65 11.14 5.44 4.31 5.19 5.89 4.55 6.77

after heating SS

Maximum

after absorption waveheating length (nm)** 341 7.96 alanine 11.37 335 s*** arginine 315 7.94 SS asparagine 4.61 338 SS aspartic acid 6.82 355 SS cysteine 349 4.75 glutamlne s 2.87 SS 300 glutamic acid 7.41 345 SS glycine 7.72 7.13 histidine 343 SS 7.74 SS 6.71 338 isoleucine 6.57 leucine 8.41 SS 338 9.63 lysine 9.61 335 s 6.42 methionine 8.35 SS 349 phenylalanine 5.83 7.72 SS 320 proline 6.51 8.78 355 s serine 7.63 SS 6.38 335 threonine 6.24 8.32 SS 352 tryptophan 6.40 SS 7.69 295 tyrosine 6.48 SS 8.08 335 valine 6.49 SS 7.39 345 All chiral amino acids used were the L-isomers

OD** value 0.0433 0.0089 0.0219 0.0072 0.0316 0.0142 0.0005 0.0174 0.0188 0.0170 0.0047 0.0248 0.0028 0.0180 0.0053 0.0192 0.0007 0.0533 0.0095 0.0116

Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS : slightly soluble S: soluble

498 determination of the relative concentration of the Maillard reaction products) are also shown in Table 1 to Table 7. When the sugars or amino acids were heated individually in PG or glycerol, the OD values of the heated solutions were found to be low. Significantly Increasing in OD values were found when each amino acid was mixed with D-glucose or D-xylose and microwave heated in PG or glycerol which indicated Maillard reactions, occurred during microwave heating.

Table 3 Some properties of various amino acids in glycerol Solubility pH Solubility pH Amino acid* alanine arginine asparagine aspartic acid cysteine glutamine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine

before heating SS*** SS SS SS SS SS SS SS SS SS SS

s SS SS

s SS SS SS SS

before heating 6.74 10.54 7.57 3.41 5.45 5.74 3.75 6.32 7.69 6.98 6.78 10.09 6.19 5.43 6.54 6.43 6.12 6.09 5.94 6.07

after heating

s*** S SS S S S S SS

s s s s s s s s s s SS

Maximum

absorption waveafter length (nm)** heating 288 8.76 288 9.46 290 8.76 288 6.72 291 9.11 7.21 288 3.06 8.50 8.47 8.90 8.78 10.12 9.46 8.92 9.66 9.28 9.02 8.99 8.31

288 342 292 288 345 285 318 288 288 318 328 295 288

OD** value 0.0046 0.0531 0.1049 0.0445 0.0647 0.0219 0.0017 0.0670 0.0823 0.0546 0.0215 0.0812 0.0380 0.0273 0.0607 0.2168 0.0579 0.1799 0.0363 0.0547

SS 9.11 285 s All chiral amino acids used were the L-isomers Maximum absorption wavelength (nm): the heated solutions were measured after 200 x dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS : slightly soluble S: soluble

valine

499 As shown in Table 4 and Table 5, L-tryptophan had the highest OD value among the annino acids in the systems of amino acid plus D-glucose or D-xylose and heated in PG. These results agreed with the Hunter "L" value measurements shown in Table 8. As shown in Table 3, L-tryptophan had the lowest Hunter "L" value among amino acids plus D-glucose or xylose and heated in PG. This result also

Table 4 Some properties of the mixtures of D-glucose and various amino acids in propylene glycQi OD** Maximum Solubility Solubility pH pH Amino acid* alanine arginine asparagine aspartic acid cysteine glutamine

before heating SS*** SS SS SS SS SS

before heating 6.04 10.89 5.60 4.41 4.82 5.45

after heating

s*** S S S S S

4.97 S SS glutamic acid 5.92 SS S glycine 6.79 SS S histidine 5.99 SS isoleucine S 6.14 SS leucine S 8.93 SS lysine S 5.80 SS methionine S 5.24 paste phenylalanine S 6.10 SS proline S 5.45 SS serine S SS threonine 5.43 S tryptophan SS 5.69 S SS tyrosine 5.88 S valine 6.12 SS S All chiral amino acids used were the L-isomers

after heating 6.23 7.72 5.08 5.21 5.26 4.12 4.21 5.76 6.28 6.68 5.98 8.84 6.30 4.65 7.23 5.74 6.15 5.65 4.86 6.72

bsorption wavelength (nm)** 301 295 292 295 292 298 295 300 295 300 298 298 295 299 300 300 302 345 300 302

value 0.6075 0.4813 0.2464 0.3939 0.2757 0.3426 0.4658 0.5754 0.5274 0.5322 0.7063 0.5364 0.4459 0.5636 0.9662 0.8057 0.6213 1.5643 0.6606 0.5260

Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS: slightly soluble S: soluble

500 indicated that L-tryptophan generated the darkest appearance among all the amino acids; L-tryptophan had the highest OD value among the amino acids in the systems of amino acid plus D-glucose used in this study when heated with D-glucose or Dxylose in PG.

Table 5 Some properties of the mixtures of D-xylose and various amino acids in propylene glycol pH Maximum OD** Solubility pH Solubility after absorption wavevalue Amino acid* before before after heating length (nm)^ heating heating heating 0.6124 308 5.83 6.52 SS*** alanine s*** 0.5135 300 8.96 S 10.80 SS arginine 5.27 0.3399 298 S 6.38 SS asparagine 0.5590 300 5.06 5.32 S SS aspartic acid 0.4507 302 5.32 6.48 S SS cysteine 0.5647 310 3.83 S 7.38 SS glutamine 0.3341 300 3.53 S 4.68 SS glutamic acid 5.74 0.7187 305 S 5.29 SS glycine 305 6.45 S 6.25 SS histidine 0.6503 isoleucine 305 6.80 S 5.96 SS 0.6243 6.31 S 5.80 SS leucine 0.5514 305 8.94 8.45 lysine 0.7654 308 S s 0.5661 5.39 S 5.79 SS methionine 300 5.40 paste phenylalanine 6.96 S 0.8987 310 5.73 proline 317 7.50 S S 1.0325 5.57 SS serine S 310 5.98 0.8292 threonine 5.98 SS 6.68 S 305 0.6130 tryptophan 6.41 7.50 SS S 345 1.6023 tyrosine 8.04 SS S 5.23 306 0.8310 valine 6.50 SS 5.27 S 295 0.7125 All chiral amino acids used were the L-isomers Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS: slightly soluble S: soluble

501 As shown in Table 6 and Table 7, L-tyrosine had the highest OD value among the amino acids in the system of amino acid plus D-glucose or D-xylose and heated in glycerol. These results agreed with the Hunter "L" value measurements shown in Table 8. As shown in Table 8, L-tyrosine had the lowest Hunter "L" value among amino acids in the system of amino acid plus D-glucose or xylose and heated in

Table 6 Some properties of the mixtures of D-gl ucose and various amino acids in glycerol Maximum OD** Solubility Solubility pH PH before heating 8.21 10.35 5.48 3.37 4.69 5.49

after heating

alanine arginine asparagine aspartic acid cysteine glutamine

before heating SS*** SS SS SS SS SS

glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine

SS SS SS SS SS SS SS SS SS SS SS SS SS SS

4.21 5.04 6.68 6.56 6.50 9.49 7.68 5.68 5.95 5.44 5.65 5.93 6.17 6.32

s s s s s s s s s s s s s s

Amino acid*

after absorption waveheating length (nm)** 6.50 305 335 9.14 325 6.78 305 6.25 315 6.91 350 5.22 360 4.22 345 6.79 330 7.42 315 6.31 310 5.60 340 8.76 325 6.43 310 6.01 335 8.32 330 6.13 350 6.63 335 6.21 335 5.41 315 7.14

s*** S S S S S

value 0.7735 0.7734 0.6607 0.8591 0.7852 0.7533 0.9727 1.5959 1.0852 0.6413 0.5557 0.9561 0.9855 0.5592 1.3099 1.4007 1.5524 1.7309 1.7316 1.0629

All chiral amino acids used were the L-isomers Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS: slightly soluble

S: soluble

502 glycerol. L-tryptophan generated the darkest appearance and had the highest OD value among all the amino acids. 3.4. Aroma descriptions of sugars and/or amino acids Aroma descriptions of microwave heated D-glucose and/or amino acids in PG or

Table 7 Some properties of the mixtures of D-xylose and various amino acids in glycerol Solubility pH Solubility pH Maximum

OD**

Amino acid*

value

alanine arginine asparagine aspartic acid cysteine glutamine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine

before heating SS*** SS SS SS SS SS SS SS SS SS SS SS SS SS SS SS SS SS SS SS

before heating 5.88

after heating

10.1 5.20 3.74 4.33 4.74 4.14 5.13 6.5 5.84 5.70 9.49 5.13 4.84 5.61 5.23 5.51 5.8 6.18 6.01

S S S S S S S S S S S S S

s***

s s s s s s

after absorption waveheating length (nm)** 300 7.06 320 9.18 300 7.48 305 6.8 300 7.35 6.24 315 310 4.48 6.83 305 330 7.24 6.62 310 6.44 300 9.12 305 300 6.96 6.36 295 8.22 315 6.64 305 7.04 335 6.79 340 6.55 345 6.76 315

1.0985 1.0128 0.4789 0.5784 0.4966 0.7028 0.8252 0.8762 1.0029 0.7912 0.4983 0.7107 0.6285 0.4715 0.8043 1.2451 0.8663 0.9314 1.3563 0.6049

All chiral amino acids used were the L-isomers Maximum absorption wavelength (nm): the heated solutions were measured after 200 X dilution OD value : the heated solutions were measured at 420 nm after 200 x dilution SS: slightly soluble S: soluble

503 glycerol are shown in Table 9 and Table 10. Aroma descriptions of microwave heated D-xylose and/or amino acids in PG or glycerol are shown in Table 11 and Table 12. As shown in Table 9 to Table 12, most of the amino acids when heated with D-glucose or D-xylose had caramellic and burned type aroma. The flavor generated from the reaction of D-glucose or D-xylose with the following amino acids

Table 8 Hunter "L" values of the reaction solutions of D-glucose (Glu) or D-xylose (Xyl) and various amino acids in propylene glycol (PG) or glycerol (Gly) (measured after 200 x dilution). Glu in PG Xyl in PG Glu in Gly Xyl in Gly in PG in Gly ND ND* 24.45 22.18 25.22 21.94 L-alanine ND ND 27.05 23.78 26.46 27.28 L-arginine ND ND 25.83 27.22 29.11 30.63 L-asparagine ND ND 24.81 24.35 25.34 27.16 L-aspartic acid ND 22.62 26.87 29.77 ND 21.99 L-cysteine 23.77 ND ND 25.08 26.89 28.16 L-glutamine ND ND 23.35 25.18 19.56 27.09 L-glutamic acid ND ND 24.31 23.34 20.43 23.48 glycine ND 21.52 21.02 20.81 25.47 L-histidine ND ND 26.94 24.21 ND 27.73 24.26 L-isoleucine ND 31.06 29.16 24.54 24.86 L-leucine ND ND 24.37 26.61 25.81 25.26 L-lyslne ND ND 23.92 28.13 L-methionine 25.20 23.59 ND 21.65 L-phenylalanine ND 30.13 26.63 17.80 ND L-proline 23.00 23.67 ND 25.35 21.33 ND 20.04 L-serine 18.97 17.31 19.55 ND ND 23.51 L-threonine 21.93 24.73 ND 23.80 ND L-tryptophan 15.26 14.05 ND 20.68 25.61 ND L-tyrosine 20.79 17.34 16.78 16.45 ND ND L-valine 24.52 26.14 23.30 ND 27.76 ND D-glucose ND ND ND ND 34.49 35.62 D-xylose ND ND ND ND 34.27 33.22 * ND: no data

504 were found stronger and more characteristic, they are: L-cysteine, L-glutamine, Lisoleucine, L-leucine, L-methionine, L-phenylalanine, and L-proline. The systems of heated L-cysteine with D-glucose or D-xylose in PG or in glycerol had roast barley, roast meaty and popcorn-like flavor. The systems of heated L-glutamine with Dglucose or D-xylose in PG or in glycerol had caramellic, gourd melon drink like

Table 9 Odor descriptions of the reaction solutions of D-glucose and various amino acids in propylene glycol Odor Description Amino Acid* caramellic, earthy, nutty alanine caramellic, baked taro-like, nutty arginine caramellic asparagine caramellic, earthy aspartic acid burned, roast barley & popcorn-like, roast meaty, nutty cysteine caramellic, gourd melon drink-like glutamine slightly caramellic with slightly acetaldehyde and glutamic acid ethyl acetate top note caramellic glycine caramellic histidine green, floral, sour earthy, slightly tomato odor isoleucine green, jasmin-like, cocoa-like, tomato-like leucine caramellic, baked bakery & baked taro-like lysine fermented radish or cabbage-like, baked potato-like methionine sweet-floral, honey-like phenylalanine caramellic, earthy, baked bakery & baked taro-like proline caramellic, baked taro-like, chocolate-like serine caramellic, baked taro-like, chocolate-like threonine caramellic, earthy, urea-like tryptophan caramellic, nutty tyrosine green, floral, chocolate-like, green tomato-like valine ' All chiral amino acids used were the L-isomers.

505 flavor. The systems of heated L-isoleucine with D-glucose or D-xylose in PG or in glycerol had green, floral, slightly tomato-like flavor. The systems of heated Lleucine with D-glucose or D-xylose in PG or in glycerol had green, jasmine-like, cocoa-like, and tomato-like flavor. The systems of heated L-methionine with Dglucose or D-xylose in PG or in glycerol had fermented radish or fermented

Table 10 Odor descriptions of the reaction solutions of D-glucose and various amino acids in glycerol Odor Description Amino Acid* earthy, burned, nutty alanine baked taro-like, burned, nutty arginine caramellic asparagine earthy, caramellic aspartic acid burned, roast barley & popcorn-like, roast meaty, nutty cysteine caramellic, gourd melon-like glutamine caramellic, caramel candy-like glutamic acid caramellic, slightly earthy glycine caramellic histidine green, floral, sour earthy, slightly tomato odor isoleucine leucine green, jasmine-like, cocoa-like, tomato-like lysine caramellic, slightly baked taro-like methionine fermented radish or cabbage-like, baked potato-like phenylalanine sweet-floral, honey-like, cinnamon-like proline burned, earthy, baked bakery, gourd melon-like serine caramellic, baked taro-like, chocolate-like threonine caramellic, baked taro-like, chocolate-like tryptophan urea-like, animal odor, burned tyrosine animal odor, burned valine green, floral, cocoa-like, green tomato-like * All chiral amino acids used were the L-isomers.

506 cabbage-like, and baked potato-like flavor. The systems of heated L-phenylalanine with D-glucose or D-xylose in PG or in glycerol had sweet-floral, and honey-like flavor. The systems of heated L-proline with D-glucose or D-xylose in PG in glycerol had caramellic, baked bakery, and baked taro-like flavor. When the amino acids were microwave-heated alone in PG or glycerol, the color and odors generated were not so intense.

Table 11 Odor descriptions of the reaction solutions of D-xylose and various amino acids in propylene glycol Odor Description Amino Acid* caramellic, earthy, nutty alanine caramellic, baked taro-like, nutty arginine caramellic asparagine aspartic acid caramellic, earthy cysteine burned, roast barley & roast flour odor caramellic, gourd melon drink-like glutamine slightly caramellic with slightly acetaldehyde and glutamic acid ethyl acetate top note caramellic glycine caramellic histidine green, floral, sour earthy, slightly tomato odor isoleucine green, jasmin-like, cocoa-like, tomato-like leucine caramellic, baked bakery & baked taro-like lysine fermented radish or cabbage-like, baked potato-like methionine sweet-floral, honey-like phenylalanine caramellic, earthy, baked bakery & baked taro-like proline caramellic, baked taro-like, chocolate-like serine caramellic, baked taro-like, chocolate-like threonine caramellic, earthy, urea-like tryptophan caramellic tyrosine green, floral, chocolate-like, green tomato-like valine * All chiral amino acids used were the L-isomers.

507 Table 12 Odor descriptions of the reaction solutions of D-xylose and various amino acids in glycerol Amino Acid*

Odor Description

alanine

earthy, burned, baked-taro like

arginine

baked taro-IIke, burned, nutty

asparagine

burned odor

aspartic acid

earthy, burned odor

cysteine

burned, roast barley& roast flour note

glutamine

burned, gourd melon-like

glutamic acid

caramellic, caramel candy-like

glycine

burned, earthy

histidine

burned

isoleucine

green, floral, sour earthy, slightly tomato odor

leucine

green, jasmine-like, cocoa-like, tomato-like

lysine

burned

methionine

fermented radish or cabbage-like, baked potato note

phenylalanine

sweet-floral, honey-like, cinnamon-like

proline

burned, baked bakery, baked taro note

serine

caramellic, baked taro-like

threonine

caramellic, baked taro-like

tryptophan

urea-like, earthy, burned

tyrosine

animal odor, burned

valine

green, floral, cocoa-like, green tomato-like

' All chiral amino acids used were the L-isomers.

4. CONCLUSIONS In this paper, the color and flavor fornnation through the interactions of various amino acids with D-glucose or D-xylose in P G or glycerol were presented. Very intense colors and flavors were generated from the microwave-heated samples. The results of this contribution could provide information for those who wish to resolve

508 the problems of weak color and flavor of microwave heated foods. The flavor characteristics shown in this study also provide information for designing the desired flavors through the combinations of various amino acids and sugars followed by microwave heating in PG or in glycerol.

5. REFERENCES 1 2

T.V. Eijk, Dragoco Report, 1 (1991) 3. T.V. Eijk in:Thermally Generated Flavors - Malllard, Microwave, and Extrusion Processes, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 395. 3 R.F. Heinze, Cereal Foods World, 34 (1989) 334. 4 T.R. Lindstrom and T.H. Parliment in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 405. 5 M.A. Stanford and R.J. McGorrin in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 414. 6 C. Whorton and G.A. Reinecciusus in:Thermal Generation of Aroma, T.H. Parliment, R.J. McGorrin and C.-T. Ho, ACS Symposium Series No 409 (1989) 526. 7 J.A. Steinke, C. Frick, K. Strassburger and J. Gallagher. Cereal Food World, 34(1989)330. 8 J.A. Steinke, CM. Frick, J.A. Gallagher and K.J. Strassburger in:Thermal Generation of Aroma, T.H. Parliment, R.J. McGorrin and C.-T. Ho, ACS Symposium Series No 409 (1989) 519. 9 T.R. Schiffmann in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 386. 10 E. Graf and K.B.D. Roos in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 437. 11 V.A. Yaylanyan, N.G. Forage and S. Madeville in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 449. 12 T. Shibamoto and H. Yeo in:Thermally Generated Flavors - Maillard, Microwave, and Extrusion Process, T.H. Parliament, M.J. Morello and R.J. McGorrin eds., ACS Symposium Series No 543, ACS, Washington, DC (1994) 457.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

509

Characterization of intermediate 3-oxazolines and 3-thiazolines from the reaction of 3-hydroxy-2-butanone and ammonium sulfide Chi-Tang Ho^ Junwu Xi^ Hui-Yin Fu^ and Tzou-Chi Huang' ^Department of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA department of Food Sanitation, Ta Jen Pharmaceutical Junior College, Pingtung, Taiwan, ROC ^Department of Food Science and Technology, National Pingtung Polytechnic Institute, Pingtung, Taiwan, ROC

Abstract Volatile compounds formed from the reaction of 3-hydroxy-2-butanone/ ammonium sulfide at 25, 50 and 70°C were investigated. Two well-known aroma compounds, 2,4,5trimethylthiazole and 2,4,5-trimethyl-3-thiazoline were identified in addition to 2,4,5trimethyloxazole and 2,4,5-trimethyl-3-oxazoline. Four interesting intermediate compounds, 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline, 2-(l-mercaptoethyl)-2,4,5-trimethyl-3-oxazoline, 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-thiazoline and 2-(l-mercaptoethyl)-2,4,5-trimethyl-3-thiazoline were identified by GC-EIMS and GC-CIMS. All intermediates were formed at a reaction temperature below 25°C. On the other hand, tetramethylpyrazine was the major product when the reaction temperature was higher than 70°C.

1. INTRODUCTION Thiazoles and thiazolines are heterocyclic compounds containing both sulfur and nitrogen atoms. They possess potent sensory quality at low concentration and are generally described as green, nutty and vegetable-like [1-2]. The occurrence of thiazoles in food flavor has been reviewed [3-4]. They have been identified in a variety of processed foods such as baked potato [5], roasted peanuts [6], peanut butter [7], cocoa butter [8] and fried chicken [9]. Thiazole have also been reported in various model system reactions involving either degradation of glucose in the presence of hydrogen sulfide and ammonia [10-11], or more frequently, fragmentation of cysteine or cystine [12-13], or reaction of these with reducing sugars [14-15] or furaneol [16]. It has been proposed that thiazolines may be formed in foods by the interaction of adicarbonyl compounds, aldehydes, ammonia and hydrogen sulfide [17]. In fact, thiazoles and thiazolines have been identified from the reaction of 2,3-pentanedione, acetaldehyde, ammonia and hydrogen sulfide [18]. In recent study on the reaction of 3-hydroxy-2-

510 butanone with ammonium acetate at low temperature, an interesting intermediate compound, 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline was isolated and identified [19-20]. The purpose of the present study was to isolate and identify the flavor precursors, thiazolines, from the reaction of 3-hydroxy-2-butanone/ammonium sulfide model system at low temperature.

2. EXPERIMENTAL PROCEDURES 2.1. Materials 3-Hydroxy-2-butanone and ammonium sulfide (20 wt. % solution in water) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Methylene chloride and w-tridecane was obtained from Fisher Scientific Co. (Pittsburgh, PA). 2.2. Sample Preparation A total of 0.88 g (0.01 mol) of 3-hydroxy-2-butanone and 6.8 mL (0.02 mol) of ammonium sulfide were mixed into 25 mL distilled water, and the pH value was adjusted to 5.5 using 6N and IN HCl. The mixture was transferred into a 0.3-L Hoke SS-DOT sample cylinder, and the cylinder was sealed and heated at 25, 50, 75, 100, 125 and 150°C for 2 hours. One mL 1000 ppm w-tridecane was added into the reaction mixture as an internal standard. The reaction mixture was then extracted with 50 mL of methylene chloride. The combined extract was dried over anhydrous sodium sulfate and concentrated to a final volume of 1 mL by blowing gently with nitrogen gas. 1 |iL of extract was injected into the GC. 2.3. Quatitation and Characterization of Volatile Compounds 2.3.1. Gas Chromatography GC analysis was accomplished by using a Varian 3400 gas chromatograph. A fijsed silica capillary column (30 m x 0.25 mm i.d., film thickness 0.25 |am, DB-1701; J&W) was used to analyze the volatile compounds. The operating conditions were as follows: injector and detector temperatures, 250 and 270°C, respectively; helium carrier flow rate, 1 mL/min; GC temperature program, 40-260°C at 3 °C/min followed by an isothermal hold at 260°C for 10 min. 2.3.2. Gas Chromatography-Mass Spectrometry Analysis EI mass spectra were obtained using a Hewlett-Packard 5790 gas chromatograph coupled with a Hewlett-Packard 5970A MSD detector electron ionization at 70 eV and an ion source of temperature 250°C. The operation conditions were the same as those used in the GC analysis described above. The data were recorded and analyzed using Hewlett-Packard MS ChemStation data with NIST/EPA/MSDC mass spectral database. CI mass spectra were performed on a Finnigan ITS-40 Magnum ion trap mass spectrometer coupled with a Varian 3400 gas chromatograph and reactant gas (isobutane) was used. A fused silica capillary column (30 m x 0.25 mm i.d., film thickness 0.25 fim, DB-5, J&W) was used. The operating conditions were as follows: injector temperature, 260°C; transfer line temperature, 260°C; helium carrier flow rate, 1 mL/min; GC temperature program, 60-260°C at 6°C/min followed by an isothermal hold at 260°C for 12 min.

511

3. RESULTS AND DISCUSSION The GC-mass chromatogram of volatile compounds formed in the 3-hydroxy-2butanone/ammonium sulfide model system at 75°C is shown in Figure 1. Eleven compounds were tentatively identified by GC-MS (EI and CI). Their identities and retention times are listed in Table 1.

Table 1. Volatile compounds identified in the reaction of 3-hydroxy-2-butanone/ammonium sulfide at 75°C No.

1 2 3 4 5 6 7 8 9 10 11

Retention Time (min) 18.40 19.01 24.10 27.50 27.89 29.29 29.95 38.64 39.07 40.49 41.05

Compounds 2,4,5-trimethylthiazole 2,4,5-trimethyl-3-thiazoline Tetramethylpyrazine 2-( 1 -hydroxy ethyl)-2,4,5 -trimethy 1-3 -oxazoline 2-(l-hydroxy ethyl)-2,4,5-trimethy 1-3-oxazoline 2-( 1 -mercaptoethyl)-2,4,5 -trimethy 1-3 -oxazoline 2-(l-mercaptoethyl)-2,4,5-trimethyl-3-oxazoline 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-thiazoline 2-( 1 -hydroxy ethy l)-2,4,5-trimethyl-3 -thiazoline 2-( 1 -mercaptoethyl)-2,4,5-trimethy 1-3 -thiazoline 2-( 1 -mercaptoethyl)-2,4,5-trimethy 1-3 -thiazoline

Compounds 4 and 5 have a molecular weight of 157 as determined by CI-MS. They have the same EI-MS spectra as shown in Figure 2. This mass spectrum matches well with the spectral data published previously by Shu and Lawrence [19] and Fu and Ho [20]. These two peaks were, therefore, identified as isomers of 2-(l-hydroxyethyl)-2,4,5-trimethy 1-3oxazoline. Shu and Lawrence [19] have also observed the isomers of this compound in their studies. They have described the flavor characteristics of this compound as mild aroma, yeasty, nutty, and bread-crust-like. Compounds 6 and 7 have a molecular weight of 173 as determined by CI-MS. They also have the same EI-MS spectra as shown in Figure 3. This mass spectrum is extremely similar to that of 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline. These two compounds were proposed to be the isomers of 2-(l-mercaptoethyl)-2,4,5-trimethy 1-3-oxazoline. Compounds 8 and 9 also have a molecular weight of 173. Their EI-MS spectrum shown in Figure 4 suggested that they are sulftir analogs of compounds 4 and 5. These two compounds were proposed to be the isomers of 2-(l-hydroxyethyl)-2,4,5-trimethyl-3thiazoline. Compounds 10 and 11 have a molecular weight of 189 as determined by CI-MS. Their EI-MS spectrum shown in Figure 5 suggested that they are the isomer of 2-(lmercaptoethyl)-2,4,5-trimethyl-3-thiazoline. Figure 6 shows the structures and formation of these newly identified oxazolines and thiazolines in the current model systems. 3-Hydroxy-2-butanone may react with ammonia and hydrogen sulfide to form the 3-hydroxy-2-aminobutane, 3-mercapto-2-butanone and 3-

512

67

10 11

89

lluL-iU

^^JUJLJLAJ

VJUUUJA--^ "T 50 40

—!—r-T—I—\—I—I—I—I—1—[-

20 Figure 1.

30

min.

GC-Mass chromatogram of volatile compounds formed at.75 °C-

513

100 71 80 112 60 42 40

20 -t

20

30

98

57 .iilL '59

31

¥ 40

50

jiT.3

60

70

84 80

124 90

100

110

120

130

Figure 2. EI mass spectrum of 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline (Peaks 4 and 5).

100 -j

4|3

80 50

71 112

40 i 20

41

55

32 30

68

llL 40

50

60

70

82 80

99 88 96, ' I ' ' ' ' I' 90 100

109

—r—I—I—I—r

110

Figure 3. EI mass spectrum of 2-(l-mercaptoethyl)-2,4,5-trimethyl-3-oxazoline (Peaks 6 and 7).

120

514

100-

817

80 128

60 1

43 53

40 i

72

42

114

20 96

127 11B

:i 60

40

80

100

140

120

160

Figure 4. EI mass spectrum of 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-thiazoline (Peaks 8 and 9).

100 -1

817

80 I

128

60

40 4%5

53

114

20

71

96

31 0

.

j

;

20

I

I

!

I '•

30

I

•••' ' [ T T f i - j - i

40

50

•!

,

.

I

•

60

•

•

•

(

i

70

.

.

.

I

xJL I

80

,

:

4-16 •

,

.

90

I

.

.

)

.

100

•

•

I

I

'

110

•

•

'

140 i

•

120

;

.

.

,

.

•

'

•

I

•

130 140

Figure 5. EI mass spectrum of 2-(l-mercaptoethyl)-2,4,5-trimethyl-3-thiazoline (Peaks 10 and 11).

241-HYDROXY-ETHYLk2.4.5TRIMETHYL-3-THIAZOLINE

t OH

t

C&

t

SH I + C&-CH -C=

OH C&-CH-C=

24.1-MERCAPTO-ETHYLb2.4.5TRIMETHYL-3-THIAZOLINE

CH-CH-CH3

C&

OH

CH-CH-CHs I SH

OH

2-(1-HYDROXY-ETHYLb2.4.5TRIMETHYL-3-OXAZOLINE

2-(l-MERCAPTO-ETHYLI-2.4.5TRIMETHYL-3-OXAZOLINE

Structures and formation of oxazolines and thiazolines in the reaction of 3-hydroxy-2-butanone Figure 6. and ammonium sulfide.

c w!

w!

516 mercapto-2-aminobutane. The interaction of these compounds will eventually lead to the formation of 1-hydroxyethyl- and 1-mercaptoethyl-oxazoles and thiazoles. Six temperatures (25, 50, 75, 100, 125, 150°C) were investigated in this model system. Quantitation of volatile compounds is summarized in Table 2. In the study of Fu and Ho [20], it was observed that in the reaction of 3-hydroxy-2-butanone with ammonium acetate, 2(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline formed predominately below 25°C, whereas tetramethylpyrazine was the major product at a reaction temperature higher than 85°C. Their study also supported the proposal of Shu and Lawrence [19] that at higher temperatures or under prolonged storage, 2-(l-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline underwent a reversible reaction and which lead to the formation of tetramethylpyrazine. From the current study, it seemed that in the presence of hydrogen sulfide, the formation of tetramethylpyrazine was reduced at higher temperatures. 2-(l-Hydroxyethyl)-2,4,5trimethyl-3-thiazoline and 2-(l-mercapto-ethyl)-2,4,5-trimethyl-3-thiazoline may also be more stable than 2-(l-hydroxy-ethyl)-2,4,5-trimethyl-3-oxazoline at higher temperatures. At temperatures higher than 100 °C, the formation of 2,4,5-trimethylthiazole and 2,4,5trimethyl-3-thiazoline were significantly increased. Table 2. Quantitaion of Identified Volatile Compounds from the 3-Hydroxy-2butanone/Ammonium Sulfide Model System. Compound identified

2,4,5-trimethylthiazole 2,4,5-trimethyl-3-thiazoline Tetramethylpyrazine 2-(l-hydroxyethyl)-2,4,5trimethy 1-3 -oxazoline 2-(l-mercaptoethyl)-2,4,5trimethy 1-3 -oxazoline 2-(l-hydroxyethyl)-2,4,5trimethy 1-3 -thiazoline 2-(l-mercaptoethyl)-2,4,5trimethy 1-3 -thiazoline

Quantity (mg/g acetoin) 25°C 50°C 75°C 100°C 125°C 150°C 0.09 tr tr

0.10 tr 0.09

0.21 0.15 1.31

0.69 0.37 11.31

2.78 1.67 66.38

4.31 1.90 97.47

26.48 24.39

11.92

3.81

0.26

0.78

0.57

0.81

0.17

0.17

0.02

tr

0.18

1.40

2.64

1.47

1.70

0.86

0.20

2.48

3.71

2.63

2.36

2.02

4. REFERENCES 1 2 3 4

A.O. Pittet and D.E. Hruza, J. Agric. Food Chem., 22 (1974) 264. C.-T. Ho and Q.Z Jin, Perfumer & Flavorist, 9(6) (1984) 15. J.A. Maga, Crit. Rev. Food Sci. Nutr., 6 (1975) 153. G. Vernin (ed.). The Chemistry of Heterocyclic Flavoring and Aroma Compounds, Ellis Horwood Publishers, Chicherster, UK, 1982. 5 B.C. Coleman, C.-T. Ho and S.S. Chang, J. Agric. Food Chem., 29 (1981) 42. 6 C.-T. Ho, Q.Z. Jin, M.H. Lee and S.S. Chang, J. Agric. Food Chem., 31 (1983) 1384.

517 7 8 9 10 11 12 13 14 15 16 17 18 19 20

K. Joo and C.-T. Ho, Biosci. Biotech. Biochem., 61 (1997) 171. C.-T. Ho, Q.Z. Jin, K.N. Lee and J.T. Carlin, J. Food Sci., 48 (1983) 1570. J. Tang, Q.Z. Jin, G.H. Shen, C.-T. Ho and S.S. Chang, J. Agric. Food Chem., 31 (1983) 1287. T. Shibamoto and G.F. Russell, J. Agric. Food Chem., 24 (1976) 843. T. Shibamoto and G.F. Russell, J. Agric. Food Chem., 25 (1976) 110. C.K. Shu, M.L. Hagedorn, B.D. Mookherjee and C.-T. Ho, J. Agric. Food Chem., 33 (1985)438. C.K. Shu, M.L. Hagedorn, B.D. Mookherjee and C.-T. Ho, J. Agric. Food Chem., 33 (1985)442. F. Ledl and T. Severin, Mikrobiol. Technol. Lebensm., 2 (1973) 155. H. Kato, T. Kurata and M. Fujimaki, Agric. Biol. Chem., 37 (1973) 539. C.K. Shu and C.-T. Ho, J. Agric. Food Chem., 36 (1988) 801. C.J. Mussinan, R.A. Wilson, I. Katz, A. Hruza and M.H. Vock, ACS Symp. Ser., 26 (1976) 133. H.J. Takken, L.M. van der Linde, P.J. de Valois, H.M. van Dort and M. Boelens, ACS Symp. Ser., 26(1976)114. C.K. Shu and B.M. Lawrence, J. Agric. Food Chem., 43 (1995) 2922. H.Y. Fu and C.-T. Ho, J. Agric. Food Chem., 45 (1997) 1878.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

519

Mechanistic Studies on the Formation of Thiazolidine and Structurely Related Volatiles in Cysteamine /Carbonyls Model System Tzou-Chi Huang', Y-M. Su', L.Z. Huang' and Chi-Tang Ho'* 'Department of Food Science and Technology, National Pingtung Polytechnic Institute, 912, Pingtung, Taiwan 'T^epartment of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA

Abstract Phosphate was found to dramatically enhance the formation of thiazolidine in a cysteamine/carbonyl model system. Phosphate tends to stabilize the primary carbocation formation which may lead to the completion of the cyclization by attacking the amino nitrogen on the activated carbon. Protic solvent further enhances thiazolidine formation by removing the water molecule. Thiazolidine formation is completed by combining the phosphate buffer with the protic solvent. The redox reaction catalyzed by phosphate ions results in the conversion of thiazolidine to the corresponding thiazoline through hydride transfer. The conversion of 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to 5-acetyl-2,3-dihydro-l,4-thiazine via a proton transfer reaction catalyzed by azodicarbonamide was evidenced as well. A formation mechanism for thiazolidine and structurely related tetrahydro-l,4-thiazine and 2,3-dihydro-l,4thiazine is proposed.

1.

EVTRODUCTION

Schiff base formation between the amino group and the aldehyde group has been the subject of numerous studies [1]. The amino group on a cysteamine may react with an aldehyde group to form a Schiff base as well. In addition to the Schiff base formation, a subsequent ring closing reaction leading to the formation of a thiazolidine deserves special interest. Thiazolidines generally possess a characteristic popcorn flavor [2]. Model systems composed of D-glucose and L-cysteine have long been used to study the thermal generation of thiazolines and thiazines [3-4]. The reaction between cysteamine, the decarboxylated cysteine and 2,3-butanedione, a glucose degradation product, may lead to the formation of 2-acetyl-2methylthiazolidine [5]. Recently, a thiazolidine derivative method for the determination of trace aldehydes in foods and beverages has been developed [6-7]. These methods are based on the reaction of volatile carbonyl compounds with cysteamine (2-aminoethanethiol) to form stable thiazolidine derivatives under mild conditions (room temperature and neutral pH). The thiazolidine

520

derivatives formed were subsequently determined by gas chromatography. However, the formation pathways of thiazolidines are not yet well documented. On the other hand, intense roasted, popcorn-like odorant 5-acetyl-2,3-dihydro-l,4thiazine was identified in the D-ribose/L-cysteine model system [8-9]. It was proposed that a SchifF base is formed from the condensation between the amino group in cysteamine and the carbonyl group in 2,3-butanedione. Tautomerization and subsequent cyclization by a Michaeltype nucleophilic attack of the thiol group at the activated methyl carbon atom yield 5-(2hydroxyethenyl)-2,3,6-trihydro-l,4-thiazine. Oxidation of this enaminol results in 5-acetyl-2,3dihydro-l,4-thiazine which, due to the electronegativity of the sulfiir atom, tautomerizes into the more stable 5-acetyl-2,3-dihydro-l,4-thiazine, which is structurely related to 2-acetyl-2methylthiazolidine [8]. This paper focuses on the reactivity of cysteamine to a carbonyl compound involving 2,3butanedione and aliphatic short-chain aldehydes. A discussion on the formation mechanism of thiazolidine and structurally related volatile compounds will be provided.

2.

THE EFFECT OF A PHOSPHATE BUFFER SYSTEM ON THIAZOLmiNE FORMATION

Quantitative data obtained revealed that phosphate is a very effective buffer system for the promotion of the thiazolidine formation. The addition of a phosphate ion resulted in a 16fold, 12-fold and 21-fold increase for 2-acetyl-2-methylthiazolidine, 5-acetyl-2,3-dihydro-l,4thiazine and 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine formation respectively as compared to a water at pH 7.2 in cysteamine/2,3-butanedione system (Figure 1). The phosphate may act as both a hydrogen acceptor and donor, which catalyzes the Schiflf base formation during the generation of a thiazolidine and thiazines. Formation of thiazolidines was affected dramatically by the concentration of the phosphate buffer. Figure 2 shows the effect of different buffers on the formation of alkylthiazolidines in a cysteamine/aldehydes model system. A limited amount of unsubstituted thiazolidine was detected in the model system of cysteamine/aldehydes (pH 7.2) without phosphate. Concentrations of individual alkylthiazolidines increased with the increasing chain length of the alkyl group. The molar recovery of the five thiazolidines formed from the corresponding aldehyde and cysteamine were found to be quite low. They were 13%, 5.4%, 18.8%, 27.2% and 37.2% for unsubstituted thiazolidine, 2-methylthiazolidine, 2ethylthiazolidine, 2-propylthiazolidine and 2-butylthiazolidine, respectively. The reactivity of the aldehydes increased with the increasing alkyl chain length as shown in Figures 2A. Quantitative data obtained in this experiment revealed that phosphate was an effective buffer system for the formation of a thiazolidine. The addition of the phosphate buffer results in a 32-fold, 11-fold, 3.8-fold, 3.2-fold and 3.2-fold increases for unsubstituted thiazolidine, 2methylthiazolidine, 2-ethylthiazolidine, 2-propylthiazolidine and 2-butylthiazolidine respectively as compared with that in an aqueous system at pH 7.2 in Figure 2D. And the molar recovery for all of the five thiazolidines increased with increasing phosphate concentration linearly from 0.025 M to 0.2 M as shown in Table 1. This observation correlates better with the higher reactivity of the aldehydes larger than C3 (C3-C5) than those from formaldehyde and acetaldehyde in the preparation of thiazolidine from various aldehydes and cysteamine [10].

X

I

X ID

a CO

O

CD

K

I 5

A)!sue)U| eA|)e|a^

o 6=0

"o

\_/^ o

1^

CO

o

CM

10

5 £ c o c

I

521

no

E

0

C/3

>%

C/5

0)

>. H

(I> 0 ed (N

c "O

>^

1

X5

3

D 0 N c cd 0 - C "O 0) •^-^ tJ cd

£

1i)

>.

cd OJ c«

£

c

1 ro v 0)

1

0 (N

0 C C

(U

«*0 0

^ '^

N Cd

c

ed

£ u

cH a;

0 u

c ,__r 1 0

0

1

Z3 - C X5 0) 1 ed m (N

x:

x: (U a. u

c+- cd 0 u^ "0 T 3 C ed

,

4;

c

w

N cd J=

^

,__

<—'

COrf

U-

522

Acetate buffer, pH 4.7 B

i C

Citrate buffer, pH 4.7 fl

>

2 |3

7

,5

9

Carbonate buffer, pH 10.3 4

6

18

I D

Phosphate buffer, pH 7.2

Water, pH 7.2

10

30

15 20 25 Retention Tlme(mm)

35

Figure 2. Gas chromatogram of derivatives from aldehydes: Peaks: 1 = 2-methylthiazoline from acetaldehyde; 2 = thiazolidine from formaldehyde; 3 = 2-methylthiazolidine from acetaldehyde; 4 = 2-ethylthiazoline from propionaldehyde; 5 = 2-ethylthiazolidine from propionaldehyde; 6 = 2-propylthiazodine from butyraldehyde; 7 = 2-propylthiazolidine from butyraldehyde; 8 = 2-butylthiazoline from valeraldehyde; and 8 = 2-butylthiazolidine from valeraldehyde.

Table 1. Effect of phosphate buflFer concentration on thiazolidine formation

Phosphate buffer (M)

0.025

0.05

Concentration (mM) 0.1 0.2

0.20 (3.0y 0.66(9.9) Thiazolidine 0.42(9.2) 1.01(22.2) 2-Methylthiazolidine 0.71(20.6) 1.16(33.6) 2-Ethylthiazolidine 0.88(31.7) 1.21(43.5) 2-Propylthiazolidine 0.94(40.3) 1.27(54.5) 2-Butylthiazolidine "* value in parenthesis are molar recovery (%)

1.68(25.2) 1.79(39.3) 1.69(49.0) 1.64(59.0) 1.67(71.7)

2.78(41.7) 2.64(58.0) 2.53(73.3) 2.43 (87.4) 2.67(114.6)

nil 0.09(1.3) 0,25(5.5) 0.65(18.8) 0.76 (27.2) 0.87(37.3)

523 3. OXIDO-REDOX SYSTEM

REACTION

IN A CYSTEAMINE/CABONYLS

MODEL

Considerable amounts of 2-methylthiazoIine, 2-ethylthiazoline, 2-propylthiazoline and 2butylthiazoline were characterized in the heated cysteamine/aldehydes model system with a carbonate buffer (pH 10.3, 0.2 M) as shown in Figure 2C. These compounds were eiuted before the corresponding thiazolidine. No detectable amounts of thiazolines were observed in the experimental condition without buffer sah. The effect of azodicarbonamide, a well known hydrogen acceptor, was added to the reacted model system to study the influence of the oxido-redox reaction on the formation of 5acetyl-2,3-dihydro-l,4-thiazine. The formation of 5-acetyl-2,3-dihydro-l,4-thiazine was found to increase lineariy with increasing concentrations of azodicarbonamide in the range of 1 to 3 mM. The amount of 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine decreased with increasing concentration of azodicarbonamide. The formation of 2-acetyl-2-methylthiazolidine was found to be independent of azodicarbonamide as shown in Figure 3. The redox reaction seems to lead to the conversion of 5-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to 5-acetyl-2,3-dihydro-l,4thiazine via a proton transfer reaction similar to that in the conversion of tetramethyldihydropyrazine to tetramethylpyrazine [11].

Azodicarbonamide 3m M

0)

c

I

L ..

1

1

rV 1"

^

L ..

Azodicarbonamide 2mM

c-c o 1

11 ^r. r Azodicarbonamide 1mM

1

^

,

10

J-

,..!::—• .11111

n-yr-i A.I

15

20

/s

f,r^

25

n

r

nj

30

Retention Time(min)

Figure 3. Effect of azodicarbonamide on the formation of 2-acetyl-2-methylthiazondine, 2acetyl-2,3,5,6-tetrahydro-l,4-thiazine and 5-acetyI-2,3-dihydro-l,4-thiazine in cysteamine/2,3butanedione model system.

524 The redox reaction was proposed by Huyghues-Despointes and Yaylayan [12] in a Maillard model system composed of D-glucose and proline. They reported that an adiketone/enediol redox couple participated in the generation of oxidation-reduction products in the Maillard system. Oxidation of thiazolidines in the presence of atmospheric oxygen may lead to the formation of the corresponding thiazoline [13]. Similarly it was also observed in a cysteamine/methylglyoxal model system [8]. They attributed methylglyoxal as a proton acceptor in the formation of 2-acetyl-2-thiazoline from 2-acetylthiazolidine. Two volatile compounds, 2-acetyl-2-thiazoline and 2-acetylthiazole were characterized in a heated aqueous L-cysteine solution [13]. They postulated that these two acetyl derivatives were the dehydrogenation products of 2-acetylthiazolidine. A significant amount of 2-acetylthiazolidine has also been observed in the reaction mixture of aldehydes and cysteamine by Hayashi et al. [6]. A redox reaction catalyzed either by phosphate or azodicarbonamide may facilitate the proton transfer from 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to form a 5-acetyl-2,3-dihydro1,4-thiazine and from thiazolidines to the corresponding thiazolines. The phosphate or azodicarbonamide may act as both hydrogen acceptor and donor, which catalyzes the Schifif base formation during the generation of a thiazolidine and thiazines. 4. THE COMBBVED EFFECTS OF A BUFFER AND SOLVENT ON THIAZOLIDINE FORMATION IN A CYSTEAMBVE/CARBONYLS MODEL SYSTEM An interesting effect of combining the phosphate buffer and protic solvent on thiazolidine formation was found. As shown in Figure 4, a 1.13-fold increase of the unsubstituted thiazolidine was observed when 60 % ethanol in a phosphate buffer was utilized as the reaction medium as compared with that without ethanol. More ethanol did not enhance thiazolidine formation. A similar effect on the formation of 2-methylthiazolidine, 2-ethylthiazolidine, 2propylthiazolidine and 2-butylthiazolidine was observed as well when the solvent and buffer were combined as shown in Figure 4. These findings are in complete accordance with those published by Lin et al. [14]. An aqueous solution of 50% methanol containing 100 mM phosphate at pH 7.0 was found to be very effective for converting an orange pigment into a red amino acid pigment. A Schiff base was formed between the a-amino nitrogen of the amino acid and the carbonyl carbon of the orange Monascus pigment. A high yield of 60-70% of l-deoxy-l-p-toluino-D-fiuctose was obtained by Rosen et al. [15] although no detailed elucidation of the mechanism was provided in their paper. The phosphate buffer system served as either a proton donor or acceptor, which catalyzed the Schiff base formation [16]. Extra phosphate may also have catalyzed the dehydrogenation reaction. The protic solvent attracted the water molecule, which led to completion of the Schiff base formation.

525

2-butyithia2clidlne 2-propyithia2oiidine 2-ethyithiazoiidine methyithiazolidine thiazolidine

Ethanoi concantraticn (%)

Figure 4. Effect of ethanoi on the formation of thiazolidines in aldehydes/cysteamine model system.

5.

PROPOSED MECHANISM FOR THIAZOLIDINE FORMATION

The proposed mechanism for the formation of thiazolidine in a cysteamine/carbonyl model system is shown in Figure 5. In a reaction medium with a buffer, the nucleophilic amino group on a cysteamine molecule tends to attack the positively induced carbonyl carbon. A general acid catalyzed the elimination of a water molecule which gives a secondary carbocation ion for 2,3-butanedione and aliphatic aldehydes with a chain length longer than C2. Conversely, formaldehyde gives an extremely unstable primary carbocation. Phosphate tends to stabilize this primary carbocation. Another nucleophilic attack of the thiolate on carbocationic carbon leads to the formation of a thiazolidine.

6.

REFERENCES P.M.T. de Kok and E.A.E. Rosing, In Thermally Generated Flavors, Maillard, Microwave and Extrusion Process, American Chemical Society, Washington, DC, Series, (1994) 158-179. C.H. Yeo and T. Shibamoto, J. Agric. Food Chem., 39 (1991) 370-373.

526

NHj

R2-C-R1 II 0-H2P04

C

H Ri

r OH H2P04' f H Ri ^N-<jj-R2 r *0H2

^

H2O

H Ri ^N-
aldehydes R-fsH, R2=0H, Ci-C2 2,3-butanedione Ri=CH3, R2= C—CH3 !l O

H

c Figure 5. Proposed formation mechanism for thiazolidines from cysteamine and carbonyl compounds.

527 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

R.A. Scanlan, S.C. Kayer, L.M. Libbey and M.E. Morgan, J. Agric. Food Chem, 21 (1973) 673-675. EJ. Mulders, Z. Lebensm. Unters. Forsch. 152 (1973) 193-201. K. Umano, Y. Hagi, K. Nakahara, A. Shyoji and T. Shibamoto, J. Agric. Food Chem. 43 (1995)2212-2218. T. Hayashi, C.A. Reece and T. Shibamoto, J. Assoc. Off. Anal. Chem. 69 (1986) 101105. K. Miyashifa, K Kanda and T. Takagi, J. Assoc. Off. Anal. Chem. 68 (1991) 748-751. T. Hofmann and P. Schieberle, J. Agric. Food Chem. 43 (1995) 2187-2194. T. Hofmann, R. Hassner and P. Schieberle, J. Agric. Food Chem. 43 (1995) 2195-2198. A. Yasuhara and T. Shibamoto, J. Chromatogr. 547 (1991) 291-298. T.C. Huang, H.Y. Fu and C.-T. Ho, J. Agric. Food Chem. 44 (1996) 240-246. A. Huyghues-Despointes and V.A. Yaylayan, J. Agric. Food Chem. 44 (1996) 672-681. S.A. Sheldon and T. Shibamoto, Agric Biol. Chem. 51 (1987) 2473-2477. T.F. Lin, K. Yakushijin, G.H. Buchi and AL. Demain, J. Indust. Micro. (1992) 173-179. L. Rosen, J.W. Woods and W. Pigman, J. Amer. Chem. Soc. 80 (1958) 4697-4702. T.C. Huang, Biosci. Biotech. Biochem. 61 (1997) 1013-1015.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

529

Effect of antioxidants on the formation of volatilesfromthe Maillard Reaction. A. Amoldi, M. Negroni, and A. D'Agostina, Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, 20133 Milano, Italy Abstract The aim of this report was to study the involvement of free radicals on the formation of volatiles from the Maillard reaction. An aqueous model system of glucose/lysine was heated in the presence of different amounts of antioxidants, such as atocopherol, 2,6-di-^er^butyl-4-methylphenol (BHT), and rosemary extract, or of a,a'azobis-isobutyronitrile (ADBN) which is a pro-oxidant (free radical initiator). The differences in volatile formation between the standard model system and those with additives are relatively small. BHT, at pH 4, decreases pyrazine, while it increases slightly 2-methylpyrazine and 2,5-dimethylpyrazine and much more 2-acetylpyrrole; at pH 6 it produces a decrease in furaneol. a-Tocopherol, at pH 4, decreases pyrazine,fiiraneol,2,3dihydroxy-6-methyl-4H-pyran-4-one, and 2-acetylpyrrole, while at pH 6 it decreases 2furanmethanol, and furaneol. Rosemary extract, at pH 4, decreases all the pyrazines and 2fliranmethanol and 2-acetylpyrrole; at pH 6 it produces a decrease of furaneol and 2,3dihydroxy-6-methyl-4H-pyran-4-one. AIBN at both pH's decreases the pyrazines and increases 2-acetylpyrrole.

1. UNTRODUCTION The reaction between amino acids and sugars (the Maillard reaction) produces many volatile heterocyclic compounds whose structures, odor thresholds, and concentrations affect the aroma of a food [1]. As part of an investigation on the effects of lipids in the Maillard reaction we studied the formation of volatile compounds from an aqueous glucose/lysine model system heated in the presence of either an antioxidant or a pro-oxidant (free radical initiator). a-Tocopherol, 2,6-di-?^r^butyl-4-methylphenol (BHT), and rosemary extract [2] were used as antioxidants and a,a'-azobis-isobutyronitrile (AIBN) as a free radical initiator. A previous paper [3] from our laboratory has presented some data on this same subject. However, recently the experimental methodology was changed because the Maillard reaction is very sensitive to pH. Previous model systems were heated at 120 and 100 °C in closed tubes after having set the pH to the desired value. Under these conditions, the pH slowly decreases to reach values below pH 4. To prevent the drop in pH, model systems were heated at 100 °C in a flask equipped with an autoclavable electrode; pH was monitored during all heating steps and maintained constant by the addition of dilute base using a procedure proposed by Ames and coworker [4].

530

2. EXPERIMENTAL Mixtures containing equimolar amounts of xylose and lysine (70 mL of 0.5 M water solution) and 60 mg of additive were heated under reflux for 2 h in a flask equipped Avith an autoclavable electrode (ATI Russel). During this time the pH was monitored and kept constant at the value of 4.0 and 6.0 by the addition of diluted sodium hydroxide (NaOH). At the end of the heating time, the pH was adjusted to 8.0. Tetradecane was added as a first internal standard, and the volatile compounds were recovered by continuous extraction with dichloromethane (CH2CI2). After careful concentration of the solvent to 1 mL, pentadecane was added as a second internal standard and the samples were quantified by gas chromatography-flame ionization detection (GC-FID) on a DANI 86.10 gaschromatographer. Two internal standards, tetradecane and pentadecane, were used. Compound concentration was calculated after determination of the correction factors and were expressed in "mg / model system". Peaks were tentatively identified by GC-mass spectrometry (GC-MS) on a Shimadzu QP-5000 by comparison with the NIST62 spectra library and commercial standards as far as possible. Ions were generated by EI at 70 eV. A capillary column SPB-1701 (30 m x 0.2 mm, film 1 ^m) was used. Temperature program: 37 °C x 10 min, 4 °C to 200 °C, then isothermal. Each experiment was repeated at least three times. Drawings from the same model systems (one every 15 min) were analyzed with an UV-visible spectrometer at 420 nm in order to detect the color development.

3. RESULTS AND DISCUSSION The overall effect of the anti or pro-oxidants on the Maillard reaction can be drawn from color development seen at 420 nm. Figure 1 shows the increase in the absorbance at 420 nm in model systems at pH 4 and 6. BHT at the concentration used in these experiments appears to favor browning at both pH values while a-tocopherol and rosemary extract seem to have no effect at pH 4 and are weak inhibitors at pH 6. AIBN inhibits browning at pH 4 and has no effect at pH 6 by 120 min. The discussion on the volatile compounds will be limited to selected compounds. Figures 2 and 3 compare the amount of these volatiles extracted from the systems at pH 6 and 4, respectively. The differences in volatile formation between the standard model systems and those with additives are relatively small especially when considering the experimental error that is rather large. While these data will be treated with suitable statistical methods in the fixture, here the discussion will be limited to volatiles that are affected at a greater degree. BHT, at pH 4, decreases pyrazine, while it increases slightly 2-methylpyrazine (2MP) and 2,5-dimethylpyrazine (2,5-MP) and much more 2-acetylpyrrole (2-AP). At pH 6, BHT produces a decrease in furaneol. a-Tocopherol decreases pyrazine, furaneol, 2,3dihydroxy-6-methyl-4H-pyran-4-one (DHP), and 2-acetylpyrrole (2-AP) at pH 4, while at pH 6 it decreases 2-furanmethanol (FM), and fiiraneol. Rosemary extract, at pH 4, decreases all the pyrazines and 2-furanmethanol (FM) and 2-acetylpyrrole (2-AP); at pH 6 it produces a decrease of furaneol and 2,3-dihydroxy-6-methyl-4H-pyran-4-one (DHP). AIBN at both pH's decreases the pyrazines and increases 2-acetylpyrrole (2-AP).

531

- reference -pH6+BHT -pHd+tocoph. -pH6+rosm. -PH6+AIBN

45

60

75

120

Time (min)

0,6 ~

pH4

0,50,4X

J7^

S0,3< 0,2 1 0.1 0<

(\

15

30

45

60

75

90

105

Time (min)

Fig.1: Colour formation (absorbance at 420 nm) as a function of time

120

—•— reference --^pH4+BHT -o—pH4+tocoph. —A—pH4+rosm. --0-PH6+AIBN

532

4.5 n

pyrazine • reference B 6 H T Dtocoph. Drosm. BAIBN

0,C35n 0,04 0,03 0,020,01

m!^ m

1 ^^

2,3-DMP | S reference DBHT Dtocoph. E3rosm. BAIBN |

®1 5 4 ?3-

T

WK^ 2 1 0^

W/.

Y-

^ ^ —

furaned 01 reference BBHT Dtocoph. Qrosm. QAIBN

|Dreference DBHT Dtocoph. Drosm. HAIBN [

DHP nreference eaBHT Dtocoph. Drosm. BAIBN

Fig. 2: Comparison of volatlles In model system at pH 6 with the indication of the confidence Interval (P=0.1) 2-MP=2-methylpyrazlne, 2,5-DMP=2,5-dimethylpyrazine, 2,3-DMP=2,3-dlmethylpyrazlne, DHP=2,3-dlhydroxy-6-methyl-4H-pyran-4-one, FM=2-furanmethanol, 2-AP=2-acetylpyrrole

533

0,09 0,08 0,07

0,9 n 0,8 0,7 0,6

T-

0.0,5

'0.4 0,3 0,2 0,1 0-1

r

.I '••••••• — • - - - "

•

•-------•

1

Dvrazine

= reference

HBHT

Dtocoph.

Drosm. fiSAIBN |

a reference

0.035 ^

HBHT

2-MP Dtocoph

Grosm.

HAIBN \

n

003 0,025 a>0,02^ ^0,015 0,01 0,005

B reference

m 1^M

S ^^^ E9BHT

2,5-DMP Itocoph Drosm.

BAIBN 1

0,03

furaneol nreference

SBHT

Dtocoph

Drosm.

DAIBN

1,4 n 1,2 1 o. 0,8 e 0.6

^

0,4 0,2-

[|[[||[

1 Preference

DBHT

DHP Dtocoph.

2-AP Drosm.

SAIBN [

Ireferer^ce

DBHT

Dtocoph

Drosm.

BAIBN

: impossible to quantify Fig. 3: Comparison of volatiles in model system at pH 4 with the Indication of the confidence interval (P=0.1). 2-MP=2-methylpyrazine;2,5-DMP=2,5-dimethylpyrazine;2-FM=2-furanmethanol; 2-AP=2-acetylpyrrole; DHP=2,3-dihydroxy-6-methyl-4H-pyran-4-one;

534 There are some relevant diflferences between the results of this work and our previous research [3]. The methodology was changed in an important way in that now the pH is kept constant during all thermal treatments. This produces a depletion of the compounds formed because while the pH varies, the mechanism of the Maillard reaction changes and different compounds are more or less favored [5]; for example, the formation of 2-furancarboxyaldehyde takes place only at low pH. Therefore, the observed discrepancies can be explained by the fact that the dependence on pH is so relevant that small differences in its value during heating can conceal or be confused for the additive effect. This underlines how carefiilly experiments must be planned in this field to obtain consistent results. The free radical initiator AIBN is thermally decomposed to give gaseous nitrogen (N^) and two alkyl radicals that can initiate a free radical chain. The presence of radicals in a reaction mixture has an inhibitory effect on the Maillard reaction (MR) as demonstrated by the slower formation of color at 420 nm and by reduction of pyrazines. The other compounds are less sensitive. BHT at the concentration used in the model system (0.27 mmol/model system) seems to be favorable for browning while this does not happen with the other two antioxidants. The effects on aroma are less clear and depend on the pH. In conclusion free radicals may be involved in some way in the Maillard reaction, but the effects are moderate in the model systems and difficult to discuss. Antioxidants may play a role, but more experimental work is necessary for a practical exploitation of these information's.

These researches were financed by the European Union: project FAIR, contract

€796-1080.

4. REFERENCES 1. S. Fors, in "The Maillard Reaction in Foods and Nutrition", G.R. Waller and M.S. Feather (eds), ACS Symposium Series 215, American Chemical Society, Washington DC, 1983, p. 185. 2. S.S. Chang, B. Ostric-Matijasevic, O.A. Hsieh, C.-L. Huang, J. Food. Sci. 42 (1977), 1102 3. A. Amoldi, E. Corain, in "Flavour Science. Recent Developments". A.J. Taylor, D.S. Mottram (eds). The Royal Society of Chemistry, Cambridge, 1996,217. 4. J. M. Ames, A. Apriyantono, in "Thermally Generated Flavors. Maillard, Microwave and Extrusion Processes". T.H. Parliment, M.J. Morello, R.J. McGorrin (eds), ACS Symposium Series 543, American Chemical Society, Washington, DC, 1994,228. 5. F. Ledl, E. Schleicher, Angew. Chem. Int. Ed. 29 (1990), 565

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

535

T h e u s e of r o a s t i n g k i n e t i c s data t o c h a r a c t e r i z e n a t u r a l a n d artificial c h o c o l a t e aroma precursors G. P. Rizzi a n d P . R. Bunke Procter & Gamble Company, Miami Valley Laboratories Cincinnati, Ohio 45239-8707 (U.S.A.)

Abstract Chocolate aroma precursors were investigated in conjunction with a study aimed at delivering enhanced chocolate flavor in thin layer roasting of homogenized raw cocoa beans. Chocolate aroma precursors were isolated from fermented cacao beans and characterized by kinetic data obtained in controlled roasting studies. A rate-limiting first order loss of amino nitrogen was observed which is consistent with a bimolecular amine/carbonyl reaction mechanism. Rate constants and Arrhenius parameters served as a guide for design and development of simplified, artificial precursor systems. Fractionation of natural precursors with ion exchange extraction and activated carbon adsorption established the significance of hydrophobic amino acids Uke leu, ileu, val, tyr, trp and phe in chocolate aroma formation. A synthetic precursor mixture containing hydrophobic amino acids and reducing sugars generated authentic chocolate aroma and provided the basis for the development of a practical chocolate flavor enhancer. 1.

INTRODUCTION

A study of chocolate aroma precursors was undertaken as a part of related process work aimed at enhancing aroma production during continuous raw bean Hquor roasting. The objective of our study was to define a simplified, synthetic precursor mixture which could serve as an adjunct source of chocolate flavor in contemporary roasting processes. Traditionally, chocolate aroma is produced by roasting cocoa beans which have previously undergone fermentative and drying treatments. During fermentation, flavor precursors are formed which react at roast temperatures to form endproduct flavor compounds. In conventional processing roasted cocoa beans are macerated to form chocolate Hquor or "mass" which is further processed to produce cocoa butter, cocoa powder or confectionary chocolate [1]. In traditional batch processing, large variations in bean sizes and low thermal conductivity can result in uneven roasting of individual beans and less than optimum flavor production.

536

In recent years traditional batch processing of beans has partially been replaced by theoretically more uniform raw liquor roasting [2]. In continuous, raw liquor processing, raw (pre-fermented) beans are finely ground and uniformly roasted in thin-layer roasters. In thin layers, fast heat transfer is possible and optimum aroma is formed in a few seconds. Thin-layer roasters also provide the opportunity to add supplemental precursors to the raw liquor before roasting. For example, in the so-called LSCP process [3], raw Uquors are modified by reducing sugar addition and by enzyme pretreatment. Extensive research has led to the conclusion that chocolate aroma is a result of complex chemical reactions of amino acids, peptides, sugars and possibly flavonoid compounds during roasting [4, 5]. It was suggested [6,7] and later proven by l^C-tracer studies [8] that Strecker degradations of amino acids (probably fueled by Maillard reaction derived dicarbonyls) are the source of volatile aldehydes, a key ingredient of chocolate aroma. Also, model studies suggest that other important chocolate aroma components Hke aliphatic acids, a, P-unsaturated aldehydes, sulfur compounds and alkylpyrazines all originate as secondary products of the Strecker degradation [9]. Recently, it has also been shown in model studies that Amadori compound decomposition contributes directly to the formation of chocolate aroma and that the composition of aroma compounds formed in these reactions is a function of water activity [10]. Knowledge of roasting chemistry has already led to formulations of synthetic precursor mixtures that generate chocolate aroma under simulated roasting conditions [11]. Quantitative changes of amino acids during roasting correlate well with the formation of chocolate aroma volatiles. IQnetic studies on cacao roasting done with whole beans showed an apparently linear (zero order) loss of free amino acids with time and concomitant production of aldehydes and pyrazines [12, 13]. Chocolate aroma is also formed without the bean matrix when isolated natural precursors are heated under roasting conditions [7]. Changes in total amino acid level during bean extract roasting were shown to depend on time, temperature and initial moisture content; and subsequently, time/temperature data obtained in the model studies were used to obtain optimized chocolate flavor in factory scale, whole bean roasting. Based on related studies on synthetic amino acid/sugar mixtures [7], it was also proposed that chocolate aroma can be afi'ected by the way individual amino acids decompose at different rates at a given temperature. The goal of our experimentation was to gather roasting ]dnetic data in the early stages of bean extract roasting and to use this data to guide the development of a simplified, synthetic chocolate aroma precursor system. 2. MATERIALS AND METHODS Commercially available fermented/dried cocoa beans were used. Activated carbon was Merck U.S.P. activated charcoal.All analytical reagents, standard reference compounds and solvents were commercial materials of analytical grade

537

purity and used as received. 2.1. Cocoa bean extraction Beans, including shells were dry-pulverized to < 1 mm diameter particle size in a Waring blender and extracted with 80-20 v/v methanol-water for 18 hrs. at 22^C. Resulting slurries were filtered, methanol was evaporated under vacuum and the aqueous residues were fireeze-dried to obtain 7-12 % yields of dry,powdered extracts. Low fermented beans like Arriba and Sanchez gave deep purple extracts characteristic of intact anthocyanin pigments; however, extracts of well fermented beans Hke Ghana and Bahia were brown, apparently as a result of the action of pol5rphenoloxidase and oxygen. 2.2. Bean extract roasting Prior to roasting, freeze-dried extracts were stored in glass chambers provided with fixed relative humidity to obtain samples with definite, equilibrium water content (Table 2). For organoleptic evaluation, 0.1 gm. samples were oven heated at various temperatures and times in closed, screwcapped (2 oz.) glass bottles. After reaching room temperature, the roast aromas were evaluated by sniffing, and results are also summarized in Table 2. For kinetic measurements, 10 mL. glass ampules containing ca. 0.05 gm. of bean extracts or synthetic precursor mixtures were equilibrated to a ^ 0.13, flame-sealed and heated (totally submerged) in a thermostated oil bath. To obtain rate data, several identical samples were thermostated at t = 0 time. Then, samples were removed sequentially at time intervals and cooled rapidly to room temperature before analysis. On analysis, the ampules were opened and contents were dissolved/dispersed in water by sonication. Soluble products were isolated by filtration and aUquots of filtrates were analyzed for total amino nitrogen content. Optionally, solutes of aqueous solutions were prepared for roasting by freezedrying the samples directly in bottles or ampules prior to the equilibration step. 2.3. Fractionation of Ghana bean extract (GBE) GBE was washed with ethyl acetate to remove residual lipids, stirred in methanol and centrifuged to separate relatively high molecular weight (insoluble) materials. The methanoHc supernatent was evaporated and the residue was dissolved in 5:95 v/v acetic acid-water before passing it through a glass column containing a cation exchange resin (sulfonated polystyrene, H"*"form). Vacuum concentration of the eluant yielded "neutral plus acidic material", GBE/NA. Further elution of the column with 10:90 v/v pyridine-water followed by concentration and freeze-drying provided a "basic" fraction, GBE/B. 2.4. Activated carbon adsorption treatment of GBE basic fraction A 0.5% aqueous solution of GBE/B was treated with 1% activated carbon and stirred for 6 hrs. at 22^0 (ambient pH 3.98). Carbon was removed by filtration and the filtrate was evaporated to yield a white solid, GBE/B 1 (38.7% recovery based on GBE/B). The recovered carbon was sequentially extracted by stirring it

538 at 220C, first with (a) 20:80 v/v pyridine-water (pH 7.64) and then with (b) 100 % acetic acid. Each extract was isolated by filtration and vacuum concentrated to yield residues GBE/B2 and GBE/B3 representing 49.6% and 8.0% recovery (based on GBE/B) ficom (a) and (b) repectively. The proximate analysis of subficactions B, B l and B2 are presented in Table 3. 2.5. Analytical methods Individual amino acids were separated and quantified by high performance liquid chromatography (HPLC) analysis after reaction with o-phthalaldehyde to form fluorescent derivatives. Several unidentified amino compounds were observed which may have been Amadori rearrangement products [10]. A fluorescence emission detector was used to quantify the results. Total amino nitrogen was determined colorimetrically with a ninhydrin reagent [14] using a glycine standard on aliquots adjusted to pH 5.0 with a citrate buffer. Sugars and glycerol were analyzed directly by HPLC on an IBM amino column using 80:20 acetonitrile-water as eluant and a refractive index detector. HPLC was also used to quantify methyl xanthines (theobromine and caffeine) and epicatechin. Karl Fischer water and Kjeldahl nitrogen were assayed by automated procedures. Total phenols were determined by a standard procedure using Folin-Ciocalteu reagent [15] and results are expressed in terms of gallic acid. 3.

RESULTS AND DISCUSSION

3.1. Isolation of chocolate aroma precursors Four varieties of dried cocoa beans representing a wide range of prefermentation were extracted with aqueous methanol and freeze-dried to obtain apparently dry powders containing aroma precursors (Table 1). Extracts of the more fermented (Bahia and Ghana) beans contained more free amino acids, total amino N and less sucrose, than the lower fermented Arriba and Sanchez varieties. Both Bahia and Ghana contained less epicatechin, an indicator of fermentation-degree, probably due to degradation by polyphenoloxidase. All four extracts contained about the same levels of total reducing sugars, however the mole ratios of amino N/reducing sugars were clearly higher for Bahia and Ghana. Fructose was the dominant reducing sugar in all four bean extracts. Preliminary roasting experiments showed that the strongest, most authentic chocolate aromas were generated by heating Bahia or Ghana extracts to 120-130OC for 3060 min. at an initial moisture content adjusted to 4.4-4.7 % ( a ^ 0.13), Table 2. Based on organoleptic performance, we selected Ghana extract as our model for synthetic precursor design. 3.2. Roasting characteristics of cacao bean extracts Kinetic measurements of extract roasting were based on loss of amino N versus time since aroma formation is known to be tightly coupled to amino acid/peptide reactions in whole bean roasting. Amino N content initially dropped rapidly during roasting (0-60 min), but its rate of decrease slowed considerably at longer roast times. We found, in agreement with published data, that in

539 Table 1 Partial composition of cocoa bean extracts Component

Arriba

Bahia

Ghana

Sanchez

aspartic acid* glutamic acid serine histidine glutamine glycine arginine alanine tyrosine tryptophane methionine valine phenylalanine isoleucine leucine lysine

0.06% 0.33 0.07 tr 0 0.02 0.05 0.29 0.17 tr tr 0.22 0.29 0.14 0.41 tr

0.08% 0.40 0.14 tr 0 0.02 0.14 0.46 0.46 0.08 0.03 0.44 0.86 0.29 0.98 0.13

0.12% 0.66 0.16 tr 0 0.06 0.12 0.63 0.54 0.08 tr 0.61 0.95 0.37 1.09 0.10

0.05% 0.31 0.10 tr 0 0.04 0.07 0.31 0.27 tr tr 0.28 0.39 0.21 0.51 tr

glycerin fructose glucose sucrose

2.0 4.9 3.3 7.1

0.8 4.0 1.9 0.4

1.3 6.2 2.1 3.6

2.4 4.3 5.1 7.8

water Kjeldahl N amino N epicatechin

3.73 3.99 0.34 2.1

3.26 5.45 0.59 3.0

4.07 5.49 0.72 1.4

3.02 3.31 0.38 4.3

mol amino N/mol reducing sugars

0.53

1.28

1.12

0.52

* Amino acids listed in order of elution during HPLC analysis.

general, reducing sugars disappeared almost completely during roasting leaving some amino N unreacted at long roast times. During the first sixty minutes of roasting, amino N loss was well described by the integrated rate equation (1). ln[(Ct-C60)/Co-C60)] = - k i t

(r2 > 0.9)

(1)

540 Table 2 Effect of water activity (a^) on aromas generated during bean extract roasting

Extract

Water Activity, a ^

K. F. Water (%)

Aroma

Bahia

0.0 0.13 0.53 0.75

1.52 4.43 10.24 19.13

strong chocolate strong chocolate weak chocolate, winey weak chocolate, musty

Ghana

0.0

1.16

0.13 0.53 0.75

4.69 11.90 23.34

chocolate, hot chocolate milk chocolate, malty very weak chocolate very weak. mushroomy

Arriba

0.0 0.13 0.53 0.75

1.04 4.19 10.18 22.13

Sanchez

0.0 0.13

1.54 4.29

0.53 0.75

11.54 20.41

sweet chocolate chocolate hquor very weak chocolate very weak, si. burnt strong chocolate sharp, chocolate Uquor musty, fecal very weak, musty

Roasting conditions: 120^0 for 60 min.

where CQ, CQQ and C^ represent percent amino N in the roasted sample at times 0, 60 and t min. respectively, t is the roasting time in min. and k^ is a first order rate constant in m i n ' l . Rate constants were calculated from roasting kinetic data as the negative slope of the Hnear regression Unes obtained from plots of In [concentration ratio] versus roasting time. Roasting half-time (ti/2) in min. is equal to In 2/ki. The rate datajoer se tells us Uttle about reaction mechanism except to say that the rate of amino N loss with time is proportional to remaining amino N in the 0-60 min. roasting range. However, the kinetic order of amino N loss observed is consistent with the accepted mechanisms for the earliest stages of the Maillard reaction or the Strecker degradation. Kinetic roasting data for bean extracts spanning a 90-135^0 temperature range are summarized in Table 3. Predictably, the rates of amino N loss

541 Table 3 Roasting kinetic data for cocoa bean extracts at 90, 120 and 135^C with initial a ^ 0.13

ki (min-1), [ti/2 (min) ] Extract Bahia Ghana Arriba Sanchez

90OC 0.0070, 0.0076, 0.0077, 0.0062,

[99] [91] [90] [112]

120OC 0.023, 0.035, 0.045, 0.032,

[30] [20] [15] [22]

1350C 0.037, 0.039, 0.043, 0.040,

[19] [18] [16] [17]

Ea (KCalM-1) 11.1 11.5 12.4 12.8

increased markedly with roasting temperature, however more rate increase was observed between 90 and 120^0 than between 120 and 135^C. A less obvious variation was observed by comparing rate constants at the same temperature for a variety of extracts. At 90 and 135^0, k^ did not vary much across bean varieties. However, at 120^0, we observed as much as two-fold rate differences among the four bean extracts. To us this was an indication that an aroma precursor system based on amino acid/pep tide chemistry was highly operative in fermented extracts at 120^C. Since optimum chocolate aroma development also took place at 120^0, we also concluded that a k i of ca. 0.025 m i n ' l was a suitable design value for synthetic precursor development in this temperature range. The sensitivity of roasting chemistry to temperature change is also quantified by the activation energy parameter Ea defined in the Arrehnius equation (2). Inki = - Ea/RT + Inkg

(2)

In this equation, k l is a rate constant and Ea is an activation energy whose unit is defined by the units of R and T. A plot of Inkj versus 1/T, where T = roast temperature in ^C + 273 produced straight Unes in the 90-135^C roasting range (r2 > 0.9) whose slopes times -R (1.987 Cal-deg'l-mole"l) gave Ea in Cal-mole"l. For bean extract roasting (Table 3) extracts of highly fermented beans (Bahia and Ghana) showed less temperature effect on roasting rate (Ea, ca. 11 KCalmole'l) than lesser fermented bean extracts (Ea, ca. 12-13 KCal-mole"l). 3.3. Synthetic chocolate aroma precursor systems Ghana bean extract (GBE) was fractionated to identify components that are most responsible for producing chocolate aroma during roasting. Fractions were tested for aroma generating potential by roasting them both with and without added sugars (Table 5). Starting with a Uterature-suggested procedure [4] we isolated an aromagenic "basic fraction" (GBE/B) using cation-exchange

542

Table 4 Partial analysis of Ghana bean basic fraction (GBE/B) and results of partitioning by activated carbon.

Not adsorbed by carbon

Adsorbed by carbon

Component

Initial GBE/B

GBE/B 1

GBE/B2

unk-1* unk-2 glycine glutamine asparagine aspartic acid tryptophane unk-3 serine threonine isoleucine glutamic acid tyrosine unk-4 valine alanine phenylalanine leucine

0.0 relative % 0.0 0.5 0.5 0.5 1.4 1.4 2.4 2.4 2.4 6.3 7.7 7.7 9.6 9.6 11.5 14.9 21.2

0.0 relative % 0.0 0.9 0.4 0.4 1.5 0.0 2.8 3.1 3.7 8.3 8.7 0.7 10.8 12.2 14.4 2.6 29.6

0.8 relative % 0.8 0.0 0.0 0.0 0.8 3.1 0.8 0.8 0.8 3.1 2.3 23.7 3.8 2.3 2.3 45.0 9.2

theobromine caffeine total phenols** total amino N ash

16.0 absolute % 2.9 15.3 3.3 0.68

0.6 absolute % 0.2 1.6 6.4 1.21

29.0 absolute % 5.4 10.2 2.2 0.41

* Amino acids and related unknowns Usted in increasing amounts found in GBE/B. ** As gallic acid. chromatography. Fraction GBE/B contained free amino acids, methylxanthines and phenolic materials of unknown structure (Table 4). Because GBE/B contained viable precursors, it was fractionated further by adsorption onto activated carbon. Material not adsorbed to carbon, GBE/B 1 failed to produce chocolate aroma; however, GBE/B2 isolated from carbon by aqueous-pyridine extraction did produce chocolate aroma in conjunction with sugars.

543

Table 5 Identification of chocolate aroma precursors in Ghana bean fi-actions

ADDED REDUCING SUGAR YES NO 1 GHANA BEAN EXTRACT (GBE)

X

CHOC. AROMA FORMED YES NO X

1

cation exchange

1 1 1

BASIC FRACTION (GBE/B)

X X

X X

1 activated carbon adsorption

1

1

NON-ADSORBED MATERIAL

1

(GBE/Bl)

X

X

X

X

1 1 ADSORBED MATERIAL 1 (GBE/B2)

X X

X X

Freeze-dried mixtures roasted at 120^C for 30 min at initial a ^ 0.13. Sugars = mixture of fructose, glucose and sucrose in same relative proportions found in Ghana bean extract; mole ratio of amino N/reducing sugars 1:1.

Interestingly, GBE/B2 contained a predominance of hydrophobic amino acids, in particular the benzenoid derivatives tyrosine, phenylalanine and tryptophane. In addition to amino acids, the carbon adsorbant also retained most of the methylxanthines and some phenoUc materials. A sixteen component synthetic precursor mixture was formulated based on the ratio of amino acids and methylxanthines found in GBE/B2 and a mixture of sugars in the same relative proportions observed in a Ghana bean extract (Table 6). On roasting at 120^0 at initial a ^ 0.13, the mixture developed a strong aldehydic, chocolate-like aroma in 30 min. Roasting kinetics data closely resembled those of natural extract roasting; however, k j for the synthetic was somewhat more sensitive to temperature variation (Ea, 14.9 KCal-mole-1) than the naturals. Differences in aroma quahty were seen between synthetic and

544

Table 1 aoie 6b Sixteen-component synthetic precursor mix and related roasting kinetics Component phenylalanine tyrosine leucine tryptophane isoleucine alanine glutamic acid aspartic acid serine threonine lysine AMINO ACIDS (total) fructose glucose sucrose SUGARS (total) theobromine caffeine XANTHINES (total)

Relative % of classes 45.0 23.7 9.2 3.1 3.1 2.3 2.3 0.76 0.76 0.76 0.76 100

Absolute % in mixture 13.4 7.1 2.7 0.92 0.92 0.69 0.69 0.23 0.23 0.23 0.23 29.8

66.7 12.8 20.5 100

28.6 5.5 8.8 42.9

85.0 15.0 100

23.2 4.1 27.3

mM/gm of mixture 0.81 0.39 0.21 0.045 0.070 0.077 0.047 0.017 0.022 0.019 0.016 1.72 1.59 0.31 0.49 1.90 (reducing only)

Roasting Kinetics Data Roasting temp, "C 90 120 135 Ea (KCalM-1)

Rate constants, ki (min'^) 0.0051 0.029 0.039 14.9

natural systems which may have been due to the unidentified phenolic components in GBE/B2. In retrospect, it was found later that a stiU better chocolate aroma could be generated by heating the sixteen component mixture for very short times (5-15 min.) at ca. 180^0.

545 A further attempt toward simplification led to a nine-component synthetic mixture (Table 7) containing only four amino acids. During roasting, the kinetic data of this mixture closely paralleled those of natural extracts and reasonably good chocolate-like aroma was formed at 120-130^C.

Table 7 Nine-component, simplified precursor system and related roasting kinetics data

Relative % of classes

Component

Absolute % in mixture

mM/gm in mixture

tyrosine phenylalanine tryptophane leucine AMINO ACIDS(total) fructose glucose sucrose SUGARS (total)

42.0 34.0 17.0 7.0 100

13.6 11.0 5.5 2.3 32.4

0.75 0.67 0.27 0.18 1.87

67.0 13.0 20.0 100

27.7 5.4 8.3 41.4

1.54 0.30 0.24 1.84 (reducing)

caffeine theobromine XANTHINES (total)

15.0 85.0 100

3.9 22.3 26.2

Roasting Kinetics Data

Roasting temp, OQ

Rate constants, ki (min'l)

90 120 135

0.0056 0.026 0.041

Ea(KCalM-l)

13.1

546 4.

ACKNOWLEDGEMENTS The authors thank L. V. Haynes and L. Ngo for their analytical support.

5.

REFERENCES

1 2 3 4 5

R. A. Martin, Jr., Adv. Food Res., 31, (1987) 211. G. Ziegleder, Lebensmittelchem. Gerichtl. Chem., 37, (1983), 63. J. Kleinert, Rev. Int. Choc, 26 (1971) 2. T. A. Rohan, Gordian, 69/12, (1969) 587. W. Mohr, E. Landschreiber and Th. Severin, Fette Seifen Anstrichm., 78, (1976) 88. S. D. Bailey, D. G. MitcheU, M. L. Bazinet and C. Weurman, J. Food Sci., 27, (1962) 165. T. Rohan and T. Stewart, J. Food Sci., 32, (1967) 625. R. R. Darsley and V. C. Quesnel, J. Sci. Fd. Agric, 23, (1972) 215. J. C. Hoskin and P. S. Dimick, Process Biochem., 19, (1984) 92. M. Heinzler and K. Eichner, Z. Lebensm. Unters. Forsch., 192, (1991) 445. K. H. Ney, Gordian, 84/11 (1984) 218-221. A. Pinto and C. O. Chichester, J. Food Sci., 31 (1966) 726. G. A. Reineccius, P. G. Keeney and W. Weissberger, J. Agr. Food Chem., 20 (1972) 202. H. Rosen, Arch. Biochem. Biophys., 67, (1957) 10. V. L. Singleton and J. A. Rossi, Jr., Am. J. Enol. Viticulture, 16 (1965) 144.

6 7 8 9 10 11 12 13 14 15

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

547

Contribution of muscle and microbial aminopeptidases to flavor development in dry-cured meat products. ^^ M. Flores^ Y. Sanz^ A.M. Spanier^ M-C. Aristoy'and F. Toldra^ '^Instituto de Agroquimica y Tecnologia de Alimentos (CSIC) Apt. 73, 46100 Burjassot, Valencia Spain. ^United States Department of Agriculture, Agricultural Research Center, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124, USA.

ABSTRACT Dry-cured meat products possess characteristic flavors due to the volatile and non-volatile compounds developed during processing. Amino acids are non-volatile compounds that contribute to the improvement of both meat taste and aroma. During the processing of dry-cured meat products the activity of enzymes such as aminopeptidases produce a noticeable change in free amino acid composition making an impact on the final flavor. The main aminopeptidases involved in this process are of muscle origin and include alanyl and arginyl aminopeptidases (RAP). The exception is the case of sausages where microbial aminopeptidases such as API and AP2 from Lactobacillus sake can also contribute to the increment of free amino acids. Sodium chloride, one of the main curing agents, is shown to be an important factor in activating RAP and AP2.

1. INTRODUCTION An increase in the concentration of free amino acids has been reported in postmortem muscle during meat aging [1-3] and in meat products such as dry-cured ham and cooked ham [4-6]. This increase in free amino acids has been attributed to the action of muscle aminopeptidases active at neutral pH [2, 3]. The contribution of peptides and amino acids to the improvement of meat taste have been reported in meat prepared at different cooking temperatures [7-9]. Also, the contribution of free amino acids accumulated during processing is relevant for the development of the specific dry-cured flavor [10]. Accumulation of amino acids is of great importance due not only to their specific tastes [77], but also for their involvement in further Strecker and Maillard reactions generating volatileflavorcompounds [72]. This is the case for dry-cured ham where many volatile compounds are generated through lipid oxidation [13, 14], and other compounds, such as sulfur compounds, pyrazines, and methyl branched aldehydes and alcohols, are generated through Strecker degradation of amino acids [75]. *Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. All communication should be with Dr. Monica Flores Phone: 34 6 390 0022 Fax: 34 6 36-36-301 E-mail: [email protected]

548

Aminopeptidases are enzymes that degrade peptides from proteins into single amino acids sequentially from the N-terminal side [16]. Two aminopeptidases, alanyl and arginyl [17-18], have been successfully purified and characterized from pork skeletal muscle; these enzymes have also been named/characterized as leucyl and pyroglutamyl aminopeptidases [6]. Aminopeptidase activity has also been detected in lactic acid bacteria involved in meat fermentation [19] and, recently, two aminopeptidases (API and AP2) from Lactobacillus sake have been purified and characterized [20, 21]. The objective of this paper was to elucidate the role of muscle and microbial aminopeptidases in the generation of free amino acids during the processing of drycured meat products such as ham, loin, and sausages.

2. METHODOLOGY 2.1. Processing technologies for dry-cured meat products. The processing of dry-cured ham involves three stages, salting, post-salting, and drying/aging [22]. During the salting stage a mixture of curing ingredients, mainly salt, nitrate, and nitrite are rubbed onto the lean surface of the ham. The hams are left fat side down to allow the curing ingredients to diffuse into the ham under the conditions indicated in Table 1. After washing, or during the post-salting stage, the hams are left in chambers for a complete and homogeneous distribution of the curing agents through the entire piece. In order to develop the characteristic dry-cured flavor the hams are dried and allowed to ripen at different time-temperature conditions (Table 1) [23]. Dry-cured loins are made of an intact/whole muscle piece as in the case of dry-cured hams. An entire loin (Longissimuss dorsi) is soaked in a wet mixture of curing ingredients mainly salt, nitrate, and nitrite and other spices such as paprika and garlic [24]. The loins are left in this mixture for at least 1 week with refrigeration (Table 1). The loins are then stuffed into casings, natural or collagen made, and hung to allow the development of the flavor by drying/aging. The end of the process is achieved when the loins reach a 35-45% weight loss in two stages as indicated in Table 1. The processing of sausages differs from that of dry-cured meats in that this process uses minced meat and fat. The curing ingredients, salt, nitrate and nitrite, and other adjuncts such as ascorbic acid, sugars, flavoring agents, enhancers, and starter cultures are mixed with the minced meat and fat under vacuum. The mixture is stuffed into casings and hung under controlled conditions (Table 1) [25]. This temperature-time period permits interactions that facilitate the development of the microflora responsible for the ripening [22]. The development of the final flavor is due to a combined microbiological, physico-chemical, and biochemical processes. As previously described, dry-cured ham and loin are made from the intact/whole muscle while sausage consists of a mixture of ground fat and lean meat; microorganisms play an important role in the latter. Another difference is the length of the processing being longest for dry-cured ham (Table 1). The generation of the products involved in the development of the final flavor of dry-cured meat products (amino acids and peptides) is due to the action of enzymes such as aminopeptidases of both muscle and microbial origin [6]\ these aminopeptidases are active during the long processing period for these meat products.

549 Table 1. Processes of dry-cured meat products Dry-cured ham Salting 4 Time (days) 6° Temp CQ 95-75 RH (%) Time (days) Temp (°C) RH (%)

Dry-cured Loin Salting 4 6° 95-75 Postsalting

Sausages Fermentation 2-3 22-24° 95-90

40-60 0-6° 95-70 Drying/Aging

r ' phase Time (days) Temp (°C) RH (%) 2"^^ phase Time (days) Temp CO RH (%) y^ phase Time (days) Temp (°C) RH (%) 4"^ phase Time (days) Temp (°C) RH (%) Total time (days)

>45 6-16° 95-70

30 8° 82-78

3-4 18° 90-85

>35 16-24° 95-70

15 14-16° 80-70

30-40 15° 90-75

49

47

>30 24-34° 95-70 >35 12-20° 95-70 >215

2.2. Measurement of enzyme activities. Muscle enzyme extracts were prepared by homogenizing 5 g of meat in 25 mL of 50 mM phosphate buffer, pH 7.5, containing 5 mM EGTA by using a Folytron^M homogenizer 3 times X 10 sec at 27,000 rpm. The extract was centrifuged for 20 min at 10,000xg at 4°C; the supernatant was filtered through glass wool and collected for further purification of alanyl, arginyl, leucyl and pyroglutamyl aminopeptidases [17, 18]. Muscle aminopeptidase activities were measured by fluorometric assays using aminoacyl-7-amido-4-methyl coumarin as substrates (aa-AMC). Alanyl aminopeptidase was assayed by using 0.1 mM alanine-AMC as substrate in 100 mM phosphate buffer, pH 6.5, with 2 mM 2-mercaptoethanol [18]. Arginyl aminopeptidase was assayed by using 0.1 mM arginine-AMC in 50 mM phosphate buffer, pH 6.5, with 0.2 M NaCl [17]. Leucyl aminopeptidase activity was assayed by using 0.25 mM leucine-AMC in 50

550 mM borate-NaOH buffer, pH 9.5, with 5 mM magnesium chloride. Pyroglutamyl aminopeptidase was assayed with 0.1 mM pyroglutamic-AMC in 50 mM borate-HCl, pH 8.5, containin 1 mM dithiothreitol (DTT). Microbial aminopeptidases were also measured by fluorometric assay. The cell-free extract was obtained from 400 mL batch cultures of MRS broth inoculated at 5% after the Lactobacillus sake CECT4808 had been subcultured twice. Cells were harvested at stationary phase by centrifugation (10,000xg, 30 min, 4° C), washed twice in 50 mM Tris-HCl buffer, pH 7.5, and then resuspended in the same buffer containing 5 mg/mL lysozyme and 0.45 M sucrose. After incubating at 30° C for 90 min, the cell-wall fraction was removed by centrifugation (15,000xg, 30 min, 4° C). The pellet was washed, resuspended in 50 mM Tris-HCl buffer, pH 7.5, and sonicated for 15 min. Cell debris was removed by centrifugation (20,000x^, 30 min, 4° C) and the supernatant constituted the cell-free extract. API and AP2 were further purified according to methods previously described [20,21]. The reaction buffer of API consisted of 50 mM TrisHCl, pH 7.5, containing 0.1 mM leucine-AMC as substrate. For AP2, the reaction buffer consisted of 50 mM phosphate buffer, pH 7.0, containing 0.1 mM arginine-AMC as substrate. The reaction mixture, consisting of 250 \iL of respective reaction buffer and 50 jiL of enzyme, was incubated 10 min for microbial and 15 min for muscle aminopeptidases. The fluorescence was measured after incubation at 355 nm and 460 nm as excitation and emision wavelength, respectively in a Fluoroskan nfluorophotometer(Labsystems, Helsinki, Finland) equipped with a thermostatted compartment at 37°C. Four measurements were made for each experimental point and the initial fluorescence at time zero was used as the blank. The effect of salt was tested by incubating the enzyme extract in the standard assay medium for each specific enzyme and in the presence of the following NaCl concentrations: 2,4, and 6 %. Controls with the absence of salt were simultaneously run. The activity was expressed as a percentage of the control in the absence of salt. 2.3. Free amino acids analysis. Samples were homogenized (1:4) in 0.01 N HCl in a Stomacher^M for 8 min at 4°C and deproteinated with acetonitrile [4]. The deproteinized samples were derivatized with phenylisothiocyanate according to the method of Bidlingmeyer et al., (1987) [45]. The derivatized amino acids were analyzed by reverse-phase HPLC in a Waters Nova Pak C18 colunm (300 x 3.9 mm) and monitored at 254 nm. Hydroxyproline was added as internal standard before derivatization. 3. ENZYMOLOGY OF DRY-CURED MEAT PRODUCTS. Many enzymes are involved in the degradation of proteins during the postmortem storage of meat. Enzymes such as cathepsins, calpains and other muscle proteinases have been deeply studied due to their contribution to tenderness [26-28]. The proteolysis in dry-cured ham has been attributed to the action of lysosomal proteinases during the initial period of maturation [29]. Cathepsins B, H, and L are stable along the entire process [30] but the contribution of calpains is very limited because their activity is lost after the salting stage [31]. In sausages, the proteolysis during ripening is predominantly due to the action of muscle cathepsin D-like

551 enzymes that are activated because of the pH drop [32, 33]. Moreover, bacterial enzymes seems to be particularly important in the degradation of peptides to free amino acids [33-34]. On the other hand, there are few reports about the role of other peptidases such as aminopeptidases involved in the development or generation of the characteristic flavor of dry-cured meat products [6, 29]. Table 2. Substrate specificity and conditions for optimal activity of muscle and microbial aminopeptidases (AP). Muscle AP Microbial AP LAP RAP PGAP API AP2 AAP Aac-AMC^ n.h.'^ n.h. 2.5 93.0 100.0^ Alan.h. 100.0 n.h. n.h. Arg64.0 100.0 0.2 100.0 n.h. 98.0 100.0 Leun.h. n.h. 42.0 n.h. 130.0 Lys40.6 n.h. n.h. 3.4 n.h. 5.0 n.h. Gly2.1 50.0 n.h. 124.0 40.0 Metn.h. n.h. 0.6 n.h. 7.3 n.h. Sern.h. n.h. n.h. 10.7 12.0 Tyr8.0 n.h. 210.0 0.3 Phe22.6 n.h. n.h. 5.8 5.1 n.h. Pro4.3 n.h. n.h. n.h. 6.0 n.h. Y-Glun.h. n.h. n.h. n.h. n.h. 100.0 p-Glu n.h. n.h. n.h. n.h. 3.3 25.6 Val37° 37° 37° 37° 50° Temp. Opt 37° 6.5 9.5 7.5 7.5 6.5 pHopt. 7.0 ^Aminopeptidase activity was measured against fluorescence substrates (aac-AMC) at 37°C [17, 18, 21]. ''Activity is expressed as a percentage against respective standard substrate. '^ n.h. not hydrolyzed.

Aminopeptidases are enzymes that hydrolyze peptide bonds at the N-terminus of proteins and polypeptides [16]. Their role is in the latter stages of protein degradation where they remove single amino acid residues sequentially from the N terminus. These enzymes are of great significance in the in vivo activity of the cell [35, 36]. Aminopeptidases are classified in many different ways; however, the most usual manner is by their substrate specificity. In some cases, a rather broad substrate specificity has given rise to the occurrence of several different names for the same enzyme. The most relevant aminopeptidase found in porcine skeletal muscle is Alanyl aminopeptidase (EC 3.4.11.14) (AAP) a soluble enzyme found in the cytosolic fraction [18]. The enzyme has a molecular mass of 106 KDa, exhibits a maximum activity at pH 6.5, 50°C, and shows a broad substrate specificity hydrolyzing aromatic, aliphatic, and basic aminoacyl bonds (Table 2). Arginyl aminopeptidase (RAP), also named aminopeptidase B (EC 3.4.11.6), is also present in

552 the cytosolic fraction of porcine skeletal muscle [17]. This enzyme has a molecular mass of 76 KDa, presents maximum activity at pH 6.5, 37°C, and has a substrate specificity against basic aminoacyl bonds (Table 2). Two other aminopeptidases present in porcine skeletal muscle are leucyl (LAP) and pyroglutamyl aminopeptidases (PGAP) [6,37,38]. LAP (EC 3.4.11.1) is a zinc metallo enzyme located in the cytosol with a molecular mass of 324 KDa and an optimal alkaline pH (Table 2). LAP catalyzes the release of leucine and methionine as well as other hydrophobic amino acids from the N terminus of the proteins or polypeptides [39, 40]. PGAP (EC 3.4.19.3) is widely distributed in the cytosol and has a molecular mass of 24KDa and an optimal pH around 7.5. This enzyme shows a high specificity against pyroglutamic acid at the N-terminal end of the proteins or polypeptides [16, 41, 42]. The characteristics of the aminopeptidases currently purified from the cell-extract of Lactobacillus sake are shown in Table 2. API is the major aminopeptidase detected in this species. This enzyme is a 35-36 KDa monomer, has optimal acitivity at 37°C and pH 7.5 and shows broad substrate specificity except for basic amino acids. In contrast, AP2 mainly hydrolyzes basic amino acids and shows optimal activity at 37°C and pH 7.0. 4. GENERATION OF FREE AMINO ACIDS IN DRY-CURED MEAT PRODUCTS. The proteolytic enzymes involved in the dry-curing process produce an increase in free amino acid concentrations (Table 3). It is important to examine the different composition of amino acids in raw meat such as that observed between Longissimus dorsi and Biceps femoris. As shown in Table 3, the amino acid increase is higher in dry-cured ham as a consequence of its longer processing time (see Table 1). The larger increases were for aspartic and glutamic acids, alanine, valine, leucine, arginine, and lysine. AAP could be the responsible for these increments due to its broad substrate specificity although RAP would contribute to the release of basic amino acids. On the other hand, the optimum activity of LAP at basic pH makes its contribution minimal because the pH of dry-curing ham is around 5.8-6.3. PGAP also has a restricted contribution because of its specificity against pyroglutamic acid. This amino acid is not usually found in muscle proteins, but is common in neuropeptides being considered a source of glutamic acid in the brain [43]. Dr>"-cured loin and sausages seem to have similar final contents of free anAno ^cids per 100 g of product. However, the fat content (around 30-50 %) in sausages makes its concentration higher than in loin when expressed per 100 % lean meat. In dry-cured loin, the larger increases were for glutamic acid, alanine, valine, leucine and lysine. These amino acids also showed the greatest increase in dry-cured ham, where enzymatic activity is essentially of muscle origin. However, in sausages the increase in free amino acid concentration is not only due to the action of muscle aminopeptidases, such as AAP and RAP, but also to microbial aminopeptidases [34]. For loin, the larger increases were for glutamic acid, taurine, alanine, arginine, valine, leucine, phenylalanine, and lysine. API could be involved in the release of amino acids such as leucine, alanine and valine while AP2 will contribute to the release of basic amino acids together with RAP. The contribution of both muscle and microbial aminopeptidases is primarily affected by the acid pH reached in this product.

553

^ en en o\ ~ . en ON en

^

00 ^ O

I . ON

g

9 §

q5

r-

00

^

43

C/3

•c

1

13 O o o. 6 ^ Q iS

ON r-

Tj-

O ON en en vd" en m

- S •^ -

c

S3

0 rj- t^ to ON J8 3 S -. ;^ CN n- »n cn Tf ^^ (N ^ . t^ en T-H r-" vcT 00" 21 ^ "^ 2 ^^

^ ^

S ^ *^

^ ^

U-i
r - i n i n N 6 ^ g g 5 o :$

o

5

;:)

^

00


2

-H"

CM «r) j5r m

as

»o

^

oo o

u-i

^

00

en m

cx)

^

Tt

in

CN VO r-H
m VO CM 00 r l n ' - : OS. cs 00 in

-H gj ^

ON CS ^ . en ••" ^

iX

^

o

t^

ON

Tt

xri

ON l > 2 ^ 0 ^ 0 0 ON

en

cs

»o u^^ IQ

-^ >n 00 Tj- CN in o »o

^ ^ eN (N• "^ Tf 00-^ CN ^ - ' (N NO CN Tf (N en

5^ I I

00 cs en 00 eN ^

e n O N O " " * ^ ^ ^ ^ ^ ^

XT)

^ ^4

rsi

T ON

5 ^ ^ -' ^

^

10 vo Tt c^ ^. '-. o <^^ 5^ go
oC

-^ ^ --0 O in [C!

N5

en ^^ ^

ON O g O ON ^- r^

W3

ON O" ^ (N en NO

SS§2

^^ONCNOr^NOi^r^en'^in'^Tt^-^.en J^ ON ON ^„ " ^ 00 "^ en — 00' — r4^ ^ ' -^- " oi g r ^ e n ^ N O r ^ m l Z N O ^ e s c S T t v o e n c N ^ T^ - HNO (N Tt in ON en — ^ 04 en CM

s ^* ^" d

O e n O ' ^ O N O N O ^ O v o o o

csjvovocST-(cnt^~^inNo^in O" '^'^ O" O" ^ 06 CS 22 O" O" rf

0 0 ±^ NO ^

O a


NO ON

t^c^cs^en
'1

vo m in ON rin r- in vo CN 0 in (N ON ON NO*^ 00 Tf 0^ 00 NO T-H 0 0 en rin in Tf en CN Tt cs rf en 0 rf"

S '^

^^

en -^ 00^ S

'"^

^^

D^

ONinONt^inNor^ONTi-ooenJC^'-HoooN Or-enr-HO\ONVOT-HCSen^(N^incNen o ' o" o^ o" o^ "^"^ o^ in o^ o" ^ JQ o" o" ^ 0

ON e n

CN '-H

cN

en^ O P« 0\ ^^ o" cN r i o" in

^ ^ ,—T '-'; " ^ _ r en

2 S -. =

0

0

0

0

0

i-i m

' - H g c N C S ^ ^ r - H i o H I S i H c N O ^ H ^ '

< o S < O O c Q . H

0

0

ON CN ON

ON

o*^ cS

0

3

554 5. EFFECT OF SALT AS THE MAIN CURING AGENT. Salt is one of the main curing agents in addition to nitrates and nitrites. The effect of NaCl on aminopeptidase activity was studied because of the higher quantities found in these products (Figure 1). The NaCl content varies depending on the product. Dry-cured ham contains around 5 to 6 % NaCl and dry-cured loin and sausages between 3 to 4 %. For this reason, we assayed three different sodium chloride concentrations, 2,4 and 6% (Figure 1). Only LAP is not affected by the NaCl concentrations used. RAP is highly activated at these concentrations, almost 3 times at 2% NaCl, and AP2 is activated or not affected by 2-4 % NaCl. On the other hand, AAP, PGAP and API are inhibited as the NaCl content increases. The broad substrate specificity of AAP could be responsible for the high increment of free amino acids such as lysine, arginine, phenylalanine, alanine, leucine, proline, valine, serine, glycine, and isoleucine that are found in all dry-cured products, but specially in dry-cured ham. However, it should be remembered that high NaCl contents can limit its activity. RAP also contributes to the release of arginine and lysine because of its specificity and its activation in the presence of NaCl. The latter is important in all dry-cured meat products, particularly at high levels of NaCl (6 %) since it is activated 2 times at this concentration. On the other hand, the contribution of LAP and PGAP is minor due to their specificity towards leucine and pyroglutamic acid, respectively, and because LAP is active at basic pH which is not a usual condition in meat products. Even though salt concentration seems to control PGAP activity, it could contribute to the increase in free glutamic acid because of the tranformation of pyroglutamic to glutamic acid. In sausages, microbial aminopeptidases also contribute to the generation of free amino acids. API could be involved in the release of amino acids such as leucine, alanine and valine which are present in large quantities in the finished product. The role of API in the generation of amino acids during the curing process may be limited by sodium chloride although this enzyme still retains 40 % of its optimal activity at 4 % NaCl. AP2 is stimulated or not affected at NaCl concentrations typically present in sausages. This aminopeptidase could be involved in the release of basic amino acids together with RAP. Salt concentration regulates the aminopeptidase activity inside the muscle or meat product by promoting the release of specific amino acids. Moreover, the amino acids generated through proteolysis can regulate this degradation via a feedback inhibition [44]. 6. CONCLUSION The contribution of each individual muscle and microbial aminopeptidase is difficult to predict. The main aminopeptidases involved in the generation of free amino acids in dry-cured meat products are of muscle origin and include AAP and RAP; this is true except in the case of sausages where microbial aminopeptidases such as API and AP2 from Lactobacillus sake can contribute to the increment in free amino acids. Careful consideration must also be given to the activating effect of sodium chloride on RAP and AP2 which promotes the activity of these enzymes.

555 Acknowledgements Grant ALI97-0353 from the CICYT (Spain) is acknowledged. The MEC contract to MF and FPI/MEC scholarship to YS are also acknowledged. The USDA ICD/RESD project SP05 to AMS is also gratefully acknowledged for its contribution to the successful completion of this research.

7. 1. 2. 3. 4. 5. 6.

REFERENCES

A. Okitani, Y. Otsuka, R. Katakai, Y. Kondo, H. Kato, J. Food Sci., 46 (1981) 47. T. Nishimura, MR. Rhue, A. Okitani, H. Kato, Agric. Biol. Chem., 52 (1988) 2323. T. Nishimura, A. Okitani, MR. Rhue, H. Kato, Agric. Biol. Chem., 54 (1990) 2769. MC. Aristoy, and F. Toldra, J. Agric. Food Chem., 39, (1991) 1792. S. Buscailhon, G. Monin, M. Comet, and J. Bousset, Meat Sci., 37, (1994) 449. F. Toldra, M. Flores, MC. Aristoy, Li: Recent developments in food science and nutrition. (G. Charalambous, Ed.), Elsevier Science Pub. B.V., Amsterdam, The Netherlands. 1995, 1303. 7. AM. Spanier, JV. Edwards, HP. Dupuy, Food Tech., 42 (1988) 110. 8. AM. Spanier, and J A. Miller, In : Food, Flavor and Safety. Molecular analysis and Design. (AM. Spanier, H. Okai, M. Tamura, Eds.) #528 ACS Books hic. Washington D.C., 1993, 78. 9. AM. Spanier, and JA. Miller, J. Muscle Foods, 7 (1996) 355. 10. M. Flores, MC. Aristoy, AM. Spanier, F. Toldra, J. Food Sci. (1997) In press. 11. T. Nishimura, and H. Kato, Food Rev. Int., 4 (1988) 175. 12. F. Shahidi, LJ. Rubin, LA. D'Souza, CRC Crit. Rev. Food Sci. Nutr., 24 (1986) 141. 13. JL. Berdague, C. Denoyer, JL. La Quere, E. Semon, J. Agric. Food Chem., 39 (1991) 1257. 14. G. Barbieri, L. Bolzoni, G. Parolari, R. Virgili, R. Buttini, M. Careri, A. Mangia, J. Agric. Food Chem., 40 (1992) 2389. 15. M. Flores, CC. Grimm, F. Toldra, AM. Spanier, J Agric. Food Chem., 45 (1997) 2178. 16. JK. McDonald and AJ. Barret, (eds.). Mammalian proteases: a glossary and bibligraphy. Vol. 2. Exopeptidases. Academic Press, London, 1986. 17. M. Flores, MC. Aristoy, and F. Toldra, Biochimie, 75 (1993) 861. 18. M. Flores, MC. Aristoy, and F. Toldra, J. Agric. Food Chem., 44 (1996) 2578. 19. MC. Montel, R. Talon, M. Cantonnet, JL. Berdague, In: Les bacteries lactiques, (G. Novel and JF. Le Querler, Coord.), Ardie Normandia, Caen, 1992, 67. 20. Y Sanz, Purificacion y caracterizacion de exopeptidasas de Lactobacillus sake IATA115 aislado de embutidos curados. PhD Thesis. Universidad de Valencia 1996, Valencia, Spain. 21. Y. Sanz, and F. Toldra, J. Agric. Food Chem. 45 (1997) 1552. 22. J. Flores, and F. Toldra, In: Encyclopedia of Food Science, Food Technology and Nutrition (R. Macrae, R. Robinson, M. Sadler and G. Fullerlove, eds) Academic Press. London, 1993, 1277.

556 23. F. Toldra, M. Flores, J.L. Navarro, M-C. Aristoy and J. Flores, In: Chemistry of novel foods (H. Okai, O. Mills, A.M. Spanier and M. Tamura, Eds.) Allured Pub. Co., Carol Stream IL, 1997, 259. 24. J. Flores, In: L'Encyclopedie de la charcuterie, Soussana, Orly, Paris, 1990,481. 25. F. Toldra, and A. Verplaetse, In: Composition of meat in relation to processing, nutritional and sensory quality (K. Lundstrom, I. Hansson and E. Wiklund, Eds.) ECCEAMST, Utrecht, 1995,41. 26. C. Valin, and A. Ouali, In: New Technologies for Meat and Meat Products. (F.J.M. Smulders, F. Toldra, J. Flores and M. Prieto, Eds) Audet, Nijmegen, 1992, 163. 27. A. Ouali, J. Muscle Foods, 1 (1990) 129. 28. M. Koohmaraie, Biochimie, 74 (1992) 279. 29. F. Toldra, M. Flores, and Y. Sanz, Food Chem., 59 (1997) 523. 30. F. Toldra, E. Rico, and J. Flores, J. Sci. Food Agric, 62,(1993) 157. 31. CM. Rosell and F. Toldra, Z. Lebensm. Unters. Forsch., 203 (1996) 320. 32. D. Demeyer, Food Chem., 59 (1997) 491. 33. K. Molly, D. Demeyer, G. Johansson, M. Raemaekers, M. GhisteHnck, and I Geenen, Food Chem., 59 (1997) 539. 34. A. Verplaetse, In: Proc. 40*^ Int. Congr. Meat Sci. Technol., The Hague, The Netherlands, 1994,45. 35. GJ. Sanderik, Y. Artur, and GJ. Siest, CH. Chem. Clin. Biochem., 26 (1988) 795. 36. A. Taylor, TIBS, 18 (1993) 167. 37. F. Toldra, M. Flores, MC. Aristoy, R. Virgih, and G. Parolari, J. Sci. Food Agric, 71 (1996) 124. 38. PJ. Blanchard and D. Mantle, J. Sci. Food Agric, 71 (1996) 83. 39. SK. Burley, PR. David, RM. Sweet, A. Taylor, WN. Lipscomb, J. Mol. Biol., 224 (1992) 113. 40. N. Ledeme, O. Vincent-Fiquet, G. Hennon, R. Plaquet, Biochimie, 65 (1983) 397. 41. B. Lauffart, and D. Mantle, Biochim. Biophys. Acta, 956 (1988) 300. 42. D. Mantle, Clin. Chim. Acta, 196 (1991) 135. 43. C. Moret, and M. Briley, TIPS, 9 (1988) 278. 44. F. Toldra, G. Falkous, M. Flores, D. Mantle, Comp. Biochem. Physiol., 115B (1996) in press. 45. BA.Bidlingmeyer, SA. Cohen, TL. Tarvin, BA. Frost, J. Assoc. Off Anal. Chem. 70 (1987) 241.

557

200 ^ — 2% NaCl iaiiii!iiiite;a 4 % N a C l 150

-8 < >

—

6% NaCl

100

AAP LAP PGAP API

RAP

AP2

Figure 1. Effect of sodium chloride on aminopeptidase activity.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

559

Effect of adding free amino acids to Cheddar cheese curd on flavor development J. M. Wallace^ and. P. F. Fox^ ^Department of Chemistry, Moorepark, Fermoy, Co. Cork, Ireland ^Department of Food Chemistry, University College, Cork, Ireland. Abstract Amino acids were added to Cheddar cheese curd to determine the effect of free amino acid concentration on the development of cheese flavor during ripening. Five cheeses were manufactured with added cas-amino acids and a further eleven cheeses were supplemented with mixtures of selected amino acids in concentrations which would be normally present in a good quahty mature Cheddar cheese. All cheeses were manufactured in dupUcate. It was found that intermediate levels of cas-amino acids (42mmol kg-i) or selected mixtures which included high levels of sulphur amino acids (Met and Cys or Met alone) improved cheese flavor while all other treatments had either no effect or a deleterious effect on cheese flavor quahty. Cheeses with added sulphur amino acids also showed accelerated flavor development and had clean extra-mature flavors after only 6 months ripening. 1. INTRODUCTION Although hpolysis and glycolysis have a vital role to play in flavor development in Cheddar cheese, proteolysis is thought to be the principal biochemical event which occurs during ripening (1). Proteolysis results in the degradation of the principal milk protein, casein to small peptides and free amino acids through the action of proteolytic and peptidolytic enzymes. Chymosin (from calf stomachs or genetically manufactured chymosin), plasmin (which is a natural milk proteinase), starter ceU proteinases and peptidases and non-starter ceU proteolytic enzymes all contribute to the production of free amino acids from casein in Cheddar cheese. Levels of free amino acids in cheese have long been associated with flavor formation (2-5). Although amino acids themselves contribute to cheese flavor, particularly Glu acid and Leu (4), their principal contribution to cheese flavor is as precursors for the cataboUc formation of volatile flavor compounds.

560 Since proteolysis appears to be the limiting factor in cheese ripening, many authors have attempted to speed up proteolysis thereby accelerating the ripening process and achieving a flavor quality comparable to that of a mature Cheddar. Methods which have been used to accelerate ripening include elevation of the ripening temperature (6,7), addition of exogenous enzymes to the cheese curd (8), the use of attenuated or genetically modified starters (911), addition of non-starter lactic acid bacteria (NSLAB) as adjunct cultures (4,12,13), the addition of cheese slurries which contain bacteria, enzymes and cofactors to the curd, and finally prepressing (14). Most of these methods have been successful to a certain extent. Increased proteolysis and rapid flavor development in experimental cheeses were closely associated with increases in the total concentration of free amino acids (2-5). It was thought that since free amino acids were the end products of proteolysis, their addition to the cheese during manufacture may accelerate flavor development. This study attempts to determine the effect of free amino acids on flavor development. In addition we wish to assess the patterns of formation and degradation of free amino acids throughout the ripening period which will provide further understanding of the role of amino acids in Cheddar cheese flavor.

2. MATERIALS AND METHODS 2.1. Cheese Manufacture Cheddar cheese was manufactured from pasteurized (74°C X 15s) bulk herd milk (100 L) using Lactococcus lactis subsp. cremoris 223 (Hansen's Laboratories, Little Island, Cork, Ireland) as starter and standard calf rennet (Hansen's) as coagulant (0.3ml L^ milk). The normal Cheddar cheesemaking protocol (tempering milk, rennet and starter cultures (30°C), addition of starter and rennet, cutting, cooking, draining, cheddaring, milling, salting, pressing, packing and ripening) was followed until the salting step. For cheeses with cas-amino acids added, the milled curd was divided into five 2kg lots. One portion was salted (2.5% NaCl w/w) as normal and used as a control. Cas amino acids (DIFCO Laboratories Inc., Michigan, USA) at concentrations of 5 to 31 g per 2 kg were added with the salt to the remaining four portions to give an expected 1.12, 1.25, 1.5 and 1.75 fold increase in the concentration of free amino acids (FAA) in the cheeses (Table 1). Quantities were calculated according to expected concentrations of free amino acids in mature Cheddar cheese (84mmol kg-1) (15).

561

Table 1 Concentration of cas-amino acids added to individual cheese

Cheese type

Cas-amino •< ^cids added

Control (A) Experimental cheese B Experimental cheese C Experimental cheese D Experimental cheese E

mg/kg curd 0 1420 2850 5700 8540

mmol /kg curd 0.0 10.5 21.0 42.0 63.0

Fold increase in free amino acids expected 1.00 1.12 1.25 1.50 1.75

Reprinted from Wallace and Fox. International Dairy Journal 7, 1997 p 157. Eleven cheeses containing selected amino acids were manufactured in duplicate. Amino acids which are generally found in highest proportions in mature Cheddar cheese were selected and their quantities chosen by averaging the amino acid concentrations at one day and 5 weeks ripening in a cheese with added cas-amino acids which received a high flavor score (Cheese D. The chosen concentrations (per kg curd) were: Glu (440mg), Leu (345mg), Phe (171mg), Arg (104mg), He (80mg), Ser(71mg), Met (48mg), Cys (94mg) and *Met (150mg). At milling the curd was divided into 2kg lots and the free amino acids were added with the salt as described in Table 2. The 5 cheeses with added cas-amino acids were manufactured in duplicate on two consecutive days using different milk suppUes each day, and the 22 cheeses containing added selected amino acids were manufactured 8 months later on four consecutive days from a different milk supply on each day. The days of manufacture are represented in Table 3. Table 2 Concentration of selected amino acids added to cheese curd. Cheese Amino acids added (mg/kg curd) F G H I J K L M N O

Control (no amino acids added) Glu(440) Glu(440) and Leu(345) Glu(440), Leu(345) and Phe(171) Glu(440), Leu(345), Phe(171) and Arg(104) Control (no amino acids added) Glu(440), Leu(345), Phe(171), Arg(104) and Ile(80) Glu(440), Leu(345), Phe(171), Arg(104), Ile(80) and Ser(71) Glu(440), Leu(345), Phe(171), Arg(104), Ile(80), Ser(71) and Met(48) Glu(440), Leu(345), Phe(171), Arg(104), Ile(80), Ser(71), Met(48) and Cys(94)

Reprinted from: J. M. Wallace and P. F. Fox, International Dairy Journal (submitted).

562 Table 3 Days of manufacture of cheeses with added cas-amino acids and selected amino acids. Day of manufacture Cheeses manufactured 1 Cheeses A to E trial 1 2 Cheeses A to E trial 2 3 Cheeses F to J trial 1 4 Cheeses F to J trial 2 5 Cheeses K to 0 trial 1 6 Cheeses K to 0 trial 2 2.2. Free amino acids analysis Water-soluble extracts were analysed for free amino acids on a Beckman 6300 analyser (Beckman Instruments Ltd., High Wycombe, UK) using a cation exchange column (Na form, 12cm X 4 mm id) as described by Wilkinson et al. (16). A standard amino acid mixture (Beckman) was used to calibrate the column and Norleucine (Sigma) was used as an internal standard. Samples and standard were eluted with sodium citrate buffers at 77°C. Amino acids were post column derivatized with ninhydrin and detected by absorbance at 570 or 440nm (Pro). Data collection and integration were with a V.G. minichrom system (V.G. Data systems, Cheshire, UK). 2.3. Sensory analysis of cheeses 2.3.1. Professional Graders Cheeses with added cas-amino acids were graded after 5,12 and 24 weeks of ripening (at 8°C) and those with added selected amino acids were graded after 4, 6, 9, 11, 13 and 15 weeks of ripening by two trained graders from the Irish Department of Agriculture, Food and Forestry on the basis of flavor and texture (Texture results are not discussed here). The maximum achievable score for flavor is 45. The graders concentrate on the 35-43 portion of this scale. Cheeses are graded as 'extra-special' if they receive a flavor score of 4243, and a 'special' cheese which is acceptable for the commercial market must receive a flavor score of at least 38 on this scale. In addition to giving flavor scores for the cheeses, the graders were asked to comment on the flavors and/or off flavors which were detected during sensory analysis. 2.3.2. Consumer Panel Cheeses K (control), O (most complex mixture of added selected amino acids) and P (Met alone added) were assessed after 15 weeks ripening by a consumer panel of 50 untrained individuals on the basis of flavor preference. The cheeses were graded by placing an X on an unmarked Une (10cm). The distance of the X from the start of the Une was taken as the flavor preference score. Most but not all assessors commented on the types of cheese which they would normally consume and on the flavors of the test cheeses.

563 3. RESULTS AND DISCUSSION 3.1. Free amino acid analysis Large losses in amino acids from cheese curds during pressing were unavoidable. The exact extent of these losses in cheeses with added cas-amino acids were not measured; however, the extent of these losses was determined in cheeses with added selected amino acids. The results for cheeses O and P are reflective of the results in the other experimental cheeses and are shown in Table 4. Table 4. Retention of free amino acids in cheeses O and P Amino acid

Amino acids added to cheese 0 (mg/kg)

Glu Leu Phe Arg He Ser Met Taken from

Amino acids retained in cheese 0 (mg/kg)

440 345 171 104 80 71 48 Wallace and

Amino acids retained in cheese 0

Amino acids added to cheese P

(mg/kg) (%) 29 128 31 106 30 51 28 30 29 23 4 5 30 15 150 Fox 1997(submitted IDJ)

Amino acids retained in cheese P (mg/kg-1)

40

Amino acids retained in cheese P

(%)

26

The concentrations of free amino acids in each cheese were measured in water soluble extracts of the cheeses extracted after 16 h of pressing. In cheeses O and P , < 30% of added amino acids were retained in the curd. A low level of catabolism at this stage was possible but it is more likely that a large proportion of added amino acids was lost either by adhering to the vat walls or in the whey during pressing. Losses of Ser appeared to be greater than that of other free amino acids; it is therefore possible that Ser was catabolised rapidly when present at high concentrations. Ser is known to be deaminated by certain moulds, producing large amounts of ammonia (17). Ser may also be deaminated by starter or non-starter bacteria in the cheese, but this process has not been previously reported. The extent of Ser catabolism in Cheddar cheese has not been investigated, but may merit further study. Only approximately 26% of Met added to cheese P appeared to be retained in the curd (Table 4). Again, this may arise from adherence of the free amino acids to the vat wall or losses during pressing. However, since Met is catabolized rapidly in the cheese by either starter or non-starter enzymes (18,19) or chemical pathways (20), its loss may be explained at least partially by these processes.

564 3.2. Changes in amino acids in experimental cheeses 3.2.1 Cas amino acid cheeses Small increases in total amino acid concentrations were observed in the control cheese (A) and cheese B (to which lowest levels of amino acids were added) during the first 5 weeks of ripening. A small decrease in iree amino acid concentrations was observed in the other experimental cheeses during the same time period. Levels of free amino acids increased significantly in all cheeses between 5 weeks and 3 months ripening and (to an even greater extent) between 3 and 6 months of ripening. Increases in the concentration of amino acids were particularly evident in cheeses to which intermediate levels of cas-amino acids had been added. Cheeses C and D (to which 21 and 42 mmol cas amino acids were added respectively) showed increases of 1.7 and 2.7g free amino acids/kg cheese between 3 and 6 months ripening. (Figure 1).

1 day

5 \A/eeks

3 months

6 months

Ripening time

Figure 1. Total free amino acid levels in cas-amino acid cheeses at various ripening times Reprinted from: Wallace and Fox, Intern Dairy J, 7, 1997, p. 157 Although the cheese with the highest concentration of added cas-amino acids (E) maintained the highest concentration up to 6 months ripening, higher levels of some amino acids (eg. He, Leu and Phe) were released in all other experimental cheeses and the control (only results for cheese D and control shown: Figure 2). (Other results can be seen in Wallace and Fox, 1997). These results suggest that while intermediate levels of added amino acids enhance peptidolytic activity, higher levels may have had an inhibitory effect on bacterial enzymes during the later stages of ripening.

565 1400 1200

A

j D1 day • 5 weeks • 3 months S 6 months

$ 1000 0
sz o

800

O)

600

t

400 200 0

dfij—mi-

linBrMHi

ii[ J i JKj J [

rfi

Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe His Lys NH Arg

Amino acid

Asp Thr Ser Glu Pro Gly Ala Val Met

lie

3

Leu Tyr Phe His Lys NH3 Arg

Amino acid Figure 2. Levels of individual free amino acids in the control cheese and experimental cheese D (to which 5,700mg cas amino acids were added per kg curd).Reprinted from Wallace and Fox 1997. Intern Dairy J, 7, p 157 The principal amino acids in all cas-amino acid cheeses after 1 day (16h) ripening were Glu, Pro, Arg and Leu. Lys and Pro were utiHzed at a faster rate than they were released (from casein) during the first five weeks of ripening. This effect was particularly evident in experimental cheeses, the extent of the losses being directly proportional to the levels of cas-amino acids added. It is possible that low increases in the levels of all amino acids during Cheddar cheese ripening is indicative of their rapid utiHzation rather than their Umited release from cheese peptides. Substantial increases in the levels of Glu, Leu and Phe during ripening agreed with the findings of many authors (4, 11, 21, 22 23). Although exact concentrations of individual amino acids in the cas-amino acids powder was not determined, it appeared that the relative concentrations of individual amino acids differed from their respective

566 proportions in Cheddar cheese. This was particularly evident in the case of Pro. Pro constituted approximately 19.5 % of the total amino acids in one day old experimental cheeses while in control cheeses it only accounted for approximately 2%. Pro was the principal amino acid in the cas-amino acid powder followed by Glu, Leu and Phe. The principal amino acids in mature Cheddar cheese are Glu, Leu and Phe. The difference between the Pro concentrations in Experimental cheeses and normal mature Cheddar cheese may have effected subsequent flavor production in cheeses with added casamino acids. The relative concentrations of the principal amino acids (Glu, Leu and Phe) were, however, similar between control and experimental cheeses. The concentration of Arg decreased between 3 and 6 months ripening in all cheeses. Degradation of Arg during the later stages of Cheddar cheese ripening was also observed by other authors (4, 24). Catabolism of Arg may be due to release of arginase and urease enzymes from non-starter bacterial cells during the later stages of ripening (Figure 2). 3.2.2. Selected amino acids added Amino acids were analyzed in cheeses K (control), O (Glu, Leu, Phe, Arg, He, Ser, Met and Cys added) and P (Met only) after 1 day, 4, 9 and 15 weeks ripening (Figure 3). After 1 day, as was expected levels of Glu, Leu, Phe, Arg, He, Ser and Met were considerably higher in the cheese to which these amino acids had been added (O) and the concentration of Met was higher in the cheese to which this amino acid was added (P) than in control cheeses. Glu, Leu and Phe were the principal amino acids in all cheeses after 6 months ripening. Higher levels of all amino acids were released (or less catabolised) in all experimental cheeses than in controls, possibly due to higher NSLAB growth. In experimental cheeses O and P (Figure 3) addition of Met appeared to result in faster cataboUsm of this amino acid than in control cheeses indicated by decreasing levels of Met in the first 4 weeks of ripening. This may have resulted from higher levels of bacterial growth in these cheeses or inhibition of peptide hydrolysis when this essential amino acid was available at a high concentration. In control cheeses with no added Met, the concentration of this amino acid increased up to 15 weeks ripening. Degradation of Lys and Arg in the first 9 weeks of ripening only occurred in the presence of high concentrations of Met (P) (Fig 3). The rate of degradation of lie and Glu during the first 4 weeks of ripening was faster than their rate of release in experimental cheese O (largest mixture of added amino acids) to which they were added but not in the other two cheeses, suggesting that addition of Glu to cheese results in increased catabolism of this amino acid or inhibition of enzymes responsible for its release fi:om casein derived peptides in the cheese.

567

Control (K) 400

10 Weeks ES 4 Weeks D 9 Weeks • 15 Weeks

I 300 + "S 200 1)100 E ^

I •ci •

ll

flti^

BJ BI • !• • I rf< •

Asp Thr Ser Glu Pro Gly Ala Val Met

Amino acid

lie Leu Tyr Phe His Lys Arg

Clieese 0 (mixture of amino acids) 400 S 300 a> " 200 % 100 E .-.^^.,,^1

jsk.

4 - a i f l l t f t i JBAL

Mk,

ll|ifi|l(l|il}

Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg

Amino acid Cheese P (Methionine only added) 400

Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg

Amino acid Figure 3. Levels of individual amino acids in control cheese, K, and experimental cheeses O (added Glu, Leu, Phe, Arg, He, Ser, Met and Cys) and P (added Met only) throughout ripening. Reprinted from Wallace and Fox 1997. Intern Dairy J, (submitted) 3.3. Flavor analysis 3.3.1. Cas-amino acid cheeses Prior to 3 months ripening off-flavors (described by the graders as burnt or yeasty) were detected in all cheeses with added cas-amino acids, but not in the control cheeses. After 6 months ripening the control cheese and experimental cheese B (1420mg/kg cheese) were described as bitter while cheese D had a

568 clean mature flavor. Cheese C was described as a good cheese but did not have the same maturity as cheese D, cheese E which had the highest level of added cas-amino acids had an intense flavor and graded well but the flavor was described as being atypical of Cheddar cheese. Cheese E received a higher flavor intensity score than C but cheese C was said by the graders to be of superior quality. These results indicate that addition of intermediate levels (2,850-5700mg/kg curd) to Cheddar cheese had a beneficial effect on its flavor attributes but once this threshold is exceeded, cheeses developed a strong, atypical flavor (Figure 4).

CO

5 Weeks

3 Months

6 Months

Ripening time Figure 4: Flavor scores awarded to 'cas-amino acid' cheeses at throughout ripening(See Table 1 for cheese descriptions) Reprinted from Wallace and Fox 1997. Intern Dairy J, 7, p 157 3.3.2. Selected amino acid cheeses bv professional graders After only 4 weeks ripening all cheeses with added selected amino acids and their respective controls had advanced flavors, and were described as 8 to 10 week old cheeses (Figure 5). Since these cheeses were ripened at 11°C rather than 8°C a shght acceleration of ripening was expected. The flavor of cheese G (Glu only) was said to be particularly advanced while cheeses L (Glu, Leu, Phe, Arg and He), 0 (Glu, Leu, Phe, Arg, He, Ser, Met and Cys) and P (Met only) had slightly unpleasant off-flavors and cheese K (control) had a slightly floral off-flavor, which may or may not have been caused by high concentrations of

569 phenylethanol or phenylacetaldehyde, produced by Strecker degradation of Phe. These compounds cause floral taints in beer and may cause similar problems in Cheddar cheese (25). After 6 weeks ripening the flavor of cheese L was described as clean, the off-flavor compounds having been either degraded or masked by other catabolites released during ripening. Cheeses I and J had developed a *dirty' off-flavor and cheeses with the largest mixture of added amino acids (O) or Met alone (P) added received high flavor scores. The highest grade was awarded to the control cheese K after 6 weeks. After 9 weeks ripening control cheese K developed a shghtly sweet off-flavor, but the other control cheese F received a high flavor score. The reason why the two control cheeses differed was possibly due to small compositional variations between cheeses manufactured on different days using different milk suppUes (Wallace and Fox 1997, Intern. Dairy J. submitted, for compositions). Cheeses M and N were downgraded due to a sweet vanilla-Uke off-flavor and cheese G developed a fruity, peachy off flavor. All other cheeses graded well after 9 weeks ripening. Sweetness in cheese is usually associated with a high concentration of Pro, but there is no reason why the concentration of Pro should be higher in cheeses M and N than in the other cheeses. Fruity flavor is one of the most common defects in Cheddar cheese and has often been associated with esters formed by fatty acid catabohsm, however, aldehydes and ketones, which can be formed by free amino acid catabohsm, can also contribute (26). The peach-Uke flavor in cheese G intensified between 9 and 11 weeks ripening and a similar flavor was reported in cheese H. All other cheeses, with the exception of F (control), O (largest mixture of selected amino acids) and P (Met only) were sUghtly bitter after 11 weeks of ripening. After 13 and 15 weeks of ripening all cheeses, except cheeses O and P, had bitter, fruity or pungent off flavors. Cheese O graded highly and was described as extra mature and cheese P had a strong, and desirable flavor. Both cheeses O and P were described as 24 week old Cheddar cheeses.

570

4 Weeks

6 weeks

9 weeks

11 weeks

13 weeks

15 weeks

Ripening time

Figure 5. Flavor scores awarded to 'selected amino acid' cheeses by professional graders (See Table 2 for cheese descriptions) Reprinted from Wallace and Fox, Intern Dairy J, (1997 submitted) 3.3.3. Selected amino acids by consumer panel Since cheeses 0 and P received high flavor scores from the professional graders, only these and their control (cheese K) were subjected to further tasting by a consumer panel after 15 weeks ripening (Figure 6). Although the average preference scores were similar, the highest number of consumers preferred cheese P (48%), followed by cheese O (34%) and the control cheese (K) (18%). The control cheese was described as having a sharp bitter aftertaste. Although preferences differed, cheese P was verbally described as having a strong Cheddar flavor both in the mouth and as an aftertaste. Cheese O was described as a mild Cheddar which was considered by a small number of graders to have a slightly acidic flavor. The control cheese was described as bland except for a sharp, bitter aftertaste.

571

K

0 Cheese type

Figure 6: Flavor preference scores awarded to ^selected amino acid' cheeses by the consumer panel (see Table 2 for description of cheeses). Reprinted from Wallace and Fox, Intern Dairy J, (submitted) 4. CONCLUSIONS Addition of 2,850 to 5,700mg cas amino acids/kg to Cheddar cheese curd improved cheese flavor, however, higher concentrations (8,450mg/kg) had a detrimental effiect on flavor quality. Cas-amino acid powder had different relative proportions of individual amino acids than mature Cheddar cheese, and high levels of Pro may have had a significant effect on biochemistry and flavor formation in experimental cheeses throughout ripening. When selected amino acids were added to the cheese, significant improvements in flavor were noted when high levels of sulphur amino acids were added [Cys and Met(O) or Met alone (P)]. Experimental cheeses exhibited stronger, more advanced flavors than controls and possible masking of off-flavors was noted. Addition of free amino acids to Cheddar cheese curd either as cas-amino acids or as mixtures of selected amino acids affected their subsequent cataboHsm during ripening, and this was particularly evident between one day and 5 weeks of ripening. 5. FOOTNOTES The above work was previously published in the form of two research papers: 1. Effect of adding free amino acids to Cheddar cheese curd on proteolysis, flavor and texture development, J. M. Wallace and P. F. Fox, Int. Dairy J. 7, 157. 2. Effect of adding selected amino acids to Cheddar cheese curd on flavor development. J. M. Wallace and P. F. Fox, Int. Dairy J. (Submitted)

572

6. REFERENCES 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

P. F. Fox, J. Law, P. L. H. McSweeney and J. Wallace, Cheese, Chemistry, Physics and Microbiology (P. F. Fox, ed.) Chapman and Hall, London (1993) 389. G. T. Lloyd, J. F. Horwood and I. Barlow, Aust. J. Dairy Technol.,35 (1980) 137. Y. Ardo and H. E. Pettersson, J. Dairy Res., 55 (1988) 239. R. Puchades, L. Lemieux and R. E. Simard, J. Food Sci., 42 (1989) 885. M. C. Broome, D. A. Krause and M. W. Hickey, Aust J. Dairy Technol., 45 (1990) 67. S. J. Cromie, J. E. Giles and J. R. Dulley, J. Dairy Res., 54 (1987) 69. B. Folkertsma, P. F. Fox and P. L. H. McSweeney, Intern. Dairy J., 6 (1996) 1117. T. P. Guinee, M. G., Mulholland, E. D. and Fox, P. F. Ir. J. Food Sci. Technol., 15 (1992) 27. H. E. Petterson and G. Sjostrom, J. Dairy Res., 42 (1975) 313. Y. Ardo, P.-O. Larsson, H. Lindmark-Manson and A. Hedenberg, Milchwissenschaft, 44 (1989) 485. M. G. Wilkinson, Cheese: Chemistry, Physics and Microbiology, (P. F. Fox ed.) Chapman and Hall, London(1993) 523. L. Lemieux, R. Puchades and R. E. Simard, J. Food Sci., 54 (1989) 1234. C . N . L a n e a n d P . F . Fox, Intern. Dairy J., 6 (1996) 715. S. Singh and T. Kristoffersen, J. Dairy Sci., 55 (1972) 744. A. F. Wood, J. W. Aston and G. K. Douglas, Aust. J. Dairy Technol., 40 (1985) 166. M. G. Wilkinson, T. P. Guinee, D. M. O' CaUaghan and P. F. Fox, Le Lait, 72 (1992) 449. D. Hemme, C. Bouillanne, F. Metro and M. J. Dezmazeaud, Sci. des AHm., 2(1982)113. A. C. Alting, W. J. N. Engels, S. van Schalwijk and F. A. Exterkate, Appl. Environ. Microbiol., 61 (1995) 4037. G. Urbach, Intern. Dairy J., 5 (1995) 877. D. J. Manning, H. R. Chapman and Z. D. Hosking, J. Dairy Res., 43 (1976) 313. B. A. Law, M. J. Castanon and M. E. Sharpe, J. Dairy Res., 43 (1976) 301. Z. Ch. Dilanian, MUchwissenschaft, 35 (1980) 614. M. W. Hickey, H. van Leeuwen, A. J. HiUier and G. R. Jago, Aust. J. Dairy Technol., 38 (1983) 110. M. C. Broome and M. W. Hickey, Aust J. Dairy Technol., 46 (1991) 19. H. C. Dunn and R. C. Lindsay, J. Dairy Sci., 68 (1985) 2859. D. D. Bills, M. E. Morgan, L. M. Libbey and E. A. Day, J. Dairy Sci., 48 (1965) 1168.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

573

The Influence of Fat on the Deterioration of Food Aroma in Model Systems during Storage M. Chen and G.A. Reineccius Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108 USA Abstract Storage studies (30 and 45°C) determining the stability of selected aroma compounds (50 ppm) in the presence of simple microcrystalline cellulose (MCC)(with or without 5% vegetable oil and water activities of 0.11, 0.33, 0.53 and 0.76) were conducted. MCC was found to exhibit substantial aroma compound binding. Binding appeared to be strongly dependent on water activity with the binding being less at higher water activities and appeared to reach a maximum at an Aw of approximately 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. Furfurylmercaptan, pyrrole, 1-methylpyrrole, 2,4-hexadienal and ethyl-2mercaptoproprionate were found to be very unstable during storage (listed in decreasing order of loss). The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so rapid that the effect was small. The degree of increase in stability in the presence of oil was dependent upon the aroma compound studied.

1. INTRODUCTION There has been a relatively long term trend in the US to develop food products which have a reduced fat content. It is well documented in the literature that reduced fat foods lack the same sensory qualities as their full fat counterparts [1,2]. These differences in products may relate to any of several factors including flavor/food matrix interactions, flavor release, flavor formation (e.g. fried products) or textural changes [2-8]. Thus we have come to accept that reduced fat foods will generally be of lesser sensory quality than the full fat food. There are other flavor issues related to reduced fat foods that may be less obvious [1]. One relates to the quality of ingredients required in reduced fat versus the full fat products. We often find that a reduced fat food will have undesirable flavor notes associated with ingredients. Fat will mask off flavors and thus when fat is taken out of a food, the off flavors associated with ingredients are freely expressed. A low fat food may well have very perceptible off flavors using the same ingredients as were used in the formulation of an acceptable flavored full fat counterpart. A similar situation exists for the susceptibility of a food to off flavor development due to contamination from the environment (e.g. water, air or packaging sources). A low fat food is quite susceptible to off flavor development from these

574 sources relative to a full fat version of the product. This may translate to needs for improved quality packaging (less residual volatiles or better barriers to organic volatiles). The work to be reported in this paper addresses the issue of shelf-life of low fat versus full fat foods. Low fat foods typically have a shorter shelf-life than full fat foods. When a food changes in flavor during storage, it is seldom clear whether this change is due to the formation of undesirable flavors (e.g. oxidative rancidity) or the loss of desirable flavors. We know that some off flavors (e.g. lipid oxidation) are quite good at masking desirable flavor notes of a food. Also the question arises whether fat content has any influence on the stability of desirable aroma constituents in a food during storage. If the flavor of a food is more stable in the presence of fat, this might account for the stability differences one observes in low fat foods. This contribution is part of a larger study in which the stability of flavor compounds in the presence various food constituents (protein, sugar fat and microcrystalline cellulose) was monitored during storage [9]. We will present data and discuss the role of fat in stabilizing food aromas during storage.

2. MATERIALS AND METHODS 2.1. Materials Microcrystalline cellulose (MCC) (FMC Corp., Newark, DE.) and Crisco oil (Procter and Gamble, Cincinnati, OH) were used to make food model systems. Systems were prepared with distilled water purchased from the Glenwood Inglewood Company (Minneapolis, MN). Four salts were used to prepare saturated solutions for adjusting water activity: lithium chloride and magnesium chloride (Mallinckrodt Inc., Paris, KY); magnesium nitrate (Sigma Chemical Company, St. Louis, MO) and sodium chloride (E.M. Science, Gibbstown, NJ). Methylene chloride (Fisher Scientific; Fair Lawn, NJ) was used as a solvent to prepare the flavor solution. Sylon - CT silanizing reagent was obtained from Supelco Inc. (Bellefonte, PA), which was used to treat glass vials. Eleven flavor compounds and an internal standard were used in our studies: Isovaleraldehyde, 1-methylpyrrole, pyrrole, 2-methylthiophene, hexenal, nonane (internal standard), furfurylmercaptan, 2,4-hexadienal, diethyldisulfide, ethyl-3-mercapto-propionate, phenylacetaldehyde, and 2-ethyl-3,5(or3,6)-dimethylpyrazine. 2.2. Sample Preparation 2.2.1. Flavor Stock Solution A 5000 ppm stock solution of most flavor compounds was prepared by adding 0.250 g of each flavor compound to about 30 mL methlene chloride in a 50 mL volumetric flask. The solution was brought to 50 mL with methlene chloride. This yielded a concentration of 5000 ppm. Ethyl-3-mercaptopropinate, phenyl acetaldehyde and 2ethyl-3,5 (or 3,6) dimethylpyrazine were diluted to 15,000 ppm since these three compounds had a poor gas chromatographic response. The flavor stock solution was wrapped with aluminum foil to limit light exposure. 2.2.2. Saturated Salt Solution Preparation Salts were dissolved in distilled water in 1 L beakers according to the solubility limits given in the Handbook of Physics and Chemistry [10]. Solutions were heated and agitated constantly. Additional salt was added until undissolved salt crystals remained. Solutions were cooled to room temperature and transferred

575 into vacuum dessicators. The following salts were used to create the different water activities (77): LiCl (Aw = 0.11 at room temperature), MgCl (Aw = 0.33), Mg(N03)2 (Aw = 0.53) and NaCl (Aw = 0.76). 2.2.3. Model System Preparation Eight hundred mL of distilled water were added to 400 g MCC (or 380 g MCC plus 20 g Crisco oil) and thoroughly mixed by hand to yield a paste. The paste was frozen at -20°C first, then freeze dried using a freeze dryer (VIRTIS; Gardiner, NY) for 48 hr. Five g dried powder was weighed into 20 mL headspace sample vials (Chrom. Tech.; Apple Valley, MN). They were then placed in vacuum desiccators at 4 different water activities and allowed to reach equilibration for about 2 months. The model systems were then prepared by adding 50 |iL of stock flavor solution into each headspace vial which contained the MCC or MCC plus oil and sealed with a septum cap (Chrom. Tech., Apple Valley, MN). Thus, the concentration of flavor compounds in each vial was 50 ppm for most flavor compounds and 150 ppm for the 3 volatiles that had a poor gas chromatographic response. 2.2.4. Storage and Sampling. Samples were stored at 30 and 45°C in the dark and removed at 0, 2, 4, 7, 14, 21, 28, 36, 43, 50 and 90 days for headspace analysis. For the starting values, samples were held for 3 hr at 45°C or 12 hr at 30°C before analysis. This was based on preliminary experiments which indicated that the headspace concentration of volatiles reached equilibrium in approximately 3 hr and 12 hr at 45°C and 30°C, respectively. 2.3. Analysis 2.3.1. Static Headspace Analysis. Static headspace concentrations of the volatiles were measured using a Hewlett Packard headspace autosampler HSS19375A (Hewlett Packard; Little Falls Site, DE). Samples were held for 38 min at 45°C in the autosampler prior to analysis. One mL headspace was introduced into the GC. 2.3.2. Gas Chromatography. Headspace volatiles were separated and quantified using a Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard; Little Falls Site, DE) equipped with a flame ionization detector and GC Chem-Station (Hewlett-Packard, Avondale, PA). A DB-5 fused siUca capillary column, 30 m long, 0.32 mm i.d., 1.00 um film thickness (J&W Scientific; Rancho Cordoba, CA), was used to separate the model system volatiles. The carrier gas was helium (2.8 mL/min) at a head pressure of 12 psi. The GC oven was temperature programmed as follows: initial temperature, 40°C; initial time, 0 min; program rate 1, 5°C/min; final temperature, 80°C; final time 1, 5 min; program rate 2, 12°C/min; final temperature 2, 200°C; and final time 2 min. 2.3.3. Reproducibility. The reproducibility of analysis was determined by conducting the following experiment: 20 \xL of flavor stock solution was added to four 20 mL headspace vials containing 5 g MCC (or MCC plus 5% Crisco oil) at a water activity of 0.11. The GC peak areas were determined by GC-HS analysis under the described experimental conditions. The arithmetic means, standard deviations and the coefficient of variance values (%CV) were calculated. 2.4. Preliminary studies Preliminary studies were conducted to determine the equilibrium time required after adding flavor solutions to the model systems. Samples were analyzed at 0, 3, 6, 12, 24 and 48 hr at 30°C and 0, 3, 6, 12 hr. at 45°C. Peak area versus equilibration time were plotted at each temperature. Flavor compounds were also added to empty headspace vials without any food

576 matrix to determine if they interacted with each other or the glass vial wall. To check for interactions with the glass vials, the vials were treated with Sylon-CT silanizing reagent, flavor solution was then added and analyzed using the same experiment conditions. 2.5. Data presentation. The GC peak areas were determined for each flavor compound in all model systems. The assumption was that zero day values represented 100%, therefore, the peak areas of all the other days were compared to the zero day values and are presented as a % remaining.

3. RESULTS AND DISCUSSION 3.1. Aroma Stability during Equilibration The data that will be presented are based on the amount of flavoring in the headspace at what was considered "zero" time. The choice of "zero" time is not all that obvious as can be appreciated from the data in Figure 1 where we were attempting to determine the optimum equilibration time to use in analysis. Time 0 on the Figure corresponds to 38 min equilibration in the headspace sampler. The initial decrease in many of the aroma compounds appears to be related to their interaction with the MCC since their headspace concentrations decreased rapidly and then "stabilized" suggesting saturation of all binding sites. Based on these data, we chose to use 12 hr GC peak areas as the zero starting time at 30°C and 3 hr values as time zero for the 45°C samples (data not shown). 120 T ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL

FURFURYLMERCAPTAN 2,4-HEXADIENAL DIETHYLDISULFIDE ETHYL-3MERCAPTOPROPIONATE PHENYLACETALALDEHY DE 2-ET-3,5-DIMEPYRAZINE 2-ET-3,6-DIMEPYRAZINE

Figure 1. The influence of equilibration time in headspace sampler (30°C) on the concentration of headspace volatiles (MCC system).

577 We were concerned about the very rapid loss of volatiles in the MCC containing vials and wanted to determine if the volatile losses were due to interactions with the MCC or to possible interactions between the glass vials and aroma compounds or reactions between aroma compounds themselves. This prompted us to add the aroma compounds to empty glass vials (silanized and unsilanized) and monitor their losses during storage (Figure 2). This experiment pointed out that 38 min was not adequate for all compounds to reach equilibrium in the headspace. The amount of phenylacetaldehyde and ethyl-3-mercaptoproprionate reached equilibrium with the headspace some time between the initial sampling time and 3 hr sampling. Also as can be seen in Figure 2, most of the aroma compounds were quite stable in the empty vials (silanized or unsilanized). Only furfurylmercaptan, exhibited substantial losses during storage during this brief period. Thus it is assumed that the MCC was involved in hastening the loss of aroma compounds. ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL -FURFURYLMERCAPTAN -2,4-HEXADIENAL -DIETHYLDISULFIDE -ETHYL-3MERCAPTOPROPIONATE -PHENYLACETALALDEHY DE - 2-ET-3,5-DIMEPYRAZINE - 2-ET-3,6-DIMEPYRAZINE

Figure 2. The influence of equilibration time in headspace sampler (30C) on the concentration of headspace volatiles (empty glass vials - not silanized).

3.2. Effect of Oil Addition on Headspace Concentration of Aroma Compounds The addition of oil to the MCC resulted in decreased headspace concentrations of all of the volatiles used in this study except phenylacetaldehyde (Figure 3). While the method of analysis was reasonably reproducible (coefficients of variation (CV) ranged from 3 to 21% with most less than 10%), the largest CV was for phenylacetaldehyde. It is possible that the data point for this compound was in error since one would have anticipated that it also decreases in the oil containing system. The decrease in headspace concentration with the addition of oil is consistent with theory and not surprising.

578

MCC control MCC + 5% Oil

^O ^

.^ ^

A^ Jy

^

« * /

^^

rJ^

rJ^

Flavor Compounds Figure 3. The influence of including oil with MCC on the concentration of headspace volatiles (ISO-isovaleraldehyde; MP: 1-methylpyrrole; PYR: pyrrole; MT: 2-methylthiophene; HEX: hexanal; FFM: furfurylmercaptan; HD: 2,4-hexadienal; DD: diethyldisulfide; EMP: ethyl-3mercaptopropionate; PA: phenylacetaldehyde; EDP(3,5): 2-ethyl-3,5-dimethylpyrazine; EDP(3,6): 2-ethyl-3,6-dimethylpyrazine.

3.3. Effect of Oil Addition on the Stability of Aroma Compounds While we could have presented data on all of the aroma compounds studied, we will present only a sampling due to manuscript limitations (45C data only and selected aroma compounds). The reader is encouraged to obtain Ms. Chen's thesis [3] for greater detail. 3.3.1. Hexanal. Hexanal was lost from the headspace to a great extent during storage in the presence of MCC (Figure 4). There appears to be an effect of water activity in that hexanal was lost the least from the highest water activity (0.75) and the greatest at the lowest water activities (0.11 and 0.33). There appears to be no difference between the two lower water activities. Hexanal losses were greatly retarded in the MCC + oil system. Losses tended to follow what one might expect from typical oxidation reactions in that losses were lowest in the highest water activity system and increased with decreasing water activity until the monolayer (0.33) and then decreased once below the monolayer (0.11). 3.3.2. 2,4-Hexadienal. Losses of 2,4-hexadienal were very rapid with nearly complete loss from the headspace in 20 days. One could not discern any dependency on water activity and the effect of oil in the model system was positive but minimal (data not shovm).

579

100 90

T

MCC

-•-0.11 -^-0.33 -A-0.53 ^^0.76

80 3)70 = 60

150

|\

\/

30 20 10 0 1

\

Days

"

50

•—1 100

Days

50

100

Figure 4. The loss of hexanal at different water activities during storage (45°C, left Figure has no oil; right has oil).

3.3.3. Furfurylmercaptan. Furfurylmercaptan was one of the most labile compounds included in this study (Figure 5). It was nearly completely lost from the MCC sample headspace in 5 days storage at 45°C. The addition of oil to the system provided substantial stability. There is again an influence of water activity with the fiirfuryl mercaptan being most stable at the highest water activities. Losses were too rapid at either storage temperature to determine if they would have been lowest at the monolayer and increased again as is typical of oxidation reactions. 30 25

MCC 45*'C

?20 15

MCC + 5% Oil 45X

-0.11 •0.33 -0.53 -0.76

10 -t

0 l a t f fltf MM^tp Days

50

=M^ 100

Days

50

100

Figure 5. The loss offtirftirylmercaptanat different water activities during storage (45°C, left Figure has no oil; right has oil).

3.3.4. 2-Methylthiophene. While the data for the 0.53 Aw in the MCC system are somewhat erratic, the trend of continuous loss (nearly linear) during storage is evident (Figure 6). There appears to be increased losses with decreasing water activity. The presence of oil in the

580

120 J 100

MCC + 5% Oil

^ * * ^ " ' '

rv5S)>—^ • •

'E

1 *^v5

I 60 0)

—•—0.11 ^ -•-0.33 -A-0.53 -><-0.76

40 20

Days 50

+

100

Days

J

1

50

100

Figure 6. The loss of 2-methylthiophene at different water activities during storage (45°C, left Figure has no oil; right has oil).

system greatly reduced losses from the headspace although they followed similar loss patterns (linear with time). The effect of water activity is clearer in this system. 3.3.5. Diethyldisulfide. Diethyldisulfide (DEDS) losses during storage were somewhat different from compounds discussed to this point. Losses were nearly linear with time and water activity dependent in the MCC system (consistent with other compounds) but extremely stable in the oil containing system (Figure 7). It appears that there was some interaction with the MCC but once the binding sites on the MCC were satisfied, the DEDS was very stable.

140 n 120

MCC 45°C

dlOO !E

80

140 n -4—0.11 -•-0.33 -A-0.53 -^<-0.76

120 0)100 c ;E 80

ieo

1 60

^40

^

MCC + 5% Oil —•—0.11 45"'C -•-0.33 -A-0.53 -K-0.76

^^*MdSf^ ^ ^'m

20

Hi

40 20

1

0 Days

50

^ 100

0 Days

\

1

50

100

Figure 7. The loss of diethyldisulfide at different water activities during storage (45°C, left Figure has no oil, right has oil).

581 3.3.6. 2-Ethyl-3,5-(limethylpyrazine. Except for losses due to initial binding with the MCC, 2-ethyl-3,5-dimethylpyrazine was stable during storage in both model systems (Figure 8). Initial binding was influenced by water activity with a trend of less binding at the higher water activities, increased binding at lower water activities to 0.33 and then decreased binding at 0.11 water activity.

M C C + 5% 45°C

Days

50

100

Days

il —#—0.11 -•-0.33 —^k-0.53 ^«-0.76

100

Figure 8. The loss of 2-ethyl-3,5-dimethylpyrazine at different water activities during storage (45°C, left Figure has no oil, right has oil)

4. DISCUSSION We were rather surprised at the high level and general nature of aroma compound binding exhibited by MCC. We expected to use the MCC as an inert support for various food components and were disappointed to observe its activity in this respect. The binding appears to be strongly dependent on water activity with the binding being less at higher water activities and appears to reach a maximum at a water activity of ca. 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. We were surprised at the very rapid loss of furfurylmercaptan during storage. This bodes badly for the coffee industry since furfurylmercaptan is considered the character impact compound of coffee. Major losses were also observed for the two pyrroles (data not presented). Their loss rates were second only to furfiirylmercaptan. 2,4-hexadienal and ethyl2-mercaptopropionate losses were 3^^^ and 4^^ , respectively. Foods which derive a portion of their characteristic flavor from these compounds would not be expected to have a good shelflife. The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so still rapid that the effect was small. The increase in stability was dependent upon the aroma compound studied. It is clear that oil has a protective effect on the stability of aroma compounds in the headspace above foods. This may translate into an increase in the shelf-life of oil-containing foods depending upon the

582 contribution of oil to lipid oxidation and the masking effect of lipids on both desirable and undesirable aroma constituents.

5. REFERENCES 1

L.C. Hatchwell, In: Flavor-Food Interactions, ACS symposium series 633. R.C. McGorrin and J.V. Leland (eds), ACS, Washington DC, 1996, 15. 2 J. Bakker, In: Ingredient Interactions, Effects on Food Quality, A. Kumar and G. Gaonkar, (EDS), Marcel Dekker, Inc., New York, 1996, 411. 3 L.J. Farmer, D.S. Mottram, and F.B. Whitfield, J. Sci. Food Agric, Vol. 49 (1989) 347. 4 D.A. Forss, J. Agric Food Chem. no. 17 (1969) 681. 5 D.A. Forss, In: Progress In The Chemistry of Fats and Other Lipids. Vol. 13. R.T. Holman(ed), Pergamon Press, London, 1972,177. 6 B.M. King, and J. Solms, J. Agric. Food Chem. Vol. 27 (1979) 133. 7 J.E. Kinsella, In: Flavor Chemistry of Lipids in Foods. DE Min and T.H. Smouse (eds) American Oil Chem. Soc, Champaign. 1989, 376. 8 S. Shamil, L.J. Wyeth, and D. Kilcast, Food Qual. Pref, Vol. 3 (1991) 51. 9 M. Chen, The Stability of Aroma Compounds in the Presence of Food Components during Storage. M.S. Thesis, Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1997. 10. Handbook of Physics and Chemistry. CRC Press, Boca Raton, FL. 1995. 11 T.P. Labuza, Course titled: "Freezing and Dehydration of Food". Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1993, 81.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences 1998 Elsevier Science B.V.

583

The effect of the addition of supplementary seeds and skins during fermentation on the chemical and sensory characteristics of red wines E. Revilla^, J.M. Ryan«, V. Kovac^ and J. Nemanic^ »Departamento de Qiiimica Agricola, Geologia y Geoqmmica, Autonoma de Madrid, 28049 Madrid, Spain.

Universidad

bTehnoloski Fakultet, 21000 Novi Sad, Federal Republic of Yugoslavia ^^Ejnetisjski Institut Slovenije, 1000 Ljubljana, Slovenia

Abstract Several industrial-scale red-tjrpe vinifications were carried out in 1995 to evaluate the effect of different pomace/must ratios on the chemical and sensory characteristics of Merlot and Frankinja wines. Resxilts have shown that the addition of supplementary grape seeds and skins during fermentation increases the levels of total phenoUcs, catechins and dimeric procyanidins, and may stabiUze wine color. The sensory ratings of wines made with higher pomace/must ratios than those conventionally used in red winemaking were similar or higher than those obtained by control wines.

1. INTRODUCTION Grapes contain a remarkable amount of different phenohc compounds, which are located in the different parts of grape cluster (seeds, pulp, skins and cluster stems), and their levels in mature grapes are affected by several agroecological factors [1-4]. PhenoHc compounds of grape pulp are mainly non-flavonoid phenoUcs, especially cinnamic a d d s derivatives, except in grape cultivars known as teinturiers, whose pulp also contains anthocyanins. On the other hand, skins, seeds and cluster stems may contain a large amount of flavonoid phenoUcs. There are important differences in the nature of flavonoids which predominate in each of these three parts of the grape cluster. Seeds usually contain relatively high levels of flavans, especially (+)-catechin, (-)-epicatechin and the procyanidins derived from them. The skins of red cultivars contain a high amount of anthocyanins, but the levels of flavans are lower than in seeds, both in red and in white cultivars. Finally, cluster stems may contain a relatively high amount of flavanoids and, in addition, significant quantities of flavonols. Furthermore,

584 seeds, skins and cluster stems contain other phenolic compounds, such as hydroxybenzoic acids (especially gallic add) and, to a lower extent, cinnamic acid derivatives. Also, skins may contain stUbenoids, especially when grapes are infected by fungi [5-6]. All these molecules possess a relatively low solubihty in water, but their solubihty greatly increases when a small amount of ethanol is present. The structures of some phenohc compounds present in red grapes are given in Figure 1. Typically, red wines are made by fermentation of crushed red grapes, which may be destemmed prior to crushing [7, 8]. During fermentation, the content of ethanol in the hquid phase (must) increases, and a significant amount of grape phenoUcs contained in the soUd phase (skins, seeds and, eventually, cluster stems, which are termed pomace as a collective term) are extracted. As a consequence, the Uquid obtained at the end of fermentation (red wine) contains a remarkable amount of different phenoUc compounds. Usually, the making of red wine involves the fermentation of must with the pomace that naturally occurs in grapes, but it is possible to modify the ratio of must/pomace in order to obtain products which differ in their content of phenoUcs extracted fi'om pomace [9-13]. The making of red wines by maceration of must with a higher quantity of pomace (skins and seeds) than that naturally present in an ordinary red-type vinification has been carried out for years in some places. A special type of dry red wine, known as vino tinto de doble pasta has been traditionally made in several Spanish regions, especially in the East of Spain, using in most cases Gamacha (Grenache) and/or Gamacha Tintorera (Grenache teinturier) grapes [14]. The process of making these red wines involves the maceration of must with a higher quantity of pomace than in ordinary red winemaking. This goal is achieved by adding supplementary pomace to the fermentation tank, or by racking away a certain volume of must some hours after the begining of fermentation, and adding crushed grapes to fill the tank. The term doble pasta makes reference to this technological approach, and may be translated into EngUsh as "double pomace". These type of wines are very deep colored and very rich in dry extract, containii^ an appreciable amoimt of phenoHc compounds. They have been extensively used to improve color and dry extract in red wines made in cooler regions in Spain and abroad. Unfortimately, scientific information about this winemaking process is very scarce. Recently, it has been shown that a winemaking procedure quite similar to that us^c i io make vinos tintos de doble pasta results in wines very rich in phenohc compounds, which contain twice the level of anthocyanins than control wines [12]. Several approaches have been carried out in order to make red wines which may contain a higher amount of flavanoids (catechins and procyanidins) than ordinary red wines [10, 11, 13]. It is reported these wine components may be endowed with protective action towards coronary heart disease, CHD, [15-18] and that they also may play a positive role in human nutrition as quenchers of fi'ee radicals [19-21]. Those approaches were made with several cultivars (Cabernet-Sauvignon, Garnacha, Merlot, Pinot noir, Tempranillo and Vranac) by doubling or tripling the quantity of seeds in pomace during fermentation, but without any change in the quantity of sldns. The experiments led to red wines

585

HO

OH

COOH

I

OH

MO _ ^ ^ C O O H

HO — C — H H _ C — OOC-^V

HO

I COOH

(II)

(I) OCHq OH OCHq

0—Glucosyl

HOi

HO,

OH

(VI)

(VII)

Figijre 1. Structures of several phenolic compounds present in red grapes. (I) caffeoyltartaric acid, (II) gallic acid, (III) malvidin-3-O-glucoside, (IV) (+)-catechin, (V) (-)-epicatechin, (VI) procyanidin Bl, (VII) procyanidin B2

586

with higher levels of catechins and procyanidins than those in control wines. Furthermore, when the quantity of seeds in pomace was doubled, wine color was stabiUzed, as shown by a shght increase in color intensity and free anthocyanin content. In addition, preliminary sensory studies indicated that red wines made by the addition of supplementary seeds usually presented more pronounced variety characteristics and more intense flavor and aroma than the control wines. Of course, "seed-enriched" wines were more tannic than control wines, and this aspect would have to be taken into account for the production of this type of wine at industrial scale, because a pronounced tannic character would be undesirable for consumers. The aim of our research has been to apply the procedure of making red wines by the addition of supplementary quantities of seeds [10, 11, 13] to industrial scale vinifications, but adding only a relatively low quantity of seeds, in order to balance the positive aspects of "seed-enriched" wines, related to their high content of flavanoids, and the possible negative aspects related to their excesive tannic character. At the same time, the effect of adding supplementary quantities of pomace (seeds and skins) has also been investigated.

2. MATERIALS AND METHODS 2.1. Wlnemaking procedures and general analyses Several industrial-scale winemaking experiments were carried out in 1995 in Agroind 1894 Cellars, Primorje, Slovenia, with Merlot grapes, and in Metlika Cellars, Posavje, Slovenia, with Frankinja grapes. Both cultivars are usually grown in Slovenia. Details of these experiments, named Me-A, Me-B and Me-C for Merlot, and Fr-A and Fr-B for Frankinja, are displayed in Tables 1 and 2. Sugar content of must, determined by refractometry, was 226 g/L for Merlot and 193 g/L for Frankinja. Merlot wines were made in rotary horizontal tanks and Frankinja wines in vertical tanks. In each experiment, the fermentation temperature was 28 "^C, and the must was pumped over the cap for an hour, four times a day.

Table 1 Details of winemaking experiments with Merlot grapes Experiments

Length of maceration (hovirs) Volume (1000 L) Supplementary pomace added

Me-A

Me-B

Me-C

92 37 -

92 37 25% seeds

144 37 30% skins and seeds

587 Table 2 Details of winemaking experiments with Frankinja grapes Experiments

Lengthof maceration (hours) Volume (1000 L) Supplementary pomace added

Fr-A

Fr-B

92 24 -

144 24 30% skins and seeds

When maceration was finished, fi-ee run wine (92%) and press wine (8%) were obtained. These wines allowed us to prepare four different Merlot wines and three different Frankinja wines, which were submitted to sensory analysis in November 1995 : * Merlot wines : - M e - 1 : fi-ee run wine and press wine fi*om experiment Me-A - Me-2 :fi^eerun wine fi-om experiment Me-B - Me-3 : 6*00 run wine firom experiment Me-C - Me-4 : press wines fi'om experiments Me-B and Me-C * Frankinja wines : - Fr-1 : fi-ee run wine and press wine fi-om experiment Fr-A - Fr-2 : firee rim wine firom experiment Fr-B - Fr-3 : press wine fi'om experiment Fr-B In March 1996, two coupages, Me-5 and Fr-5, were prepared. Wine Me-5 was prepared with wines Me-2 (46%), Me-3 (46%) and Me-4 (8%), and wine Fr-5 with wines Fr-2 (92%) and Fr-3 (8%), respectively. Afterwards, those coupages and wines Me-1 and Fr-1 were submitted to sensory analysis. The composition of these wines is presented in Tables 3 and 4. The analyses of alcohoUc degree, total acidity, volatile acidity, dry extract, reducing sugars, total SO2 and free SO2 were performed by official O.I.V. methods [22].

2.2. Color measurements and analyses of phenolic compounds Color measurements were carried out by the procedure described by Sudraud [23], that allows the calculation of color intensity and tint or hue. Free anthocyanins were measured by the procedure described by Ribereau-Gayon and Stonestreet [24], and total phenoHcs by colorimetry after their oxidation with phosphomoUbdic and phosphotungstic acids in basic medium [25]. Catechins and dimeric procyanidins were measured by HPLC after the fractionation of wine phenoUcs, following the procedures previously described [26, 27

588

Table 3 Composition of Merlot wines Wines

Alcoholic degree Total acidity (g/L) Volatile acidity (g/L) Dry extract (g/L) Reducing sugars (g/L) Total SO2 (mg/L) Free SO2 (mg/L)

Me-1

Me-2

Me-3

Me-4

Me-5

11.80 7.1 0.41 26.97 4.3 67 19

11.88 7.3 0.28 29.42 4.8 70 23

11.15 6.9 0.31 32.34 4.7 57 21

11.72 7.0 0.47 31.76 5.0 59 20

11.53 6.9 0.30 30.27 5.5 68 22

Table 4 Composition of Frankinja wines Wines

Alcoholic degree Total acidity (g/L) Volatile acidity (g/L) Dry extract (g/L) Reducing sugars (g/L) Total SO2 (mg/L) Free SO2 (mg/L)

Fr-1

Fr-2

Fr-3

Fr.4

10.47 8,0 0,41 27.84 2,6 47 15

10.31 7,5 0.51 25.16 2.7 62 10

10.68 7.7 0.38 29.68 3.8 52 15

10.32 6.6 0.54 26.86 4.0 65 30

2.3. Sensory analysis Sensory evaluation of wines was carried out by eight professional paneHsts, using the quantitative descriptive analysis proposed by W. Buxbaum for red wines [28], officially adopted in Slovenia. This procedure involves the evaluation of color and limpidity (four points), smell (foiu: points) and taste (12 points), with a total maximimi score of 20 points. The first evaluation was carried out in November 1995, for wines Me-1, Me-2, Me-3, Me-4, Fr-1, Fr-2 and Fr-3. The second evaluation was carried out for Merlot wines Me-1 and Me-5, and for Frankinja wines Fr-1 and Fr-5. One-way analyses of variance of the numerical

589 data obtained in the second sensory evaluation were carried out using the program MICROSOFT EXCEL 7.0 (Microsoft Co. U.S.A.).

3. RESULTS AND DISCUSSION 3.1. Phenolic compounds The addition of supplementary pomace during fermentation clearly affects the content of phenoUc compounds in wines, as reflected in Table 5 for Merlot and in Table 6 for Frankinja. As can be noted, the effects of the different winemaking experiments on the content of free anthocyanins are different than those observed for total phenoUcs, catechins and dimeric procyanidins.

Table 5 Content of phenohc compounds in Merlot wines

Free anthocyanins (mg/L) Total phenoUcs (mg/L) (+)-catechin (mg/L) (-)-epicatechin (mg/L) Frocyanidin B l (mg/L) Procyanidin B2 (mg/L) Total flavanoids* (mg/L)

Me-1

Me-2

Me-3

Me-4

Me-5

440 2150 39 26 24 17 106

463 2400 47 30 33 16 126

398 2680 89 73 40 23 225

323 3340 48 32 33 31 144

386 2500 63 44 35 27 169

''sum of (+)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2

Table 6 Content of phenoUc compounds in Frankinja wines

Free anthocyanins (mg/L) Total phenoUcs (mg/L) (+)-catechin (mg/L) (-)-epicatechin (mg/L) Procyanidin B l (mg/L) Procyanidin B2 (mg/L) Total flavanoids* (mg/L)

Fr-1

Fr-2

Fr-3

Fr-5

502 1770 33 21 26 13 93

585 2300 53 41 44 29 167

404 2960 55 45 53 54 207

463 2710 53 42 44 31 170

*sum of (+)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2

590 3.1.1. Free anthocyanins In Merlot wines (Table 5), the addition of supplementary seeds during fermentation led to a slight increase in the levels of free anthocyanins, as reflected by the results corresponding to wines Me-1 (control wine) and Me-2 (free run wine from experiment Me-B). This effect was observed in previous experiments doubling the quantity of seeds during fermentation [11, 13]. Nevertheless, wine Me-3 (free run wine from experiment Me-C) contained less free anthocyanins than control wine (Me-1). This effect may be explained since maceration was longer in the experiment Me-C (144 hours) than in the experiment Me-A (92 hours). It is weU known that the content of free anthocyanins in wine usually increases in the early stages of fermentation, reaching a maximimi between two and seven days after the process has begun, and then decreasing for several reasons [8]. The levels of anthocyanins in wine Me-4 (press wine from experiments Me-B and Me-C) were lower that in control wine (Me-1) and in free run wines from experiments Me-B and Me-C (Me-2 and Me-3), as would be expected. That wine (Me-4) contained higher levels of total phenoUcs than control wine and free run wines. In press wine, the combination of anthocyanins with flavanoids to give polymeric phenols could have probably taken place to a higher extent that in control wine and in free run wines. Finally, the content of free anthocyanins in wine Me-5 was lower than in control wine, and reflects that this wine was prepared by coupage of wines Me-2, Me-3, and Me-4. In the case of Frankinja wines (Table 6), the addition of supplementary skins and seeds (experiment Fr-B) resulted in a free run wine (Fr-2) which contained a higher amount of free anthocyanins than control wine (Fr-1), despite the longer maceration in the experiment Fr-B (144 hours) than in the experiment Fr-A (92 hours). This effect is different than that observed for Merlot wines Me-1 and Me-3. The difference in the degree of maturity and in varietal characteristics of each cultivar may be responsible for that effect, as the processing technology appUed was the same for Merlot and Frankinja [29]. 3.1.2. Total phenolics The addition of supplementary pomace led to higher contents of total phenoUcs in free r u n wines from experiments Me-B, Me-C and Fr-B than in Merlot and Franldnja control wines (Tables 5 and 6). As would be expected, the levels of total phenoUcs in press wines (Me-4 and Fr-3) were higher than in the corresponding free run wines (Me-2 and Me-3 for Merlot, and Fr-2 for Frankinja). The levels <^ total phenoUcs in coupage wines (Me-5 and Fr-5) were quite cimilar to the expected, considering the content of total phenoUcs of the wines involved in the coupages. 3.1.3. Catechins and dimeric procyanidins The content of (+)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2 in Frankinja and Merlot wines, and the amoimt of total flavonoids, considered as the sum of those four substances, are displayed in Tables 5 and 6. As it was

591

expected, the levels of these molecules in free run wines (Fr-2, Me-3 and Me-4) and in press wines (Fr-3 and Fr-4), made with supplementary quantities of pomace, were higher than in control wines (Fr-1 and Me-1). Wines prepared by coupage of wines made with supplementary quantities of pomace (Fr-5 and Me-5) also contained higher levels of catechins and procyanidins than control wines (Fr-1 and Me-1). In most cases, these results may be easily explained by the differences in the quantity of pomace in contact with must and in the length of maceration in different experiments, as shown in Tables 1 and 2. As has been previously reported [11], the extraction of catechins and procyanidins may be considered linear throughout the first seven days after the beginning of maceration. In the case of Merlot wines, the content of catechins and procyanidins in wine Me-2 would be about 25% higher than in wine Me-1, because the maceration lasted the same time (92 hovirs) and the amount of seeds in contact with must was 25% higher for wine Me-2 than for wine Me-1. This means that the theoretical contents of (+)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2 in wine Me-2 would be 49, 32, 30 and 21 mg/L. These theoretical values are quite similar to the experimental results for wine Me-2 (Table 5). A similar comparison can be made for wines Me-3 and Me-1, considering that for wine Me-3 maceration was longer (a factor of 1.55) and must was put in contact with more pomace (a factor of 1.3). For these reasons, the content of catechins and procyanidins in wine Me-3 should be approximately double that in wine Me-1, and the theoretical contents for (-i-)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2 would be 78, 52, 48 and 34 mg/L. Surprisingly, if these theoretical values are compared with the experimental results for wine Me-3 (Table 5), the contents of (+)-catechin and (-)-epicatechin are higher than the expected, and the contents of procyanidin B l and procyanidin B2 are lower than the theoretical data. This phenomena coxild be probably explained as a consequence of partial depolimerization of dimeric procyanidins B l and B2, leading to higher contents of monomeric flavanoids, especially in the case of (-)-epicatechin. For Frankinja wines, it could be expected that the content of catechins and procyanidins in wine Fr-2 was approximately double than in wine Fr-1, as there was more pomace in contact with must (a factor of 1.3) and maceration was longer (a factor of 1.5) for wine Fr-2, Then, the theoretical contents of (+)-catechin, (-)-epicatechin, procyanidin B l and procyanidin B2 in wine Fr-2 would be 66, 42, 52 and 26 mg/L. Although the theoretical contents of (-)-epicatechin and procyanidin B l are quite close to the experimental values found for those molecules in wine Fr-2 (41 and 29 mg/L, respectively), the concentrations of (+)-catechin and procyanidin B l in wine Fr-2 are lower than the theoretical data obtained by a factor of 2.0. The concentrations of catechins and dimeric procyanidins in press wines from experiments carried out by adding supplementary pomace (Me-4 and Fr-3) were quite similar to that expected, especially for Frankinja. Finally, the contents of these substances in coupage wines (Me-5 and Fr-5) were quite similar to the

592 expected, taking in account their contents in the wines used for preparing those coupages. 3.2. Color measurements Color intensity of Merlot wines was affected by the addition of supplementary pomace during fermentation, as shown in Table 7. This effect is similar to that previously observed for wines made by doubling or tripling the quantity of seeds in contact with must during fermentation [11, 13]. Tint values were considerably different, and easily explained by differences in the contents of free anthocyanins, catechins and dimeric procyanidins of these wines. Consequently, wine Me-2, that contained the highest amount of anthocyanins and a relatively low content of catechins and dimeric procyanidins, presented the lowest tint, the situation being the opposite to that of wine Me-4, whose tint was the highest in the series. In the case of Frankinja, color intensity was quite similar for each wine, as shown in Table 8. Once again, the values of tint were considerably different, but these differences may be easily explained taking into account the contents of free anthocyanins and flavanoids in each wine.

Table 7 Color measurements in Merlot wines Wines

Color intensity (E420 + E520) Tint (E420/E520)

Me-1

Me-2

Me-3

Me-4

Me-5

6.70 0.63

9.35 0.48

8.65 0.66

8.20 0.73

8.90 0.58

Table 8 Color measurements in Frankinja wines Wines

Color intensity (E420 + E520) Tint (E420/E520)

Fr-1

Fr-2

Fr-3

Fr-5

10.30 0.45

10.60 0.58

10.80 0.71

10.60 0.59

593 3.3. Sensory analysis Table 9 displays the mean total scores obtained by four Merlot wines and three Frankinja wines in the sensory evaluation carried out in November 1995, which is some weeks after the end of fermentation. As it can be noted, scores were quite similar for the different Merlot wines, although several panehsts considered that free run wines made by the addition of supplementary pomace (Me-2, and Me-3) presented a more complex aroma and a higher tannic character than control wine (Me-1). Similar observations had been noticed in the preUminary sensory evaluations of a nxmiber of experimental Garnacha, Tempranillo and Vranac wines made by the addition of supplementary seeds, as previously reported [10, 11, 13]. Fxnrthermore, the higher tannic character observed in wines Me-2 and Me-3 in relation to control wine (Me-1) is weU correlated with certain results obtained by chemical analysis, such as the contents of total phenoUcs, catechins and dimeric procyanidins, as shown in Table 5. As expected, wine Me-4, that consisted in the press wine obtained in the experiments Me-B and Me-C, presented an agressive aroma and was too astringent. In the case of Frankinja wines, the situation was qvdte similar. Free run wine from experiment Fr-B (wine Fr-2) was considered more tannic than control wine (Fr-1). Nose and palate characters of wine Fr-3, which was the press wine from the experiment Fr-B, were too intense. Palate characters of those Frankinja wines are well related to their content of total phenoUcs, catechins and dimeric procyanidins, that are displayed in Table 6. Wines Fr-1 and Fr-2 obtained similar mean total scores, that were relatively higher than those obtained in wine Fr-3, as would be expected for a press wine.

Table 9 Sensory analysis, November 1995. Cultivar

Wine

Total score

Observations

Merlot

Me-1 Me-2 Me-3 Me-4

16.6 16.8 16.9 16.4

Fruity aroma, low tannic taste Complex aroma, strong tannic taste Complex aroma, smooth tannic taste Hard aroma, astringent

Frankinja

Fr-1 Fr-2 Fr-3

16.7 16.6 16.1

Fruity aroma, low tannic taste Tannic character predominates Very hard aroma, astringent

Table 10 displays the mean scores for color and limpidity (eye score), aroma (nose score) and taste (palate score) and the mean total score for Merlot wines Me-1 and Me-5, and Frankinja wines Fr-1 and Fr-5 for the sensory evaluation

594 carried out in March 1996, five months after the end of fermentation. For each pair of wines, mean scores were higher for wines prepared by mixing firee run and press wines fi-om vinifications with supplementary quantities of pomace (Me5 and Fr-5) than for the corresponding control wines (Me-1 and Fr-1), Statistical analysis showed that there were not significant differences between Merlot wines, although there were significant differences between Frankinja wines for color and Hmpidity (p<0.01), taste (p<0.05) and total score (p<0.01). Several paneUsts reported that wines Me-5 and Fr-5 presented a more complex (varietal) aroma and were more bodied than control wines. These results confirm previous sensory observations in wines made by the addition of supplementary quantities of seeds, and support the hypothesis that the adddition of supplementary pomace for making red wines may improve their sensory caracteristics [10, 11, 13]. In addition, the differential palate characters of each pair of wines (Me-1 and Me-5, and Fr-1 and Fr-5) are related to the results obtained for chemical analysis of phenoHc compounds, as reflected in Tables 5 and 6.

Table 10 Sensory analysis, March 1996 Cultivar

erlot

ankinja

Wine

Mean scores

Observations

Eye

Nose

Palate Total

Me-1

3.4NS

3.3NS

10.3NS ijQm

Me-5

3.6NS

3.3NS

Fr-l

2.2**

2.9NS

10.5*

Fr-5

3.4**

2.9NS

10.9*

Pleasant aroma, mediiun to full-bodied 10.5NS 17.4NS Varietal, pleasant aroma ; full-bodied, tannic 15.6** Single, pleasant aroma; fi*esh, lack of quaUty 17.2** Varietal, pleasant aroma; medium to full-bodied.

Levels of significance : NS=no significant, p<0.05*, p<0.01*

4. CONCLUSIONS The object of this study has been to obtain red wines with a higher content of phenoUcs and with similar sensory properties as those of the wines made by a classic vinification. The experiments made with Merlot and Frankinja grapes, adding supplementary quantities of pomace, have shown that fi'ee run wines obtained by this technology contained a higher amount of total phenoUcs and flavanoids than control wines. The procedures assaied may result in a sUght

595 decrease in the levels of free anthocyanins, but color intensity of wines does not decrease. Sensory evaluation of wines showed that the addition of supplementary pomace (seeds, or skins and seeds), even when performed for an extended maceration, did not affect negatively the descriptions made by paneUsts in comparison with control wines. Coupage wines prepared by blending several free run and press wines made by the addition of supplementary pomace obtained better scores than control wines when evaluated by profesional panehsts, being more complex in nose and having improved their varietal characteristics. All these results point out that the addition of supplementary pomace during red wine fermentation may be a promising technology for obtaining wines with a higher content of several phenohc compounds, and for improving their sensory characteristics.

5. ACKNOWLEDGEMENTS Authors are grateful to Agroind 1894 Cellars, Primorje, Slovenia, and Mehtka Cellars, Posayje, Slovenia, for winemaking faciUties. J.M.R. is a recipient of the financial support of Grupo Iberdrola, S.A., Bilbao, Spain.

6. REFERENCES 1 2 3 4

5 6 7 8 9 10 11 12 13

V.L. Singleton and P. Esau, Phenohc Substances in Grapes and Wines, and Their Significance, Academic Press, New York, 1969. M. Bourzeix, D. Weyland and N. Heredia, BuU. OIV, 59 (1986) 1325. J.J. Macheix, A. Fleuriet and J. Billot, Fruit PhenoHcs, CRC Press, Boca Raton, FL., 1990 E. Revilla, E. Alonso and V. Kovac, in : T.R. Watkins (ed.). Wine : Nutritional and Therapeuthic Benefits, ACS Symposium Series 661, American Chemical Society, Washington, D.C., 1997, pp. 69-85. E. Sieman and L.L. Creasy, Am. J. Eno. Vitic, 43 (1992) 49. P. Jeandet, R. Bessi, M. Sbaghi, P. Meunier and P. TroUat, Am. J. Enol. Vitic. 46 (1995) 1. R.S. Jackson, Wine Science : Principles and Apphcations. Academic Press, New York, 1994. R.B. Boulton, V.L. Singleton, L.F. Bisson and R.E. Kunkee, Principles and Practices of Winemaking, Chapman and Hall, New York, 1995. 0 . Lamikanra and D. Garhck, Food Chem., 26 (1987) 245. V. Kovac, M. Bourzeix and E. Alonso, C. R. Acad. i ^ c . Fr., 77 (1991) 121. V. Kovac, E. Alonso, M. Bourzeix and E. Revilla, J. Agric. Food Chem., 40 (1992) 1953. V. Katahnic and G. Konja, 4 * International Symposium on Innovations in Wine Technology, Stuttgart, 1995, pp. 67-78. V. Kovac, E. Alonso and E. Revilla, Am. J. Enol. Vitic, 46 (1995) 363.

596

14 J. Mardlla, Tratado Practice de Viticultura y Enologia Espaiiolas, Tomo II, Enologia, Sodedad Espaiiola de Traductores y Autores, Madrid, 1974. 15 E.N. Frankel, A.L. Waterhoixse and P.L. Teissedre, J. Agric. Food Chem, 43 (1995) 890. 16 C.R. Pace-Asciak, S. Mahn, E.P. Diamandis, G. Soleas and D.M. Goldgerb, Clin. Chim. Acta, 235 (1995) 207. 17 J.C. Riif, J.L. Berger and S. Renaud, Artherioscler. Thromb. Vase. Biol., 15 (1995) 140. 18 J.A. Vinson, Y.A. Dabbach, M.M. Serry and J. Jang, J. Agric. Pood Chem., 43 (1995) 2800. 19 T. Ariga and M. Hamano, Agric. Biol. Chem., 54 (1990) 2499. 20 C.A. Rice-Evans, N.J. Miller, P.G. Bolwell, P.M. Bramley and J.B. Pridham, Free Rad, Res., 22 (1995) 375. 21 K. Satoh and H. Sakagami, Anticancer Res., 16 (1996) 2885. 22 Office Internationale de la Vigne et du Vin, Recueil des methodes nternationales d'analyse des vins et des mouts, Paris, 1990. 23 P. Sudraud, Anal. Technol. Agric, 7 (1958) 203. 24 P. Ribeerau-Gayon and E. Stonestreet, Bull. Soc. Chim. Fr., 9 (1965) 2649. 25 V.L. Singleton and J.A Rossi, Am. J. Enol. Vitic, 16 (1965) 144. 26 E. Revilla, E. Alonso, M. Bourzeix and N. Heredia, in : G. Charalambous (ed.). Flavours and Off-Flavors '89, Elsevier Science PubUshers, Amsterdam, 1990 27 E. Revilla, M. Bourzeix and E. Alonso, Chromatographia, 31 (1991) 465. 28 W. Buxbaum, Deutsche Weinbau (1951) 203. 29 C.W. Nagel and L.W. Wulf, Am. J. Enol. Vitic, 30 (1979) 111.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

597

Role of phenolics inflavorof rapeseed protein products M. Naczk*, R. Amarowicz^, and F. Shahidi'' ""Department of Human Nutrition, St. Francis Xavier University, P.O. Box 5000, Antigonish, Nova Scotia, B2G 2W5, Canada ^Division of Food Science, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland ''Department of Biochemistry, Memorial University of Newfoundland, St. John's, NF, AlB 3X9, Canada Abstract Utilization ofrapeseed/canola as a source offood-grade proteins is still limited by the presence of glucosinolates, phytates, hulls, and phenolics. Phenolic acids and condensed tannins are the predominant phenolic compounds found in rapeseed. The content of phenolic compounds in rapeseed/canola products is 4-3 0 times higher than that found in corressponding products obtained from other oleginous seeds. Contribution of free phenolic acids, sinapines and condensed tannins to the bitter taste and astringency of rapeseed products is important. In addition, both phenolic acids and condensed tannins may form complexes with proteins. Better understanding of factors influencing the interactions between phenolics and proteins would be beneficial in developing more efficient technological procedures for the production of phenolic-free rapeseed/canola protein products. 1.

INTRODUCTION

Rapeseed/canola are conventionally processed to oil and feed-grade meal by employing an extraction process which is an adaptation of soybean technology adjusted to small seed size, high oil content, and the presence of myrosinase [ 1 ], an enzyme which degrades glucosinolates to toxic products such as isothiocyanates, nitriles, and thiocyanates [2-4]. The use of rapeseed meal as a source of protein for human consumption has been considered for many years because the meal obtained after oil extraction contains approximately 40% protein (Nx6.25), up to 10% non-protein nitrogen, and about 7% ash [5, 6]. Futhermore, rapeseed meal has a reasonably well balanced amino acid content [7] which is not affected by the handling and processing conditions [8]. In addition, the protein efficiency ratio (PER) ofrapeseed meal was found to be 2.64 as compared to 2.19 for soybean meal [9]. The acceptability of oilseed protein products such as meal, protein isolate, and protein concentrate depends not only on their nutritive value but also on theirfiinctionalproperties and sensory quality. The functional properties ofprotein products include their water absorption (WA), oil absorption (FA), nitrogen solubility index (NSI), emulsifying capacity, emulsion stability (ES),

598 whippability and foam stability. The WA of commercial canola meal was found to be similar to that of soybean meal, but it was 15-37% lower than that of laboratory prepared canola meal. Moreover, canola meals absorbed up to 80% more soybean oil than soybean meals, but exhibited similar emulsifying properties to those of soybean meals (Table 1). These functional properties suggest that rapeseed/canola meals could be used as binders in meat products and as extenders for meat proteins [6]. Although the proximate composition, nutritive value and functional properties ofrapeseed products are comparable to corresponding soybean products, the use of rapeseed/canola protein products as a food component is still limited by the presence of undesirable components such as glucosinolates, phytates, hulls (fiber) and phenolics.

Table 1 Functional properties of canola and soybean meals Meal Canola Varieties Tower Regent Candle Altex Commercial canola Soybean I Soybean II " oil is released Adapted from: M. Naczk,

NSI (%)

WA (%)

FA (%)

ES (%)

25.3 23.5 17.5 27.6 13.7 16.3 15.5

370 343 383 377 278 311 301

188 203 190 219 134 105 96

108 102 106 93 79a 103^

99a

L.L. Diosady, and L.J. Rubin, J. Food Sci. 50, 1985, p. 1687.

Over the last two decades the composition of rapeseed has been significantly altered by both Canadian and European breeders who developed new low glucosinolate and low erucic acid rapeseed varieties. Glycosinolates upon hydrolysis produce nitriles, hydroxynitrites, isothiocyanates and thiocyanates which are responsible for goitrogenic effects. Erucic acid in the oil may cause heart lesion in certain experimental animals. In Canada, these new varieties are known as canola [10]. Nonetheless, these improved varieties of rapeseed still contain too high levels of glucosinolates to be considered as a suitable source of protein for human consumption. A number of chemical, microbial and physical as well as combination of treatments have been developed to reduce the content of glucosinolates in meals or seeds to negligible levels [2, 11, 12]. The rapeseed/canola products contain up to 4.1 % phytates [13]. Phytates are responsible for the decrease in the bioavailability of mono- and divalent cations due to complex formation [1418]. In addition, phytates are reported to delay the digestion of starch [19]. Because of this, a number of methods have been developed to remove phytic acid from rapeseed products and these have been thoroughly discussed by Thompson [20]. On the other hand, some recently published studies indicate that phytates, at low concentrations, may possess antioxidative [21] and anticarcinogenic effects [19]. Rapeseed meal contains 20-3 0% indigestible hulls, on a dry weight basis [22-24]. The rapeseed hulls consist of low-molecular-weight carbohydrates, polysaccharides, pectins, cellulose, lignin,

599 as well as proteins, polyphenols, glucosinolates and minerals. High levels of hulls limit the use of meal as feed due to lowering of its metabolizable energy [25-27] and cx)ntribute to the unattractive appearance of products containing rapeseed meal. A number of procedures for dehuUing rapeseed/canola have been proposed [28-33]. However, these methods are still not very efficient and therefore dehuUing is not a standard practice in canola processing and crushing industries. The content ofphenolic compounds in rapeseed/canola products is much higher than that found in corresponding products from other olegineous seeds. Therefore, phenolics are thought to be responsible for the dark color, undesirable flavor and lower nutritional value of rapeseed products. However, some published data also implicate residual solvent [34], residual oil [35], free amino acids [36, 37] and glucosinolates and their breakdown products [38] as contributors to the objectionable taste of rapeseed/canola products. This contribution discusses possible roles of phenolics in the flavor attributes of rapeseed/canola products.

2.

PHENOLIC ACIDS

Rapeseed/canola phenolic acids are derivatives of benzoic and cinnamic acids (Figure 1). Of these, sinapic acid is the predominant phenolic acid found in rapeseed/canola varieties. Phenolic acids are present in seeds and in corresponding protein products in the free, esterified and bound forms. The content of phenolic acids in rapeseed/canola meals is up to five times higher than those found in soybean meals. The content of phenolic acids in rapeseed flours is 10-30 times higher than that found in flours obtained from other oleginous seeds. Free and esterified phenolic acids are considered to be the principal contributors to the objectionable taste of rapeseed/canola products.

w HO-/

\-COOH

X

Y

HO - \ Z

V - C H =: CH — COOH

Acid

W

Protocatechuic

H

OH

Vanillic

OCH3

H

Syringic

OCH3

OCH3

Gallic

OH

OH

p-Hydroxybenzoic

H

H

Acid

Y

Z

p-Coumaric

H

H

Caffeic

H

OH

Ferulic

H

OCH3

Sinapic

OCH3

OCH3

Figure 1. Structures of phenolic acids found in canola and rapeseed.

600 2.1 Free Phenolic Acids Free phenolic acids constitute up to 24% ofthe total phenolic acids present in rapeseed/canola meal andfloursofdifferent cultivars [3 9,40] and approximately 15 % ofthe total phenolics present in rapeseed/canola meals (Table 2) [41]. Rapeseed protein products contain sinapic,/?-hydroxybenzoic, vanillic, gentisic, protocatechuic, syringic,/?-coumaric, cis- and trans-iQmXxc, caffeic and chlorogenic acids in thefreeform (Table 3) [40,42]. Ofthese, sinapic acids constitute 70.2 - 85.4% of the total phenolic acids present [41].

Table 2 Content of free and esterified phenolic acids of some rapeseed product (mg/lOOg of product, on dry basis) Product

Free

Esterified

Total

244 1202 1542 Tower meal' 262 1470 1837 Regent meal' 1458 1807 248 Altex meal' 98.2 Tower flour*' 982 1080.2 1196 84.5 1280.5 Candle flour^ 71.8 700 776.5 Start flour^ ' adapted from: M. Naczk and F. Shahidi, Food Chemistry, 31, 1989, p. 162. ^ adapted from: K. Krygier, F. Sosulski, and L. Hagge, J. Agric. Food Chem. 30, 1982, p. 335. "" adaptedfrom:H. Kozlowska, D. A. Rotkiewicz, and R. Zademowski, J. Am. Oil Chem. Soc. 60, 1983, p. 1121. Table 3 Free phenolic acids in rapeseed flours (ppm) Phenolic acid

Candle'

Tower'

Start^

Bronowski*'

22 5 6 p-hydroxybenzoic trace 8 6 3 3 vanillic trace 8 2 4 gentisic 14 5 6 protocatechuic 24 3 15 6 syringic 39 8 31 11 coumaric 47 18 68 33 ferulic 18 4 6 3 caffeic 517 523 739 891 sinapic ' adapted from: K. Krygier, F. Sosulski, and L. Hogge, J. Agric. Food Chem. 30, 1982, p. 335. ^ adaptedfrom:H. Kozlowska, D. A. Rotkiewicz, and R. Zademowski, J. Am. Oil Chem. Soc. 60, 1983, p. 1121. Rackis et al. [43] suggested that mixtures of phenolic acids found in soy products in trace amounts possess typical soyflavorbut pure phenolic acids were, according to them, almost tasteless.

601 On the other hand, Arai and co-workers [44] described the flavor of phenolic acids as sour, astringent, bitter and phenol-like. Later, Maga and Lorenz [45] determined the taste thresholds of 30 individual phenolic acids soluble in distilled water. The taste thresholds of phenolic acids present in oilseeds, including rapeseed, were in the range of 30 to 240 ppm. Taste threshold of sinapic acid was not determined as it was insoluble in water at concentrations required for testing. Combination of two to four phenolics resulted in much lower flavor thresholds (Table 4). These authors also sugested that combinations of seven or more phenolic acids found in oilseeds should result in lower flavor thresholds. The taste thresholds of phenolic acids are also affected by the solvent used. Dadic and Belleau [46] reported thatflavorthresholds ofphenolic acids in 5% aqueous ethanol ranged from 2 to 10 ppm, while thresholds for phenolic acids dissolved in beer ranged from 10 to 50 ppm.

Table 4 Flavor thresholds ofindividual phenolic acids and their combinations used at a 1:1 (w/w) ratio (ppm) Phenolic Acids

Individual Thresholds

Combination Thresholds

30 Protocatechuic 40 Gallic 30; 40 Vanillic + p-hydroxybenzoic 90; 40 Ferulic + p-coumaric 90; 90 Ferulic + gentisic 90; 90; 90 Ferulic + gentisic + caffeic 90; 90; 90; 240 Ferulic + gentisic + caffeic + syringic Adapted from: J.A. Maga and K. Lorenz, Cereal Sci. Today, 18, 1973, p. 328.

_ 10 25 80 60 95

Rapeseed/canola meals contain over 2000 ppm offree phenolic acids [41,47], while for flours, the reported values range from trace to about 1000 ppm [40,42]. The published data indicate that individual phenolic acids are present in rapeseed flours at subthreshold levels (see Table 4). According to Maga and Lorenz [45] all phenolics acids present in oilseeds possess astringent flavor characteristics and their combination results in synergism. In addition, Bartoschuk and Cleveland [48] demonstrated the existence of synergism for bitter stimuli present in quaternary mixtures at both subthreshold and suprathreshold levels. Thus, the results of these studies strongly suggest that although the presence of individual free phenolic acids in rapeseed products is at subthreshold levels, their contribution to the objectionable taste of rapeseed meals, due to a synergistic effect, can not be ignored. 2.2 Esterified Phenolic Acids Esterified phenolic acids constitute up to 80% of the total phenolic acids. Total content of phenolic acids liberated from esters ranged from 520 to 1196 mg per lOOg of flours [42] and up to 1458mgper 100 g ofmeal [41]. Sinapic acid was the predominant phenolic acid found in alkaline hydrolyzates of soluble esters extracted from Tower and Candle canola flours [40]. It constituted 70.9 - 96.7% ofthe phenolic acids liberated from esters [47]. Small quantities of/?-hydroxybenzoic.

602 vanillic, protocatechuic, syringic, /7-coumaric, cis- and trans-fQvwXic and cafFeic acids were also reported in soluble phenolic esters fraction [40]. Fenton et al. [49] demontrated the presence of at least seven phenolic esters soluble in 70% acetone extracts ofrapeseed meals ofMidas and Echo varieties that upon hydrolysis yielded sinapic acid. Moreover, a number of phenolic esters and glycosides have been isolated and identified in rapeseed/canola. Ofthese, the sinapines, choline esters of phenolics, were the predominant soluble esters ofphenolic acids found in rapeseed/canola varieties [50]. The chemical structure of sinapines found in rapeseed/canola is shown in Figure 2. In addition, the presence of methyl esters o^cisand trans-^QwXic acids was confirmed by mass spectroscopy [51]. Flours ofPolish rapeseed varieties contained 0.16 - 0.65 ^mol phenolic acid glycosides/g flour [52]. According to Wanasundara et al. [53], Amarowicz and Shahidi [54] and Amarowicz et al. [55], 1-0-13-D glucopyranosyl sinapate was the predominant glycoside in rapeseed/canola varieties. Moreover, presence of flavonoid glycosides containing sinapic acid bound to aglycone was reported by Durkee and Harbome [56]. Two such glycosides, namely 3-(0-sinapoyl sophoroside)-7-0-glucoside of kaempferol and 3-(0-sinapoyl glucoside)-7-0-sophoroside of kaempferol have been identified in rapeseed meal [57].

z Y —^

\ -

^CH« + / 3 OH = CH — c — CHp — CHg — N — CH3

II X

0

Sinapines

X

Y

Coumaroylcholine

H

OH

H

Feruloylcholine

H

OH

OCH3

Isoferuioylcholine

H

OCH3

OH

Sinapine

OCH3

OH

OCH3

Sinapine glucoside

OCH3

0-Glu

OCH3

Z

Y ^CH« X —^

\ - C — 0 — CHg — CHg -- N — CH« CH3 0

Sinapines

\ "^

X

Y

4-Hydroxybenzoylcholine O H

H

Hesperaiin

OCH3

0CH3

Figure 2. Structures of sinapines found in canola and rapeseed.

603 Sinapine, a choline ester of sinapic acid, the most abundant phenolic ester present in rapeseed/canola products, is both an astringent and a bitter tasting phenolic compound. Because of this, it is considered to be a major contributor to the objectionable taste of rapeeseed/canola protein products [58-60]. According to Mueller etal. [61] the sinapine content of 5. napus cultiwavs was 1.65 - 2.26%, while that ofB. campestris cultivars was 1.22 - 1.54%. These authors found a statistically significant (P<0.01) difference in sinapine content between these two rapeseed cultivars. Later, Clausen [62] reported much lower levels of sinapine in both B. napus and B. campestris cukivars. The diversity in the reported sinapine contents may not only be affected by the differences in cultivar and growing conditions, but possibly due to the existing differences in solvent systems employed for extraction and methods used for their quantification. Larsen et al. [63] suggested that other sinapines (see Figure 2) may also contribute to objectionable taste of rapeseed/canola protein products. According to Durkee and Thivierge [64], sinapine is susceptible to both enzymatic (B-glucosidase) and alkaline hydrolysis, thus producing choline and sinapic acid. Free choline was found in both rapeseed flour and rapeseed protein concentrate [36]. This indicates that some hydrolysis of sinapine takes place during processing and handling of rapeseed. It has been reported that 0.1% choline solution was sligthly bitter in taste [65]. Therefore, the free choline may also contribute to undesirable taste of rapeseed/canola products. Ismail et al. [36] used the magnitude estimation test to evaluate the bitterness of solutions containing a mixture of sinapic acid and choline chloride. They demonstrated that sinapic acid and choline chloride accounted for about 80% of the bitterness of sinapine when used at equimolar concentrations. Futhermore, these authors reported that only 50 - 94% of the bitterness perceived by tasting of water slurries of rapeseed flours and concentrates can be derived from the bitterness evoked by sinapine and free choline present in rapeseed products examined. Thus, these results suggest that not only free choline, sinapic acid and sinapine, but other rapeseed components present in rapeseed may contribute to the objectionable taste of rapeseed protein products.

3.

CONDENSED TANNINS

Condensed tannins are dimers, oligomers and polymers offlavan-3-ols.The consecutive units of condensed tannins are linked through interflavanoid bonds between C-4 and C-8 or C-6 atoms [66]. Condensed tannins upon acidic hydrolysis produce anthocyanidins and therefore are also known as proanthocyanidins. According to Salunkhe et al. [67] seed coats of cereals and legumes are the primary locations of tannins in seeds. Presence of condensed tannins in rapeseed hulls was reported by Bate-Smith and Ribereau-Gayon [68]. Durkee [69] verified thisfindingand reported the presence of cyanidin, pelargonidin and an artefact, n-butyl derivative of cyanidin in hydrolytic products ofrapeseed hulls. Later, Leung et al. [70] reported that leucocyanidin was the basic unit of condensed tannins isolated from rapeseed hulls (Figure 3 ). According to Clandinin and Heard, [71] rapeseed meals contain approximately 3% tannins assayable by the AO AC method for determination oftannins in cloves and allspice [72], but Fenwick and Hogan [73] demonstrated that this value included sinapine. Later, Blair and Reichert [74] reported that the content of tannins, as determined by the vanillin assay, was 0.09-0.39% in the defatted rapeseed cotyledons and 0.23-0.54% in the defatted canola cotyledons. In addition, Shahidi and Naczk [75] found that canola meals contained 0.68 - 0.77% condensed tannins. They also

604

OH

OH

Leucocyanidin

Figure 3. Stmctures of basic units of condensed tannins of rapeseed.

reported that the high glucosinate Midas and Hu You 9 Chinese cultivars contained 0.56% and 0.43% of tannins, respectively. The discrepancies in the reported data on tannin contents may be due to the existing differences in solvent systems employed for the extraction and assays used for quantification of tannins. The total content oftannins in selected canola and rapeseed varieties, as determined by vanillin assay, are shown in Table 5. The content of tannins in samples of canola hulls ranged from 48 to 1717 mg tannins per 1 OOg ofhulls, while hulls ofEuropean varieties contained 5 7 -1508 mg tannins per 100 g sample. These results indicate that rapeseed/canola hulls may contain up to eight times more tannins than previously reported by Mitaru et al. [76] and Leung et al. [70]. The data shown in Table 5 also demonstrate that the differences in the tannin levels within canola varieties may range from nine- to fifleen-fold. Price et al.[77] and Butler [78] reported that tannin content in mature sorghum seeds may rangefrom3 % to 93 % ofthe maximum tannin found in immature seeds. On the other hand, Radhakrishnan and Sivaprasad [79] demonstrated that the variation in tannin content of sorghum varieties grown in different locations may range up to eight-fold. Thus, these differences in tannin content within the canola varieties may be due to growing location as well as the stage of seed maturation. Recently, Amarowicz et al. [80] isolated and fractionated the tannins in canola hulls using the method of Strumeyer and Malin [81 ]. In this study, a lyophilized sample of crude tannin extracts isolatedfromCyclone canola hulls was dissolved in 95% ethanol and applied onto a SephadexLH-20 column (2.3 x 40 cm) equilibrated with 95% ethanol. The column was exhaustively washed with 95% ethanol at a flow rate of 60 mL/hr; 6 mL fractions were collected and their absorbance at 280nm was recorded. The column was then eluted with 50% acetone-water at a flow rate of 60

605 Table 5 Sinapine content ranges in some rapeseed/canola varieties (%) Cultivar

Content

Candle 0.39-0.76 Tobin 0.57-0.69 Altex 0.62-0.77 Line 0.79-1.06 Karat 0.81-0.98 Adapted from: J. Pokomy and Z. Reblova, Potrav. Vedy, 13, 1995, p. 157.

<

30

E c g20 —

1—

if)

to

d O 10 -

6

.1

(iijiinn'nm niir 10 i i i i20

^

III

IV

-

^ * i * * * ^

30

40

50

60

Tube number ( 6 ml / tube ) Figure 4. Sephadex LH-20 chromatography ofCyclone canola hull tannins using 50% (v/v) acetone: water.

mL/hr and 6 mLfractionswere collected. Each collected fraction was assayed for tannins by the modified vanillin assay ofPrice and Butler [82]. Approximately 98% ofthe original material applied on to the column was recovered. Crude tannin extract contained about 47% tannins. Figure 4 shows the elution profile ofpurified canola condensed tannins using a Sephadex LH-20 and 50% acetone as eluent. Canola tannins were separated into fourfractions.Similar fractionations of condensed tannins, eluted from Sephadex LH-20 using 70% acetone, were reported by Czochanska et al. [83 ] and Kumar and Horigome [84]. These authorsfractionatedtannins according to their molecular size. The IR spectrum of purified canola tannins obtained with KBr pellets is

606 similar to that reported by Foo [85] for class A procyanidins. Class A procyanidins are polymers that are mainly of procyanidin type with monomers having the cis configuration, i.e. are of the epicatechin type. Each fraction of canola tannins separated on a Sephadex LH-20 column using 50% acetone was examined by the TLC methodology on silica-gel (Sigma) as described by Lea [86]. The TLC chromatogram (Figure 5) revealed the presence of a number of oligomeric proanthocyanidins in Fractions III and IV and the presence of more polymerized (less retained) proanthocyanidins in Fractions I and II. Futhermore, catechins were not detected in canola tannin fractions. Absence of catechins in Cyclone canola tannin extracts was also confirmed by HPLC methodology [87]. The catechin standards used were isolated as described by Amarowicz and Shahidi [88].

1.0

0.5J

Figure 5. TLC chromatogram of Cyclone canola hull tannin fractions separated on a Sephadex LH-20 column using 50% (v/v) acetone: water. Mobile phase: toluene: acetone: formic acid (3:1:1, v/v/v). 1, (-) epigallocatechin; 2, (-) epicatechin-3-gallate; 3, (-) epigallocatechin; and 4, (-) epigallocatechin-3 -gallate.

Condensed tannins with molecular weights of 500 to 3000 Da may bring about the astringent sensation [89] because their phenolic groups are oriented into 1,2-dihydroxy and/or 1,2,3 -trihydroxy configurations [90]. Molecular interpretation offormation of astringency has recently been reported [91,92]. According to Lea [86] and Lea and Arnold [93], the bitterness and astringency of cider procyanidins was a fiinction of their molecular weights. The maximum bitterness was observed for tetramers, while the maximum of astringency corresponded to octamers. Delcour et al. [94] determined the astringency threshold values for solutions of tannic acid (14.1 ppm),(+)catechin (46.1 ppm),procyanidinB-3 917.3 ppm);quercetindihydrateplustetrameric procyanidins (8.9) and a mixture of trimeric and tetrameric proanthocyanidins with (+) catechin (3.6 ppm) in deionized water. A mixture of trimeric and tetrameric procyanidins as well as combination of this mixture with catechin resulted in three to ten times lower threshold values. The contents of total and oligomeric tannins in Cyclone canola hulls (17170 and 3434 ppm, respectively) and corresponding meals (3434 and 687 ppm, respectively) have been determined.

607 In performing this experiment, the content of oligomeric tannins was determined by extraction of aqueous crude tannin solutions with ethyl acetate. According to Porter [95] only monomeric and dimeric proanthocyanidins are highly soluble in ethyl acetate. The content of oligomeric tannins soluble in ethyl acetate was assayed by the modified vanillin method [82]. The crude extracts of Cyclone canola tannins contained 20% of tannins soluble in ethyl acetate [96]. The data shows that the content ofoligomeric tannins in canola meal is a hundred times the threshold value reported for a mixture of trimeric and tetrameric proanthocyanidins. Because of this, condensed tannins present in hulls should be considered as one of the important contributors to the objectionable taste of rapeseed products.

4.

TANNIN-PROTEIN INTERACTIONS

Proteins, one ofthe macrocomponents offood-systems, may interact withflavoringcompounds. Such interactions will influence the flavor release and perception [97- 99]. Phenolic compounds may form soluble and insoluble complexes with proteins. The phenol-protein complexes may be stabilized by covalent bonds, ionic bonds, hydrogen bonding and/or hydrophobic interactions [ 100]. It is, however, believed that phenol-protein complexes are usually the result offormation ofhydrogen bondings and hydrophobic interactions [101], particularly under acidic conditions [103]. Studies on the complexations ofpolyphenols with proteins mainly concentrated on the evaluation of factors influencing these interaction(s) and on the impact of formation of phenol-protein complexes on nutritive value of proteins. The phenol-protein interactions are affected both by the size, conformation and charge of protein molecules and also by the size, length andflexibilityof phenol molecule [100, 101, 103]. Proteins with compact globular conformation like lysozyme andribonucleaseexhibit low affinity for phenols. On the other hand, proteins with conformational^ open structure, like gelatin, readily form complexes with phenols [104]. In addition, it has been demonstrated that proanthocyanidins should have at least threeflavanolssubunits to be an effective protein precipitating agent [ 105-107]. Phenol-protein complexes precipitate from solution only when sufficient hydrophobic surface is formed on the surface of complex [108]. The exact role ofpolyphenol-protein interactions in generating objectionable taste ofrapeseed products is still unclear. Kozlowska and Zademowski [109] and Sosulski and Dabrowski [110] reported that at least seven extractions of rapeseed flour with aqueous ethanol was needed to produce bland protein concentrates containing trace amounts of phenolics. Later, Kozlowska and Zademowski [39] demonstrated the formation of phenolic-protein complexes during preparation of protein isolates. They found that the amount of soluble matter in 80% ethanol, rich in phenolics, increased as the pH of solution used for extraction of proteins increased. Also, Zademowski [52] found that alkaline hydrolyzates of albumin and globulin fractions ofrapeseed contained 6.68 ^imol sinapic acid/g albumin and 0.49 ^mol sinapic acid/g globulin. Recently, Amarowicz and Kmita-Glazewska [111] reported that phenolic acids were bound only to selected low molecular weight rapeseed proteins. Protein precipitating capacities ofcrude condensed tannin extracts isolated from selected canola hulls are shown in Table 6. The protein precipitation assay [112] measures the amount of phenolic bound with protein, whereas the dye labeled BSA assay [113] determines the amount of proteins precipitated by phenols. Protein precipitating values for canola tannins are comparable to those reported by Hagerman and Butler [112] and Asquith and Butler [113] for sorghum tannins.

608 Table 6 Protein precipitating capacities of canola tannins as determined by two assays Sample*

Westar sample 1^ sample 2** sample S""

Protein Precipitation Assay (A5,o) 1.1 4.0

Dye-Labeled BSA Assay (mgBSA/g hulls) 30.7 23.7 58.6

Cyclone 4.5 52.7 sample 1*' 4.9 sample 4** 44.2 sample 5^ 52.2 Excel'^ 2.0 33.2 Ebony*^ 5.0 49.2 3.4 PR3113'^ 45.4 * Samples codes same as in Table 5 ^ Adapted from: M. Naczk, R. Amarowicz, A. Sullivan, and F. Shahidi, Food Chemistry, submitted for publication. ^ Adapted from: M. Naczk, T. Nichols, D. Pink, and F. Sosulski, J. Agric. Food Chem. 42, 1994, p. 2198 ** Adapted from: M. Naczk, unpublished data Figure 6 shows the effect of pH on the amount of tannins precipitating with selected proteins. BSA, fetuin, gelatin and pepsin were precipitated markedly at pH values between 3.0 and 5.0, but maximum precipitation of lysozyme occurred at pH >8.0. The pH optimum for precipitation was found to be 0.3 -3.1 pH units below the isoelectric points ofproteins [114]. The results demonstrate that each protein has characteristic pH optimum for precipitation by phenolics. Therefore, phenolics may be present in food system in the free or bound form depending on the pH and the kind of proteins present in this system. This in turn will influence the contribution of condensed tannins to the undesirable taste of rapeseed products.

5.

CONCLUSIONS

The bitterness and astringency of rapeseed/canola products may result from both additivity and synergism among the different stimuli present in rapeseed/canola at subthreshold and suprathreshold levels. More detailed research is needed to determine the contribution of each stimulus to the objectionable taste of rapeseed/canola products. The influence of protein-tannins interactions on the perception of objectionable taste of rapeseed/canola product is still not well understood. More detailed research is needed to determine the contribution of these interactions to the perception of the flavor of rapeseed products. Model systems to be used in such studies

609

3

o CO

4

5

6

7

8

9

10 11 12 PH

Figure 6. pH dependence of complex formation between canola tannins and several proteins. Adapted from: M. Naczk, D. Oickle, D. Pink, and F. Shahidi, J. Agric. Food Chem. 44 (1996) 1444. should consist of rapeseed proteinfractionsto whichfreephenolic acids, esterified phenolic acids, tannins or their combinations are added. Phenolic compounds form complexes not only with proteins but also with carbohydrates and minerals [100]. However, the influence of these interactions on the flavor release and perception is still not well understood. Better understanding of factors influencing the interactions between phenolics and other rapeseed/canola components would help in the development of more efficient procedures for production of bland rapeseed protein isolates and concentrates. Acknowledgment This work was supported, in part, by a research grant (to M.N.) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. 6.

REFERENCES E.H. Unger, in: Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology, F. Shahidi (ed) pp 235-250. An AVI Book, New York 1990. K. Nishie and M.E. Daxenbichler, Food Cosmet. Toxicol, 18 (1980) 159.

610 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

G.R. Fenwick, E.A. Spinks, A.P. Wilkinson, R.K. Heaney, and MA. Legoy, J. Sci. Food Agric, 37 (1986) 735. S. Rabot, C. Guerin, L. Nugon-Baudon, and O. Szylit, in: Proc. 9th Intern. Rapeseed Congress, Cambridge, UK. Volume 1 (1995) 212. F. Shahidi, M. Naczk, L.J. Rubin, and L.L. Diosady, J. Food Prot., 51 (1988) 743. M. Naczk, L.L. Diosady, and L.J. Rubin, J. Food Sci., 50 (1985) 1685. R. Ohlson, in: Proc. 5th Intern. Rapeseed Congress, Malmo, Sweden (1978) 152. F. Shahidi, M. Naczk, D. Hall, and J. Synowiecki, Food Chemistry, 44 (1992) 283. J. Delisle, J. Amiot, G. Goulet, C. Simard, G.J. Brisson, and J.D. Jones, Qual. Plant.-Plant Foods Human Nutr., 34 (1984) 243. R.K. Downey and J.M. Bell, in: Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology, F. Shahidi (ed) pp 37-47. An AVI Book, New York 1990. L. Rubin, L. Diosady, M. Naczk, and M. Halfani, Can. Inst. Food Sci. Technol. J., 19 (1986) 57. F. Shahidi and M. Naczk, in: Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology, F. Shahidi (ed.) pp 291-301, An AVI Book, New York 1990. M. Naczk, L.L. Diosady, and L.J. Rubin, Lebensm.-Wiss.u.Technol., 19 (1986) 13. J.W. Erdman, Jr., J. Am. Oil Chem. Soc, 56 (1979) 736. M. Cheryan, CRC Critical Rev. Food Sci. Nutr., 13 (1980) 297. J.A. Maga, J. Agric. Food Chem., 30 (1982) 1. E.R. Morris, in: Phytic Acid: Chemistry and Applications, E. Graf (ed) pp 57-76. Pilatus Press, Minneapolis 1986. L. Hallberg, Scan. J. Gastroenterol., 22 (1987) 73. S.E. Rickard and L.V. Thompson, in: Antinutrients and Phytochemicals in: Food, F. Shahidi (ed) pp 294-312, ACS Symposium Series 662, ACS Washington, DC, 1997. L.U. Thompson, in: Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology, F. Shahidi (ed) pp 173-192. An AVI Book, New York 1990. F. Shahidi, unpublished data. L. A. Applequist and R. Ohlson, Rapeseed Cultivation, Composition, Processing and Utilization. Elsevier Publ. Co., Amsterdam 1972. J.M. Bell, Can. J. Animal Sci., 73 (1993) 679. S.K. Nensen, Y.G Liu, andB.O. Eggum, in: Proc. 9thlntern. Rapeseed Congress, Cambridge, UK (1995) 188. J.D. Jones and I.R. Sibbald, Poultry Sci., 58 (1979) 385. A.J. Liesle, J.D. Summers, and J.D. Jones, Can. J. Animal Sci., 53 (1973), 153. G. Sarwar, J.M. Bell, T.F. Sharby, and J.D. Jones, Can. J. Animal Sci., 61 (1981) 719. N.W. Tape, Z.J. Zabry, and K.I. Eapen, Food Sci. Technol., 3 (1970) 78. L.L. Diosady, L.J. Rubin, C.G Tar, and B. Etkin, Can. J. Chem. Eng., 64 (1986) 768. F.W. Sosulski and R Zademowski, J. Am. Oil Chem. Soc, 58 (1981) 96. J.D. Jones and J. Holme, Oilseed Processing, Canadian Patent, No. 1 117 134 (1982). J.J. Baudet, B. Greilsamer, and G. Vermeersch, in: Proc. 6thlntem. Rapeseed Congress, Paris, France (1983) 1381. F.H. Schneider, Method of Shelling Oil- and Protein-Containing Grains, Canadian Patent, No. 1062 118(1979). D.H. Honig, A. Kathleen, L. Selke, and J.J. Rackis, J. Agric. Food Chem., 6 (1979) 1363. J.J. Rackis, D.J. Sessa, and D.H. Honig, J. Am. Oil Chem. Soc, 56 (1979) 262.

611 36 F. Ismail, M. Vaisey-Genser, and B. Fyfe, J. Food Sci., 46 (1981) 1241. 37 M. Donaldson, M. Vaisey-Genser, B. Hoehn, and M. Latta, Cereal Foods World, 23 (1978) 495. 38 W. Boyd, Science, 112 (1950) 153. 39 H. Kozlowska and R. Zademowski, In: Abstracts, Third Chemical Congress ofNorth America, Toronto, June 5-10, 1982. 40 K. Krygier, F. Sosulski, and L. Hogge, J. Agric. Food Chem., 30 (1982) 334. 41 M. Naczk and F. Shahidi, Food Chemistry, 31 (1989) 159. 42 H. Kozlowska, D. A. Rotkiewicz, and R. Zademowski, J. Am. Oil Chem. Soc, 60 (1983) 119. 43 J.J. Rackis, D.J. Sessa, andD.H. Honig, Soybean protein foods, U.S. Dept. Agric, ARS-71-35, 1957, p. 100. 44 S. Arai, H. Suzuki, M. Fujimaki, and Y. Sakurai, Agric. Biol. Chem. (Tokyo), 30 (1966) 364. 45 J.A. Maga and K. Lorenz, Cereal Sci. Today, 18 (1973) 326. 46 M. Dadic and G. Belleau, Proc. Am. Soc. Brew. Chem. (1973) 107. 47 M. Naczk, J.P. Wanasundara, and F. Shahidi, J. Agric. Food Chem., 40 (1992) 444. 48 L.M. Bartoschuk and C.T. Cleveland, Sensory Processes, 1 (1977) 177. 49 T.W. Fenton, J. Leung, and D.R. Clandinin, J. Food Sci., 45 (1980) 1702. 50 J. Pokomy and Z. Reblova, Potrav. Vedy, 13 (1995) 155. 51 R. Amarowicz, F. Shahidi, and R. Pegg, J. Am. Oil Chem. Soc, submitted for publication. 52 R. Zademowski, Acta Acad. Agric Techn. 01st., 21(F) (1987) 1-55; (in polish). 53 U. Wanasundara, R. Amarowicz, and F. Shahidi, Food Chemistry, 42 (1994) 1285. 54 R. Amarowicz and F. Shahidi, J. Am. Oil Chem. Soc, 71 (1994) 551. 55 R. Amarowicz, M. Karamac, B. Rudnicka, and E. Ciska, Fat Sci. Technol., 97 (1995) 330. 56 A.B. Durkee and J.B. Harbome, Phytochemistry, 12 (1973) 1085. 57 B. Tantawy, J.P. Robin, and M.T. ToUier, in: Proc 6th Intern. Rapeseed Congress, Paris, France (1983) 1313. 58 D.R. Clandinin, Poultry Sci., 40 (1961) 484. 59 F.W. Sosulski, J. Am. Oil Chem. Soc, 56 (1979) 711. 60 F. Shahidi and M. Naczk, J. Am. Oil Chem. Soc, 69 (1992) 917. 61 M.M. Mueller, E. Ryl, T.W. Fenton, and DR. Clandinin, Can. J. Animal Sci., 58 (1978) 579. 62 S. Clausen, L.M. Larsen, A. Ploeger, and H. Sorensen, World Crops, 11 (1985) 61. 63 L.M. Larsen, O. Olsen, A. Ploeger, and H. Sorensen, in: Proc 6th Intern. Rapeseed Congress, Paris, France. (1983) 1577-1582. 64 A.B. Durkee and P.A. Thivierge, J. Food Sci., 40 (1975) 820. 65 D.J. Sessa, K. Wamer, and DiH. Hontg, J. Food Sci., 39 (1974) 69. 66 R. Hemingway in: Chemistry and Significance of Condensed Tannins, R. Hemingway and J.J. Karchesy (eds) pp 83-108, Plenum Press, New York, 1989. 67 D.K. Salunkhe, J.K. Chavan, and S.S. Kadam, Dietary tannins: consequences and remedies, CRC Press Inc., Boca Raton, FL, 1990. 68 E.C. Bate-Smith and P. Ribereau-Gayon, Qual. Plant Mater. Vegetable, 5 (1959) 189. 69 A.B. Durkee, Phytochemistry, 10 (1971) 1583. 70 J. Leung, T.W. Fenton, M.M. Mueller, and D.R. Clandinin, J. Food Sci., 44 (1979) 1313. 71 D.R. Clandinin and J. Heard, Poultry Sci., 47 (1968) 688. 72 AOAC Official Methods of Analysis. 10th ed. Association of Official Agricultural Chemists: Washington, DC, 1965. 73 R.G. Fenwick and S.A. Hoggan, Br. Poultry Sci., 17 (1976) 59.

612 74 75 76 77 78 79 80 81 82 83

R. Blair and R.D. Reichert, J. Sci. Food Agric, 35 (1984) 29. F. Shahidi and M. Naczk, J. Food Sci., 54 (1989) 1082. B.N. Mitaru, R. Blair, J.M. Bell, and R.D. Reichert, Can. J. Animal Sci., 62 (1982) 661. M.L. Price, A.M. Stromberg, and L.G. Butler, J. Agric. Food Chem., 28 (1979) 1214. L.G. Butler, J. Agric. Food Chem., 30 (1982) 1087. M.R. Radhakrishnan and J. Sivaprased, J. Agric. Food Chem., 28 (1980) 55. R. Amarowicz, M. Naczk, and F. Shahidi, unpublished data. D.H. Strumeyer and M.J. Malin, J. Agric. Food Chem., 23 (1975) 909. M.L. Price, S. Van Scoyoc, and L.G. Butler, J. Agric. Food Chem., 26 (1978) 1214. Z. Czochanska, L.Y. Foo, R.H. Newman, and L.J. Porter, J. Chem. Soc. Perkin Trans., 1 (1980) 2278. 84 R. Kumar and T. Horigome, J. Agric. Food Chem., 34 (1986) 487. 85 L.Y. Foo, Phytochemistry, 20 (1981) 1397. 86 A.G.M. Lea, J. Sci. Food Agric, 29 (1978) 471. 87 A.C. Hoefler and P. Coggan, J. Chromat., 129 (1976) 460. 88 R. Amarowicz and F. Shahidi, Food Res. International, in press. 89 E. Haslam, in: TheFlavonoids, J.B. Harbome, T.J. Mabry and E. Mabry (eds), Academic Press, New York, 1975. 90 E. Haslam, in: The Biochemistry of Plants, P.K. Stumpf and E.E. Conn (eds) Volume 7, pp 527-556. Academic Press, London, 1981. 91 E. Haslam, T.H. Lilley, and T. Azawa, Bull, de Liason du Groupe Polyphenols, 13 (1986) 352. 92 E. Haslam and T.H. Lilley, CRC Critical Rev. Food Sci. Nutr., 27 (1988) 1. 93 A.G.H. Lea and G.M. Arnold, J. Sci. Food Agric, 29 (1978) 478. 94 J. A. Delcour, M.M. Vandenberghe, P.F. Corten, and PC. Dodeyne, Am. J. Enol. Vitic, 35 (1984) 134. 95 L.J. Porter, in: Methods in Plant Biochemistry, J. Harbome (ed), Volume 1, pp 389-420. Academic Press, San Diego, 1989. 96 M. Naczk, D. Oickle, D. Pink, and F. Shahidi, J. Agric Food Chem., 44 (1996) 2144. 97 J. Bakker, in: Ingredient Interactions — Effect of Food Quality, A. G. Gaonkar (ed) pp. 411 439, Mercel Dekker, Inc., New York, 1995. 98 W. Pickenhagen, Food Technol. Europe, 2 (1996) 60. 99 N. Fisher and S. Widder, Food Technol.., 51 (1997) 68. 100 F. Shahidi and M. Naczk, Food Phenolics: Sources, Chemistry, Effects and Applications, Technomic Publishing Co. Inc., Lancaster, PA, 1995. 101 A.E. Hagerman and L.G. Butler, J. Biol. Chem., 256 (1981) 4494. 102 J.P. McManus, K.G Davis, J.E. Beart, S.H. Gaffney, T.H. Lilley, and E. Haslam, J. Chem. Soc. Perkin Trans., 2 (1985) 1429. 103 M. Naczk and F. Shahidi, in: Antinutrients and Phytochemicals in Food, F. Shahidi (ed) pp 186-208, ACS Symposium Series 662, ACS, Washington, DC, 1997. 104 L.G. Butler, in: Food Products, J. Kinsella and W.G. Soucie (eds), AOCS Press, Champaign, IL, 1989. 105 D.G. Roux, Phytochemistry, 11 (1972) 1219. 106 E.C. Bate-Smith, Phytochemistry, 12 (1973) 907. 107 W.E. Artz, P.D. Bishop, A.K. Dunker, E.G. Schanus, and B.G. Swanson, J. Agric. Food Chem., 35 (1987) 417.

613 108 J.P. McManus, K.G. Davis, T.H. Lilley, and E. Haslam, J. Chem. Soc. Chem. Comm. (1981) 309. 109 H. Kozlowska and R. Zademowski, Proc. 6th International Rapeseed Congress, Paris, France (1983), 1412. 110 K. Dabrowski and F. Sosulski, J. Agric. Food Chem., 32 (1984) 128. 111 R. Amarowicz and H. Kmita-Glazewska, Pol. J. Food Nutr. Sci., 43 (1995) 88. 112 A.E. Hagerman and L.G. Butler, J. Agric. Food Chem., 26 (1978) 809. 113 T.N. Asquith and L.G. Butler, J. Chem. EcoL, 11 (1985) 1535. 114 M. Naczk, D. Oickle, D. Pink, and F. Shahidi, J. Agric. Food Chem., 44 (1996) 1444.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

615

Effect of ethanol strength on the release of higher alcohols and aldehydes in model solutions H. Escalona-Buendia, J. R. Piggott, J. M. Conner and A. Paterson Centre for Food Quality, University of Strathclyde, Department of Bioscience and Biotechnology, 204 George Street, Glasgow G l IXW, Scotland, UK

Abstract Headspace concentrations of homologous series of higher alcohols and aldehydes dissolved in aqueous solutions at different ethanol concentrations were analysed by gas chromatography-flame ionisation detection. For each volatile, activity coefficients at all ethanol strengths were estimated and statistically compared to evaluate the effect of ethanol strength. There was a significant reduction of the activity coefficients between 10% and 20% v/v ethanol for all the volatiles studied. The reduction of the activity coefficients between 10% and 15% v/v was significant only for decanol, dodecanol, octanal and dodecanal. This confirms that there is a change in the efiect of ethanol concentration on volatile flavour compounds in aqueous solutions at 15-20% v/v ethanol.

1. INTRODUCTION Higher alcohols, aldehydes and esters are important volatiles for the aroma and flavour of wines and spirits. According to studies carried out in wine model solutions (Voilley et al. 1990, 1991; Lubbers et al. 1994a, 1994b; Langourieux and Crouzet 1994) and previous studies of whisky flavour (Conner et al. 1994a, 1994b), the release of volatiles is affected by other components in the solution and, therefore, the flavour quality of the beverage may also be affected. In model wine solutions the presence of ethanol reduced the activity coefficients of isoamyl alcohol, octanal and ethyl esters (Lubbers et al. 1994a). These volatile compounds are more soluble in ethanol than in water and when they are in alcoholic aqueous solutions, an increase of ethanol concentration also increases their solubility and, therefore, reduces their release. However, Conner et al. (1997) reported a significant reduction of the release of ethyl esters from alcoholic solutions above 17% v/v ethanol, while at lower strengths the activity coefficients remained almost constant. This behaviour may be explained by changes in the structure of ethanol-water solutions which are modified by the

616 proportions of the mixture. According to D'Angelo et al. (1994), above about 20% v/v ethanol concentration there are non-polar interactions between the hydrocarbon chains of the alcohol molecules, forming agglomerates or "pseudomicelles", and interaction with other non-polar molecules in the system is possible. In order to evaluate the effect of ethanol strength on the release of higher alcohols and aldehydes, the activity coeflScients of homologous series of both groups of volatiles (C6, C8, CIO and C12) were measured in aqueous solutions at different ethanol strengths (10%, 15% and 20% v/v). As the activity coefficient is estimated as the slope of a linear relation between the activity and the concentration of the solute, effects on release were evaluated by a statistical comparison between the slopes.

2. MATERIALS AND METHODS 2.1. Model solutions Absolute ethanol was used to prepare model solutions at 10%, 15% and 20% v/v. Water was distilled and filtered using a Millipore-Q system. For each volatile, stock solutions at 10 mg mL'^ were prepared in absolute ethanol. The volatiles studied were 1-hexanol, 1-octanol, 1-decanol, 1-dodecanol and the analogous series of aldehydes, all of them at least 95% pure. For each ethanol concentration, series of solutions from 1 mg L'^ to 12 mg L^ for every volatile (at least 6 different concentrations) were prepared adding different aliquots from the stock to the model solution. 2.2 Activity coefficients determinations The activities of higher alcohols and aldehydes were obtained by chromatographic determinations of headspace concentrations (Grant and Higuchi 1990; Conner et al. 1997) and activity coefficients calculated as described by Conner et al. (1994a). Glass vials (20 mL), fitted with PTFE fined silicone septa in plastic screw caps, were filled with 10 mL aliquots of standard ethanolic solutions of the volatile. After equilibration in a 25 °C water bath for at least 30 min, a 2.5 mL sample of headspace was withdrawn using a 5 mL gas tight syringe, preheated to 50 °C. Only one headspace c-;- column injection was made per vial and samples were analysed in duplicate on a Carlo Erba™ Mega Series gas chromatograph using a flame ionisation detector. Peak areas were calculated using Chromperfect™ integration software. Cold, on-column injection used a 0.55 mm x 0.5 m ultimetal retention gap with an external gas tight septum. For hexanal, a 0.53 mm x 12 m B P l column (df = 1) was used with helium carrier gas at 20 kPa, holding the column at 30 °C for 1 min and increasing to 50 °C at 18 °C min"\ For all the other volatiles, a 0.53 mm x 12 m BP20 column (df = 1) was used with helium carrier gas at 30 kPa, holding the column at 60 °C for 1 min and increasing to 240 °C at 18 °C min"\ The temperature of the detector was 250 °C.

617 3. RESULTS AND DISCUSSION Figure 1 shows the behaviour of octanol in aqueous solution at 10%, 15% and 20% v/v of ethanol; this was the typical behaviour of all the alcohols and aldehydes studied. A linear relation between the activities and the mole fraction of the volatile in the ethanolic solution is observed, and a gradual reduction of the slope of the curves, which is the numerical estimation of the activity coefficient, as the ethanol concentration increased.

O.OOE+00

5.00E-07

l.OOE-06

1.50E-06

2.00E-06

Octanol (mole fraction) Figure 1. Activities of increasing concentrations of octanol in 10%, 15% and 20% v/v ethanol aqueous solutions calculated from headspace concentration at 25 °C.

Table 1 shows the activity coefficients obtained for each volatile at every ethanol concentration. Statistical comparison of the slopes was carried out by calculation of the 95% confidence interval (Mead and Curnow 1983), considering a difference to be significant when there was not overlapping of the intervals. For all the volatiles, activity coefficients were significantly reduced between 10% and 20% v/v ethanol. This behaviour was expected according to the results reported by Conner et al. (1997) for ethyl esters, and agreed with the concentration range reported by D'Angelo et al. (1994) required to start deagglomeration of the alcohol molecules in ethanol-water solutions. A continuing effect would be expected at higher ethanol concentrations which are more favourable conditions for the formation of agglomerates. For the higher alcohols, the higher the number of carbons the higher is the activity coefficient. Figure 2 shows the semilogarithmic relation between the activity coefficient and the number of carbons in the alcohol. For all the alcohols there was a gradual reduction of the

618 activity coefficients as the ethanol concentration increased. However, for hexanol and octanol the difference between 10% and 15% was not significant (Table 1).

Table 1 Activity coefficients at 25 "C of higher alcohols and ethanol/water solutions. Ethanol strength Activity Standard error coefficient (% v/v) 566 29.4 10 Hexanol 497 25.0 15 13.2 393 20 3810 113.1 10 Octanol 3424 128.2 15 1926 87.8 20 2372 81746 10 Decanol 3737 58990 15 41221 1657 20 844331 67658 10 Dodecanol 47660 544778 15 24098 270845 20 87.7 1076 10 Hexanal 827 86.5 15 36.5 420 20 6360 279.3 10 Octanal 210.7 3829 15 135.9 2801 20 1747 27195 10 Decanal 1762 22281 15 16917 623 20 15614 251360 10 Dodecanal 6153 181478 15 147821 12258 20

aldehydes dissolved in 9 5 % Confidence interval 503-629 443-552 364-421 3570-4050 3138-3709 1735-2117 76578-86914 50765-67215 37667-44775 695417-993245 436964-652593 218340-323350 873-1278 628-1027 336-505 5738-6982 3380-4278 2489-3104 23389-31001 18355-26207 15545-18289 216570-286150 167935-195021 120509-175133

R^ 0.96 0.98 0.99 0.99 0.99 0.98 0.99 0.96 0.98 0.93 0.94 0.91 0.95 0.92 0.94 0.98 0.96 0.98 0.98 0.97 0.99 0.96 0.99 0.94

The relation between the activity coefficients and the number of carbons for the series of aldehydes studied is observed in Figure 3. There is the same semilogaritmic relation and similar behaviour in comparison to the higher alcohols. Activity coefficients for Hexanal and Decanal at 10% and 15% were not significantly different, and only Dodecanal, which is the less polar compound, had a significant difference between 10% and 15%, but no difference between 15% and 20%. The gradual reduction of the activity coefficients of the volatiles studied while ethanol concentration increases may be attributed to the increase of the

619 solubility of non-polar compounds by the presence of ethanol in the aqueous solution. The longer hydrocarbon chains of decanol, dodecanol, decanal and dodecanal may be more susceptible to hydrophobic interactions explaining the reduction of their volatility from 10% ethanol, which for hexanal, hexanol and octanol was from 15%.

8

10

12

Number of carbons in alcohol

Figure 2. Activity coefficients of alcohols in aqueous solution at 10%, 15% and 20% v/v ethanol.

-^10% -^15%

i 5

-i-20%

0)

o o ^

4

•r-H

1 bo o

3[ ,

8

10

12

Number of carbons in aldehyde

Figure 3. Activity coefficients of aldehydes in aqueous solution at 10%, 15% and 20% v/v ethanol.

620 4. CONCLUSIONS There was a reduction in the activity coefficients of all the higher alcohols and aldehydes studied when mixed in progressively higher ethanol concentrations. Activity coefficients of all volatiles were significantly reduced between 10% and 20% v/v ethanol; for hexanol, octanol and hexanal there was a significant reduction between 15% and 20% v/v. Thus not only the composition of the volatiles but also their interactions with the matrix must be taken into account to understand the aromatic properties of such alcoholic beverages.

5. REFERENCES Conner, J.M., Paterson, A. and Piggott, J.R. (1994a). J. Sci. Food Agric. 66, 4553. Conner, J.M., Paterson, A. and Piggott, J.R. (1994b). J. Agric. Food Chem. 42, 2231-2234. Conner, J.M., Birkmyre, L., Paterson, A. and Piggott, J.R. (1997). Headspace concentrations of ethyl esters at different alcoholic strengths. J. Sci. Food Agric. In press. D'Angelo, M., Onori, G. and Santucci, A. (1994). J. Chem. Phys. 100, 3107-3113. Langourieux, S. and Crouzet, J. (1994). Lebensmittel-Wiss. u Technol. 27, 544549. Grant, D.R. and Higuchi, T. (1990). Solubility Behaviour of Organic Compounds. John Wiley & Sons, New York. Lubbers, S., Voilley, A., Charpentier, C. and Feuillat, M. (1994a). Am. J. Enol. Vitic. 45, 29-33 Lubbers, S., Voilley, A., Feuillat, M. and Charpentier, C. (1994b). LebensmittelWiss. u Technol. 27, 108-114. Mead, R. and Curnow R. (1983). Statistical Methods in Agriculture and Experimental Biology. Chapman and Hall, London UK. Voilley A., Lamer C , Dubois P. and Feuillat M. (1990). J. Agric. Food Chem. 38, 248-251. Voilley A., Beghin V., Charpentier C. and Pe3n:*on D. (1991). Lebensmittel-Wiss. u Technol. 24, 469-472.

Acknowledgements The authors wish to acknowledge the UK Biotechnology and Biological Sciences Research Council and CONACyT-Mexico (Consejo Nacional de Ciencia y Tecnologia) for the financial support provided.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

621

Ultrasonic Inactivation of Soybean Trypsin Inhibitors H. H. Liang^^ R. D. Yang^ and K. C. Kwolc^ 'Department of Applied Biology & Chemical Technology, University, Hong Kong

Hong Kong Polytechnic

''Department of Bioengineering, South China University of Technology, Guangzhou, China.

Abstract Soybean trypsin inhibitors (TI) in soymilk were treated by heat and ultrasound of 20 kHz. The influence of several factors (temperature, time of treatment, pH, ultrasonic power, soymilk concentration, and ionic strength) on inactivation of TI was investigated. The results of the experiment shown that temperature was the major factor to affect the inactivation of TI, and treatment time was the next important factor. Under the experimental conditions of temperature 80 °C, ultrasonic power of 150 w, and pH 7.0 for 5 min treatment, TI in soymilk sample could be inactivated by 73%. The retained 27% TI was difficult to inactivate. This residual component is mainly Bowman-Birk inhibitor (BBI) which is extremely stable to heat as well as ultrasound of 20 kHz.

1. INTRODUCTION In soymilk processing, elimination of enzymic off-flavor development and destruction of growth inhibitors in raw soymilk are important concerns. Growth depression, pancreatic hypertrophy, hyperplasia and adenoma in experimental animals have been partly or fully attributed to soybean trypsin inhibitors (TI) [1]. Proper heat treatment improves the nutritional value of soymilk by inactivation of TI and by increasing the digestibility of soy proteins [2]. Previous reports on inactivation of TI in soymilk were mostly based on heat treatments [3]. Although heat is generally used to inactivate soybean TI, such inactivation is often incomplete. Heat treatment at temperatures below 100°C have shown that the inhibitors are rather heat stable, and it takes a long time to reduce the TI activity (TIA) to a satisfactory level. The use of high temperatures to destroy TI may lead to the degradation of amino acids and vitamins, development of off-flavor, and other deteriorative reactions. One of the important applications of ultrasound in food processing is its direct involvement into the processing, often in the form of high-intensity [4]. A number of beneficial effects have been reported in a broad aspects of applications, such as promotion of hydrolysis rates [5-6], assistance on diffusion [7], and destruction of micro-organisms [8]. Recently, the applications of manothermosonication (MTS), a technique of applying heat and ultrasound simultaneously under pressure, on inactivation of food spoilage enzymes have drawn more and more attentions of researchers. Lopez et al [9] studied the inactivation effects of MTS on some enzymes and found that the enzyme destruction efficiency greatly increased with

622 ultrasonic wave amplitudes, and the static pressure did not seem to significantly affect the destructive effect of the process. Lopez and Burgos [10] further investigated the effects of sonication physical parameters, pH, KCl, sugers, glycerol and enzyme concentration on the inactivation of soybean lipoxygenase (LOX), an enzyme involved in off-flavor development in vegetable products, under MTS operation. Their resuhs suggest that MTS inactivates LOX by two different mechanisms, one associated with heat and the other with ultrasound. They also found that the effect of static pressure on the inactivation of LOX is not substantial. Our objective is to investigate the feasibility of applying ultrasound in combination with a mild heat treatment on the inactivation of TI in soymilk at atmospheric pressure and the effects of various influent factors such as temperature, duration of ultrasound treatment, ultrasound power level, pH value of the solution, soy solid concentration and ionic strength.

2. MATERIALS & METHODS 2.1. Preparation of soymilk Canadian no. 1 grade soybeans were soaked in deionized water (soybean-to-water 1:7) for 14 h at 5°C. The soaked beans, along with the soak water were blended in a Waring blender at high speed for 3 min . The slurries were diluted with deionized water and filtered through a nylon filter bag and the insoluble residue was discarded. The filtrate, designated as soymilk, was lyophilized in a laboratory freeze-drier for 48 h and the dried product was stored in a screw-cap test tube at 5°C for later use. 2.2. Sample preparation 0.75 g sample of freeze dried soymilk solids were dispersed in 50 ml of deionized water and stirred using a magnetic stirrer for 3 h. The reconstituted soymilk has a pH of 6.5 and a solid content of about 3%. The pH of the soymilk was adjusted to the desired value by adding 1 M NaOH or 1 M HCL for experiments of pH effect. 2.3. Heat treatment 25 ml of the soymilk sample reconstituted from freeze dried soymilk solids was filled into a screw-cap test tube and heated in a boiling water bath. When the temperature of soymilk reached the desired temperature, the tube was transferred to another water bath previously set at the desired temperature and held for a desired length of time (holding time). At the end of the holding time, the tube was immediately transferred to a cold water bath. 2.4. Heat and ultrasonic treatment For ultrasound treatment, the sample, after heating up to the desired temperature, was immediately transferred to a glass cylinder (35 ml volume), which was then placed into a cooling cell with cold water inlet and outlet. The flow rate of cold water could be adjusted to maintain the sample temperature within ± 1°C of the desired value during ultrasound treatment. The sound probe (with a 13 mm tip) of the ultrasonic processor (Acros Chimica, model CPX 600), along with a thermocouple, were inserted into the soymilk sample, about 1 cm from the bottom of the cylinder. The desired ultrasound power level was obtained by controlling the wave amplitude. The operation mode of the ultrasound processor was set as

623 pulse with the on-off ratio of 3:2 second. At the end of the treatment, the cylinder with soymilk was immediately immersed into ice water. The treated sample was then diluted and the residual TIA was assayed. 2.5. Assay for trypsin inhibitor activity (TIA) A modification of the Kakade's procedure, developed by Smith et al. [11], using the synthetic substrate benzoyl-DL-arginine-p-nitroanilide (BAPNA), was employed to measure TIA. The method involves extraction of the inhibitors from the sample at pH 9.5 and mixing unfiltered suspensions with bovine trypsin. The activity of the remaining trypsin is then measured by offering it BAPNA under standard conditions. The p-nitroaniline released is measured spectrophotometrically at 410 nm. This provides a linear measure of the residual trypsin activity, so that the amount of pure trypsin inhibited per unit weight of sample can be calculated.

3. RESULTS i& DISCUSSION 3.1. Effect of temperature on ultrasound inactivation of TI Figure 1 gives a comparison between the effect of heat treatment alone and the combined effect of heat and ultrasound treatment on the inactivation of TI in soymilk. The results showed that the inactivation of TI was facilitated when ultrasound treatment was simultaneously applied with heat. The synergistic effect was found to be the highest at about 70 °C. One possible mechanism is that sonification gives rise to H- and OH- free radicals by decomposition of water inside the oscillating bubbles [12]. Hydroxyl radicals are very reactive and can induce the initial formation of peroxy radicals on amino acid residues, producing great losses of tryptophan, tyrosine, and other amino acids [13]. The hydroxl radicals may also cleave the disulfide bonds of the TI [14]. Another possible mechanism is that the vapor pressure inside the cavitation bubbles increases as the treatment temperature increases. The high pressure and temperature cause intensive collapse of the cavitation bubbles, that consequently increase the reaction rate of sonochemistry [15]. However, when the temperature was too high (90 °C in Figure 1), early collapse may occur due to the excessive high vapor pressure inside the cavitation bubbles, resulting in a the smaller ultrasonic effect on the inactivation of TI. 3.2. Effect of ultrasound treatment time on inactivation of TI Figure 2 shows the effect of sonification time on the inactivation of TI when the other influent factors are set as constants. It was found that the ultrasound inactivation effect was rapidly raised when the treatment time was increased from time zero to 5 min. The percentage of residual TIA declined from 100% to 32% during this period. However, sonification time longer than 5 min. shows adding no extra effect on the inactivation of TI. Experiment conducted at temperature of 80 °C also shown the similar trends. It is well known that there are two types of trypsin inhibitor in raw soybean [16], namely Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). The content of KTI in soybean is about 1.4%) and that of BBI is about 0.6%. The KTI and BBI contain two and seven disulfide bonds respectively. Since disulfide bonds stabilize the native conformations of proteins [17], thus BBI is much more stable than KTI to the effect of varying conditions

624 such as heat, acids and alkaUs. The experimental results indicated that BBI is also extremely stable towards the effect of ultrasound. Therefore, under the operation of ultrasound, about 30% of residual TIA is always difficult to inactivate.

20.8

1 UVJ

o

c«

bJ3

15.6

Xi

c H 10.4 c a,

>. ^

40

60

80

100

T E M P E R A T U R E (^C)

Figure 1. Effect of temperature on TIA when treated by: • -heat treatment; ^ -heat & ultrasound with power 150 W for 8 min.

80 -

0) r ^

20

Q

^-^ < ^

\

\

^

H 60 W P^

<:

\

40

\^v \ i

H

*~'^=«=^^=*

^ 20 0 0

2

4

6

6

TIME (mm.)

Figure 2. Effect of sonication time on TIA when treated with power 160 W and at: O - 70 °C; • - 80 °C

3.3. Effect of ultrasound power on the inactivation of TI The effect of ultrasound power on the inactivation of TI is shown in Figure 3. When the applied power is lower than 130 W, the inactivation effect of TI is readily increased as the ultrasound power increases. The effect of ultrasound on the inactivation of TI recedes as the power of 150 W or higher is applied. This may be explained as that ultrasound power of about 150 W is enough to inactivate most KTI, while BBI is not affected by ultrasound power up to 170 W at given experimental conditions. 3.4. Effect of pH on ultrasound inactivation of TI Soybean TI was found to be more heat labile under alkaline conditions [15]. Heating in alkaline solution may cause more rapid destruction of disulfide bonds, which are important in the stability of soybean TI. However this phenomenon was not observed when heat and ultrasound treatment were applied. Figure 4 shows that inactivation of TI in soymilk is more effective at neutral pH than at acidic or alkaline conditions. 3.5. Effect of cosolutes on ultrasonic inactivation of TI Previous studies have shown that the stability of proteins in aqueous solution may be affected by the presence of cosolutes [10, 18]. The effects of soymilk concentration and NaCl concentration on the inactivation of TI are shown in Figures 5 and 6 respectively. Figure 5 indicated that the effect of solid contents of soymilk on the inactivation of TI is less profound compared to other influent factors. Figure 6 shows that NaCl concentration up to 0.5 M

625 facilitated the ultrasonic inactivation of TI significantly. For soymilk, concentration of NaCl higher than 0.5 M will affect its taste and no further investigation was carried out at such high NaCl concentration.

100

0

30 60 90 120 150 180 U L T R A S O U N D P O W E R (W)

Figure 3 Effect of ultrasonic power on TIA when treated at 70 °C and pH 7.0 for 5 min.

0

1 2

3

4

5

6

7

S O Y M I L K S O L I D C O N T E N T (%)

Figure 5. Effect of soymilk concentration on TIA when treated by heat & ultrasound

Figure 4. Effect of pH on TIA when treated by heat & ultrasound at 70 °C & power 160 W for 5 min.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 N a C l C O N C E N T R A T I O N (M)

Figure 6. Effect of ionic strength on TIA when treated by heat & ultrasound

626 4. CONCLUSIONS This study shows that the appHcation of ultrasound with mild heat treatment at atmospheric pressure can facilitate the inactivation of TI. Treatment temperature is the most important factor in such operation. Other influent factors are sonification time, ultrasound power, pH of the solution and ionic strength. Under the condition of temperature 80 °C, ultrasonic power 150 W, and pH 7.0, about 73% of TI was inactivated for 5 min treatment. About 27% residual of TI was difficult to inactivate. This residual component is mainly Bowman-Birk inhibitor (BBI) which is extremely stable to the ultrasound of 20 kHz.

5.

ACKNOWLEDMENT

This work was financially supported by the Research Committee of Hong Kong Polytechnic University.

6. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

I.E. Liener and M. Kakade, Toxic Constituents of Plant Foodstuffs, Academic press. New York, 1980. A.K. Smith and S.J. Circle (eds.), Soybeans: Chemistry and Technology, Vol. I, Proteins, AVI Publishing, Westport, CT, 1972. K.C. Kwok, W.H. Qin, and J.C. Tsang, J. Food Sci. 58 (1993) 859. D. J. McClements, Trends Food Sci. & Tech., 6(9) (1995) 293. S. Barton, C. Bullock and D. Weir, Enzyme & Microbial Tech., 18(3) (1996) 190. M. Sakakibara, D. Wang, R. Takahashi, and S. Mori, Enzyme & Microbial Tech., 18(6) (1996) 444. J. D. FlorosandH. Liang, Food Tech., 48(12) (1994) 79. H. S. LiUard, Food Tech., 48(12) (1994) 72. P. Lopez, F. J. Sala, J. L. de la Fuente, S. Condon, J. Rosa and J. Burgos, J Agri. & Food Chem. 42(2) (1994) 252. P. Lopez and J. Burgos, J. Agri. & Food Chem., 43(3) (1995) 620. C. Smith, W. Van Megen, L. Twaalfhoven, and C. Hitchcock, J. Sci. Food Agric. 31 (1980)341. E.A. El'piner, A.V. Sokolskaya, and M.A. MarguHs, Nature 208 (1965) 945. K. Davies, M. Delsignore, and S. Lin, J. Biol. Chem. 262 (1987) 9902. N.K.D. Kella and J.E. Kinsella, J. Biochem. Biophys. Methods 11 (1985) 251. T. J. Mason and J. P. Lorimer, Sonochemistry, EUiis Horwood Ltd., Chichester, England, 1988. M. Jr Laskowski and I. Kato, Annu. Rev. Biochem. 49 (1980) 593. H. Neurath and R. L. Hill (eds.). The Role of sulfur in proteins (3rd ed.). Academic Press, New York, 1977. S. Timasheff, Annu. Rev. Biophys. Biomol. Struct., (1993) 67.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

627

Evaluation of shelf life of flavored dehydrated products using accelerated shelf life testing and the WeibuU Hazard sensory analysis M. Bill and P.S.Taoukis National Technical University of Athens, Department of Chemical Engineering, Laboratory of Food Chemistry and Technology, 15780 Athens, GREECE

Abstract The shelf life of foods is a function of their composition, processing, packaging and environmental factors, most notably temperature. For dehydrated foods, end of shelf life is usually signaled by an unacceptable loss of sensory attributes. Since the time to reach this level of unacceptabihty, under normal storage conditions, is targeted to be 12 to 24 months, techniques of Accelerated Shelf Life Testing (ASLT) are employed to determine the shelf life of such products within a reasonable length of time. Use of Weibull Hazard Analysis facilitates the effective application of ASLT with sensory evaluation by allowing the use of a practical panel size and easy quantitation of the results. These can be used to model the shelf life behavior and to extrapolate from accelerated to normal conditions. The degradation of the intense sweetener aspartame was studied in a gelatin-based dessert with a fruity flavor. Tests were conducted at 45, 50 and 60°C and the end of shelf life, expressed as unacceptably low level of sweetness, was determined by sensory evaluation as 70.4, 51.9 and 24.3 days respectively. An activation energy of degradation of aspartame, E^, was calculated as 15.1 kcal/mol, from which a shelf life for the product stored at 20°C of 554 days was estimated. Sensory results correlated very well with HPLC measurements of the aspartame degradation giving practically the same E^, and showing that end of shelf life coincided in all cases with 60% remaining aspartame.

1. INTRODUCTION Quality and shelf life are food product attributes that interest all parties involved namely, producers, food scientists, food manufacturers, legislators and food control authorities and consumers. Despite the wide use of these terms and a tendency to consider them as self explanatory, their definitions and the approaches to quantify them can vary considerably, often being product type specific or dependent on their intended use. An early comprehensive definition of food quality, conforming to the current attitudes and terminology, was given

628 by Kramer and Twigg [1] as "the assemblage of properties which differentiate individual units and influence the degree of acceptability of the food by the consumer or user". As foods are intrinsically active systems, both physicochemically and biologically, there is a finite time period after production during which they retain a required level of quality, organoleptically and safetywise. This is the shelf life of the food product. There are different working definitions of shelf life. High Quality Life (HQL) is the time from production of the food for a just noticeable sensory difference to develop. Practical Storage Life (PSL) is the period of proper storage after processing of an initially high quality product during which the organoleptic quality remains suitable for consumption or for the intended process. PSL can be two to three times longer than HQL. Time of minimum durability is the time during which the foodstuff retains its specific properties under reference storage conditions. This definition refers to product characteristics and not to considerations of its use. However, characteristic properties (e.g. flavors) are overlaid, and it has to be considered when the change in a certain characteristic (such as flavor loss or off flavor development) is detectable by the consumer. Any working definition has to be connected to further guidehnes. Thus the meaning of organoleptic quality has to be accurately defined with reference to appropriate methodology and criteria for specifying acceptability hmits [2]. Sensory evaluation by a trained panel, whereby the food is graded on a "standardized" hedonic scale, usually best approximates the overall quality state of the food [3]. However, there are difficulties in estabhshing a meaningful scale for each food product. Even if we can accept an expert panel's results as indicative of consumer preference [4], a cut-off level of acceptability has to be set. The time at which a predecided large percentage of panelists judge the food as being just beyond that level is the end of shelf life (PSL). Chemical, microbiological and physical tests are being used alternatively in the study of food quahty. Characteristics used by the consumer for evaluation of a product, such as flavor, color and textural properties can be measured instrumentally or chemically. The study of the chemical and biological reactions and physical changes that occur in the food during and after processing allows the recognition of those most important to its safety, integrity and overall quality. Physicochemical or microbiological attributes can be used to quantffy quality. They can be correlated to sensory measurements for the food, and values corresponding to the lowest Hmit of organoleptic quality can be established. However, correlation of values of individual chemical parameters to sensory data is often difficult or even misleading. Overall organoleptic quality is a composite of more than one changing factor [5], and the relative contribution of each to the overall quality can vary at different levels of quality or at different storage conditions. Despite the aforementioned difficulties, use of appropriate sensory methodology combined with proper appUcation of chemical kinetic principles to food quality loss allows for the efficient design of tests and the analysis of their results. This can lead to shelf life predicting models.

629 The rate of food quality change may, in general, be expressed as; a function of intrinsic factors, such as the concentration of reactive compounds, inorganic catalysts, enzymes, reaction inhibitors, pH, water activity, and microflora and extrinsic factors, such as temperature, relative humidity, total pressure and partial pressure of different gases, Ught and mechanical stress. In low moisture systems the most important factors are temperature and water activity. The latter is controlled by packaging. Loss of shelf life in a food or an individual ingredient is usually evaluated by the measurement of a characteristic quality index, A. The change of A with time, t, can be usually expressed as: f(A) = k t = kA exp(-EA /RT) t

(1)

where f(A) is the quality function of the food and k the reaction rate constant. The rate constant is an exponential function of inverse absolute temperature, T, given by the shown Arrhenius expression, where k ^ is a constant, E ^ is the activation energy of the reaction that controls quality loss and R the universal gas constant. The form of the quality function of the food depends on the apparent reaction order. To study quaUty loss of dehydrated foods that usually have long shelf Uves at ambient temperatures, sometimes in the range of two or more years. Accelerated Shelf Life Testing (ASLT) techniques are often employed. The concept of ASLT is to determine the shelf life of a food product using results from abuse conditions, thus predicting the true shelf life through the use of the Arrhenius equation with extrapolation. That cuts down substantially the testing time. ASLT principles are applicable to methods using sensory techniques in order to predict shelf life. There are two main categories of tests that may generally be used for this purpose: Difference tests (and especially paired comparison, duo-trio -usually in the variation of difference from control test- and triangle tests) and tests using an appropriate scale (hedonic or of some specified attribute). A practical approach, which effectively combines ASLT principles and sensory methodology, is the Maximum Likelihood Graphical Procedure or Weibull Hazard Analysis. The Weibull method is based on the assumption that at an early time a moderate level of probability of failure exists. This probability drops close to zero until the food approaches the true end of shelf-life, where it rises sharply. Thus, the hazard plot, which describes the failure rate, assumes the shape of a bath-tub tj^e curve [5, 6]. In this study the shelf life behavior of a fruit-flavored dehydrated dessert mix sweetened with aspartame (APM) was modeled using the described methodology. Aspartame, a-L-aspartyl-L-phenylalanine methyl ester, is an intense sweetener increasingly being formulated into a variety of commonly consumed food products. During storage aspartame degrades and a number of decomposition products are formed [7]. Aspartame has varying stabiUty in aqueous solutions with maximum degradation rates at pH 6-7, e.g. a half life of 1 week at 20°C for

630 commercially sterilized skim chocolate milk has been reported [8]. In low moisture food systems stability is dependent on water activity [9]. Most low moisture products have a long targeted shelf Ufe (usually more than 24 months). Sweetness loss due to aspartame degradation can limit the sensory shelf life of the product and is used as the basic quality index of the product. The industrial products are usually overcompensated in aspartame, to still be of acceptable sweetness when 40%-50% of the initial quantity has degraded [10].

2. ASLT AND WEIBULL HAZARD ANALYSIS The principles and the methodology for conducting effective Accelerated Shelf Life Testing (ASLT) are described by Labuza and Schmidl [11], Taoukis and Labuza [10] and Taoukis et al. [2]. The following steps summarize the ASLT design: a. Assessment of the microbiological safety factors for the studied food product and process, and determination of the basic biological and physicochemical reactions that can be used as quality loss indices. b. Selection of the appropriate package for the shelf life test. Dehydrated products as in this study should be stored so that their water activity, a^, is constant (e.g. in sealed glass vials). c. Decision on (at least two) accelerated storage temperatures and estimation for each of the total experiment time based on target shelf life at 'normal' storage temperature and expected temperature dependence (e.g. range of Q^Q value from previous studies or reports on similar products, where Q^Q is the ratio of the deterioration rate of the food at two temperatures differing by 10 C, i.e. QIO~^T+IO^T)- Determination of the minimum frequency of testing can be based on that of the highest temperature as: At2=AtiQio^T/10

(2)

where At^ is the time between tests at highest test temperature T^; At2 is the time between tests at any lower temperature T2. If Q^Q is not accurately known, the time between tests should be reduced. Besides, use of too long intervals may result in an inaccurate determination of shelf life. At each storage condition, the minimum number of data points is six, in order to minimize statistical errors. d. It is essential to plot the data while the test is still underway in order to decide whether the test frequency should be increased or decreased. e. Determination of reaction order and rate from each test storage condition and prediction of the shelf life at the desired actual storage conditions using the appropriate Arrhenius plot. The Weibull probability function has been widely used in engineering to describe failure phenomena [12]. It was proposed by Gacula and Kubala [6] for

631 shelf life testing and reviewed with step by step methodology by Labuza and Schmidl [3]. The steps they described for carrjdng out a sensory shelf-life experiment using Weibull hazard analysis are as follows: 1. The time Umit for the study is decided based on the actual or desired end of shelf life using kinetic predictions. In the case of ASLT it is the time expected for the accelerated condition, as above. The time is divided into equal or unequal segments, depending on cost and time availabihty. 2. The recommended number of paneHsts at the initial time is between 8 and 10, though as few as two can be used. The number of subjects is given the value of n. 3. The panel is given a stored sample for evaluation in any one of four ways: a) Evaluation of only a stored sample [acceptable(+)/unacceptable(-)]. In this case the panelists are not provided with a control sample and the situation is more like real consumer conditions. b) Evaluation in the previous way, but with comparison to a control sample as reference. c) Scoring of some attribute after the definition of a difference from the initial score as index of unacceptabiUty. d) Using an appropriate objective test. 4. As the data are collected, a time table with the scores from the subjects' values is filled out using a plus (+) for an acceptable sample and a minus (-) for an unacceptable one. A constant C must be selected which represents the increase in the number of the paneHsts for each next test time period. If n^ is the number of subjects at time i, n^+i = n^ + C is the number of subjects at next time i+1. C is usually given the value zero or one. 5. The method has an acceleration phase. This begins when at least 50% of the panelists identify the product as unacceptable. Then the number of the testers for the next period is n^+i = n^ + C + nf where Uf is the number of assessors who gave a minus value to the stored sample at time i. 6. The testing interval is shortened for the next time period, as the product gets closer to the end of its shelf life. 7. At the next test time, the test is terminated provided that no more paneHsts or samples are available. The rank scores are determined for use in the plot method. The plot method is based on the cumulative hazard function, H(t), derived from the WeibuU probability function: H(t)=(t/a)P or log(t)= (l/p) log(H) + log(a)

(3a) (3b)

where a is caUed the scale parameter and (3 the shape parameter. The shape parameter can be calculated as p=(l/o)(7c/6l/2), with a the standard deviation of the natural logarithm of samples that were judged expired, or directly read from the WeibuU Hazard probability paper (Team Technical and Engineering Co.,

632 NH), in which log of storage (shelf) time is plotted vs log of cumulative hazard, expressed as EH. This plot is a straight line. It was calculated that for the probability of a spoiled sample to be recognized, i.e. PQ to be 50%, the %SH value is 69.3, if P is larger than 2. This value allows the calculation of the end of shelf life time by linear regression of log(t) vs log(%EH). Following the above 7 step procedure, a rank score is assigned to each minus value starting from 1 for the bottom left one and increasing by one for the others. The WeibuU Hazard value is expressed as H=100/rank and ZH can be calculated.

3. MATERIALS AND METHODS The food studied was a dehydrated low calorie dessert mix sweetened with aspartame (APM) with ingredients : gelatin, citric acid, APM (3.4%), fruit flavor and natural coloring. The water activity of the product was 0.32. Before consumption or sensory evaluation it was diluted and prepared according to instructions. One of the most important stages in running an experiment using sensory analysis is that of panel training. In the case studied, the training focused on recognition of differences in concentration of the four basic tastes (one series with solutions in ascending order for each taste) and on recognition and differentiation between them. In the first mentioned part of training, the panel was famiharized with two different types of thresholds (detection and recognition threshold). The methods used were those proposed in ISO 3972 [13]. Particular attention was given to the sweet taste. In the tests aspartame was also used in addition to sucrose since it was expected to be the most important factor for prediction of the product's shelf Hfe. The detection and recognition threshold were calculated for each assessor and for the team with a variation of the method ASTM E-679 (1991). CosteU et al. [14] described the original method. In order to validate the selection of APM degradation as the quahty index, two experiments were conducted using the triangle test. In the first the samples were a control and an aged sample with 31.39% remaining aspartame, and in the second one the samples were a control and an aged one (remaining aspartame: 37.31%) with aspartame compensated to 100% (aspartame was quantified by HPLC in both cases). The experimental design is outHned below: -The samples were isothermally stored at 45, 50 and 60°C. -The control sample was stored at -20°C. -The sampling frequency was determined by ASLT principles. A sample of 10 g was taken every 7 days from 45°C, and a sample of 15 g every 5 days from 50°C and three times weekly from 60°C. After that, the samples were stored at -20°C until they were sensory tested. It should be mentioned that the testing frequency was shorter than the sampHng frequency and depended on the WeibuU method's phases. According to this plan, every third sample was tested at the normal

633 phase; at the acceleration phase, where the interval time is shortened, the test was made on the second sample. Three different tj^es of tests were conducted. The first one was a difference ficom control test, where the panelists had to score a stored sample as acceptable or unacceptable comparing it to a control. The answers were used for prediction of shelf life using the Maximum Likehhood Graphical Procedure. The second was an overall hking hedonic test, using a 7-point discrete scale, and the third one was a test for measuring sweetness using a 9-point continuous scale. In both hedonic and sweetness tests the panel was provided with a control sample in order to be able to make a comparison.

4. RESULTS AND DISCUSSION The methods used in training the panel resulted in ranking of the paneHsts. According to ISO 3972 [13] no statistical methods were used. The thresholds calculated for aspartame and sucrose confirmed the relative sweetness of the two sweeteners. The thresholds for detection and recognition for sucrose were 0.497 g/1 and 2.524 g/1, whereas for APM they were 0.00373 g/1 and 0.0144 g/1. This corresponds to a sweetness intensity ratio of the APM to sucrose of 133 and 175 respectively. The triangle tests verified the selection of aspartame degradation as being the basic quality index. Namely the aged sample with degraded APM was easily judged as different from the control. More importantly the compensated (to 100% APM) aged sample could not be distinguished from the control, as was shown from the statistical analysis of the paneHsts responses. The results from the shelf life experiment are shown below. The method for predicting shelf life using sensory analysis and the Weibull plot is presented for the sample stored at 45°C.

Table 1 Weibull Hazard test score table with rankings Subjects and samples Test time E F G H C D A B (days) + + + + + + + + 0 + + + + + + + + 21 + + + + + + + (44) 42 + + + + (40) 63 (W) (4) + + 77 (&) (6) (^) W

m

I

J

K

L

(^)

+

(8)

(9)

m) m)

Table 1 is the time table showing the panel's scores and Table 2 is the Weibull Hazard ranking table. Using the Weibull plot paper, the shape parameter can be calculated and a shelf life prediction can be made from the plot of log time versus

634 log(IH) (Figure 1). The shelf life, i.e. the t value corresponding to %ZH=69.3, shown graphically, and it was calculated by linear regression as 70.4 days.

Table 2 Weibull Hazard ranking table Rank Shelf time (days) H=10Q/Rank 7,14286 42 14 7,69231 63 13 8,33333 63 12 9,09091 63 11

63 77 77 77 77 77 77 77 77 77

10 9 8 7 6 5 4 3 2 1

10

11,1111 12,5 14,2857 16,6667

20 25 33,3333

50 100

IS

EH* 7,14286 14,8352 23,1685 32,2594 42,2594 53,3705 65,8705 80,1562 96,8229 116,823 141,823 175,156 225,156 325,156

'^(ZHi+1 = IHi +Hi+i, i > 0 and ZHo=0)

Similarly the end of shelf life at 50 and 60°C, expressed as unacceptable level of sweetness, was determined as 51.9 and 24.3 days respectively. From the Arrhenius plot (Figure 2) of these values an activation energy of degradation of aspartame, E^, was calculated as 15.1 kcal/mol (R2=0.996).

100

10

ZH

100

1000

Figure 1. Weibull plot for sensory data for the sample stored at 45°C.

0,00293

0,00313 1/T

0,00333

Figure 2. Arrhenius plot for shelf Hves calculated using Weibull Hazard Analysis.

635 The sensory shelf life results were compared against instrumentally measured values of APM concentration using equation (4) for the three storage temperatures and a^=0.32 of the product. This temperature (T) and water activity (a^) dependent model of aspartame degradation was developed for the same food product [15]. Multiple T and a ^ conditions of product storage were used and APM was measured with time by a reverse phase HPLC method [7]. Sensory results correlated very well with HPLC measurements of the aspartame degradation showing that end of shelf life coincided to an average of 60% remaining aspartame. ln(APM/APMo)= -ko exp(p.a^ - 1 ^ ) t Ki

(4)

where: ko=7.73 xlO^ d i, (3 =2.25 and E^ =13.6 kcal/mol. The change in hedonic and sweetness perception (AH and AS respectively) was plotted versus time (Figures 3 and 4 ) and the difference corresponding to end of shelf life was calculated using the times estimated by the Weibull method (marked on the plots as points). Average difference values of AH=3.1 and AS==3.8 as hmit of acceptabihty for overall hking and sweetness are calculated. The dependence on temperature or the rate of change of hking and sweetness scores (slopes of regression hnes) followed the Arrhenius function. The respective activation energies were 16.1 kcal/mol (R2=0.972) and 15.2 kcal/mol (R2=0.999).

0

20 40 60 SHELF TIME (DAYS)

80

Figure 3. Change in hedonic scores versus time. Regression Hnes at the three temperatures.

Figure 4. Change in sweetness scores versus time. Regression hnes at the three temperatures.

5. CONCLUSIONS A systematic approach was used that allows shelf life predictive modeUng of food systems with well-defined quahty indices, such as the disappearance of a characteristic flavor or the development of an off flavor. Combined appHcation of

636 ASLT methodology and the Weibull Hazard graphical approach allows the practical and quantitative use of sensory evaluation for long shelf life products. It is very important to use appropriate preHminary experiments to verify and justify that the selected quality index (e.g. flavor) is the main shelf life limiting factor. In this study the suitability of APM as the limiting factor was validated by the triangle sensory testing. That the sensed end points of sweetness acceptability were all close to the same instrumentally measured level of 40% APM degradation reinforces this assumption and also justifies the use of ASLT even at the high storage temperature of 60°C. Caution should be exercised for other systems of flavors where ASLT conditions above 40 to 45°C might not be advisable. The activation energies calculated for sweetness by the Weibull graphical method and from the scale rating approach were practically the same. The small difference from the temperature dependence of overall liking, although well within the statistical confidence Hmits of the estimated parameters, might indicate that besides sweetness other minor factors could be contributing to the shelf life degradation of this very stable food product at higher temperatures. Based on sweetness, a shelf fife for the product stored at 20°C of 560 days can be estimated. This is within the expected range for such products and can be easily extended to e.g. 2 years by an appropriate initial overcompensation of APM. The obtained kinetic information can also be used for the estimation of the consumed fraction of shelf life of the products under variable storage conditions and the remaining shelf life under any assumed further conditions [2].

6. REFERENCES 1 Kramer and Twigg, 1968. Measure of frozen food quality and quality changes. In "The freezing preservation of foods" 4th Ed., Vol. 2, D. K. Tressler(ed), AVI Pub. Co., Westport, Conn. 2 Taoukis, P. S., Labuza, T. P. and Saguy, I. S., 1997. Kinetics of Food Deterioration and Shelf-life Prediction. In: Handbook of Food Engineering Practice, 1st ed., CRC Press, New York, Ch. 9, pp. 361-403. 3 Labuza, T. P. and Schmidl, M. K , 1988. Use of Sensory Data in the Shelf Life Testing of Foods: Principles and Graphical Methods for Evaluation. Cereal Foods World, 33 (2): 193-194, 196-198, 200-206. 4 Mackie, I. M., Howgate, P., Laird, W. M., Ritchie, A. H. (1985) Acceptability of frozen stored consumer fish products. I.I.F. - I.I.R. - (Commisions C2, D3) 1985-4, p. 161-167. 5 Sontag-Trant, A., Pangborn, R. M., Little, A., 1981. Potential fallacy of correlating hedonic with physical and chemical measurement. Journal of Food Science, 46: 583-588. 6 Gacula, M. C. Jr. and Kubala, J. J., 1975. Statistical models for shelf-life failures. Journal of Food Science, 40: 404-409.

637 7 Stamp, J. A. and Labuza, T. P., 1989. An ion-pair high performance liquid chromatographic method for the determination of aspartame and its decomposition products. Journal of Food Science, 54: 1043. 8 Bell, L. N. and Labuza, T. P., 1990. Aspartame Degradation as a Function of Water Activity. In: Water relationships in foods, Levine, H. and Slade, L. (Eds.), Plenum, New York, pp. 337-349. 9 Bell, L. N. and Labuza, T.P., 1991. Aspartame degradation kinetics as affected by pH in intermediate and low moisture food systems. Journal of Food Science, 56: 17-20. 10 Taoukis, P. S. and Labuza, T. P., 1996. Summary: Integrative Concepts. In: Food Chemistry, 3rd ed., Fennema, 0 . (Ed.), Marcel Dekker, New York, Ch. 17, pp. 1013-1042. 11 Labuza, T. P. and Schmidl, M.K., 1985. Accelerated shelf-life testing of foods. Food Technology, 39(9): 57-62, 64. 12 Weibull, W. 1951. A statistical distribution function of wide applicability. J. AppUed Mechanics, Sept. 1951, p. 293-297. 13 ISO, 1991. Sensory analysis-Methodology-Method of investigating sensitivity of taste. ISO Standard 3972, 2st ed.. International Organization for Standardization, Geneva, Switzerland. 14 Costell, E., Pastor, M. V., Izquierdo, L. and Duran, L., 1994. Comparison of simplified methods for evaluation of sensory threshold of added substances. Journal of Sensory Studies, 9: 365-382. 15 Taoukis , P.S. and Skiadi, 0 . 1997 Shelf Hfe prediction modeUing of packaged dehydrated foods for dynamic temperature and relative humidity storage conditions. Food Quahty ModelHng Workshop. COST 915 and Copernicus CIPA-CT94-0120. EU Comission. June 4-6, Leuven, Belgium.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

639

Behavior of histamine during fermentation of fish sauce determined by an Oxygen Sensor using a purified amine oxidase N.G. Sanceda/ E. Suzuki,^ and T. Kurata' "Institute ofEnvironmental Science for Human Life, Ochanomizu, University, Tokyo 112, Japan, ^Department of Human Biological Studies, Ochanomizu University, Tokyo 112, Japan Abstract Addition of up to 2% histidine to fresh fish during fermentation did not bring any significant changes in the histamine level of the products. However, when added to spoiled fish, histamine content rose to a high level. There was a continuous decrease in the amount of histamine formed during incubation time which may suggest the presence of histaminedecomposing bacteria in the samples. In spite of the decrease in the content of histamine, the total amines in the spoiled samples continued to increase as incubation time prolonged. All the commercialfishsauces analyzed contained very low histamine levels. The increase in histamine at the initial stage, and the decrease in histidine might suggest histidine was converted to histamine by microorganism possessing the enzyme histidine decarboxylase

1.

INTRODUCTION

Biogenic amines have been found to occur during processing of foods which include fishery products and other fermented foods [1]. Amino acid decarboxylation is the main mode of biosynthesis of these amines [2]. It has been reported that many microorganisms contain amino acid decarboxylase and can produce amines from natural amino acids [3]. These compounds are generally vasoactive and can cause changes in blood pressure. Severe headache, hypertension, renal intoxication or in other severe cases, intracebral hemorrhage and eventually death [4-5] are the most common effects of these amines. Histamine which is one of these amines, results fi'om ingestion of foods containing unusually high levels of histidine [6-7]. Scombroid poisoning has occasionally broke out as a result of ingesting fish such as saury, tuna, mackerel and bonito, which are characterized to contain high levels offi'eehistidine in their tissues [8-10]. Also, non-scombroid fish belonging to the families Pomatomidae (bluefish), Coryphenaenidae (mahi mahi), Carangidae (jack mackerel, amberjacks, yellowtail), Clupidae (herring, sardines), and Engraulidae (anchovies) have occasionally been implicated [11], however, only spoiled fish of these species can cause histamine poisoning. Fish containing hazardous levels of histamine are not detected by sensory tests. High levels of histamine in the incriminated fish are generally formed via microbial decarboxylation of histidine [12-14]. It has been reported that trimethylamine, trimethylamine oxide, agmatine, and choline [15], cadaverine [16], putrescine [17] are food-borne potentiators of histamine toxicity.

640 Fish sauce is a clear brown liquid hydrolysate of salted fish obtained after about a year salting which has a characteristic odor. In Southeast Asia, it is commonly used as a condiment, but in some areas and certain classes in the region, it is the main source of protein in the diet. It contains 20 g/L nitrogen, of which 16% is in the form of amino acids; thus they may be considered an important source of protein [18]. Our previous study has shown that addition of histidine accelerated fermentation process in the manufacture offish sauce [19]. Fermented fish products which include fish sauce and fish paste contain high amounts of histamine [20]. Histamine was reported to be present in noucman, a Vietnamese fish sauce [21], however, our previous work on histamine in commercial fish sauces (unpublished report) showed very low, if any, histamine in the product. Fujii et al. [22] reported that outbreaks of scombroidfishpoisoning are caused by ingestion of frozen-thawed fish and its products, even when the viable count of histamine-forming bacteria is low. The amount of histamine accumulated in the sample depended both on the bacterial production and decomposition of histamine 2324]. Hayashi [25] found that once accumulated, histamine content may decrease or and disappear possibly due to bacterial decomposition of histamine. This study aimed to investigate the behavior of histamine during fermentation process in the manufacture offish sauce. Histidine and other amines as well as amino acids remained in the sauce were quantified and the effect of salt on histamine formation was also determined. 2.

MATERIALS AND METHODS

2.1. Materials Fish of sardine family (Sardinops melcmostictus), 13 cm in length, were used. Commercial fish sauces were obtained from their country of origin. Histidine of standard grade was obtained from Nacalai Tesque Inc., Tokyo, and 99% NaCl was purchased from a Japanese supermarket. Fungal amine oxidase for histamine assay was obtained from Yamaguchi University, Yamaguchi, Japan and Kikkoman Corp., Chiba, Japan. 2.2. Methods 2.2.1. Sample preparation Unevisceratedfishwere cut 3-4 cm long and were used for all the experiments. Three sets of experiments were carried out. In the first experiment, histidine in the concentrations of 0.1, 0.2, 0.5, 1.0 and 2.0% was added to freshfishand incubated for 4 months. The mixtures were placed in layers in glass beakers, covered loosely with a parafilm, incubated at 30°C and then the liquid was collected. In the second of experiment,fishwere allowed to spoil for about a day, fi-om which, the following mixtures were prepared: only salt was added; histidine and salt were added; and only histidine was added. All the mixtures were prepared similar to the procedure in the first experiment but incubated for 16 days. In the third experiment, freshfishwere added with 30 (control), 20, 10, 5% NaCl. The same procedure used in the previous experiments was used except that incubation was carried out at 10°C to 30°C for 49 days. 2.2.2. Histamine analysis Histamine was assayed using an Oxygen Sensor KV-101 from Oriental Electric Co. Ltd., Japan, and employing afiingalamine oxidase [26].

641 2.2.3. Statistical analysis Test of significance was done using a Student T-test [27]. 3.

RESULTS AND DISCUSSION

The formation behavior of histamine during fermentation in the manufacture offish sauce was studied. Table 1 shows the concentrations of histamine in the fish sauces added with different concentrations of histidine during fermentation. The fish used were fresh. Results showed that in both the 2 and 4 months fermented samples, the levels of histamine in the 0.1, 0.2, 0.5 and 1.0 % histidine added samples were slightly lower than the control but not significantly different. In the 2.0% histidine added sample, the histamine seemed to be numerically higher than the control but the difference was not significant. The values of histamine in both the control and the histidine added samples were low and out of danger of food poisoning caused by histamine (Table 1).

Table 1 Histamine contents (mg/mL) of a histidine added fish sauces Incubation period (months) Added histidine {Vof 0 0.1 0.2 0.5 1.0 2.0

2 0.11 0.05 0.04 0.07 0.05 0.12

±0.03 ± 0.02 ± 0.02 ±0.01 ± 0.03 ±0.04

4 0.12 0.07 0.05 0.08 0.05 0.15

±0.02 ± 0.02 ±0.01 ± 0.03 ±0.01 ±0.01

"•Results are mean values of triplicate determination ± standard deviation. ^Histidine was added to fresh fish before incubation.

The sauces were tested and no symptoms of histamine poisoning, gastrointestinal (nausea, vomiting, diarrhea, abdominal cramps), or cutaneous (rash, urticaria, edema), or neurological (flushing, itching, burning, tingling, headache), [7, 28], were observed. This suggested that addition of histidine even in large amount did not result in the formation of histamine provided that the fish were in the fresh state. The very high concentration of salt used in the mixture during fermentation might inhibit the growth of microorganisms that could decarboxylate free histidine to form histamine. According to Chin and Koehler [29], formation of histamine and tyramine in miso appeared to be inhibited by high salt concentration. Good hygiene prevents bacterial contamination and plays a role in the formation of histamine. In the study conducted onfishstored at 5C [23], histamine was accumulated, decreased and disappeared as histaminedecomposing bacteria took over when the sample putrefied, but at 30°C, histamine did not always decrease. This suggests that the capacity of histaminedecomposing bacteria might be inhibited. They further described the formation of histamine from histamine-metabolizing

642 bacterial flora in fish sauce during fermentation [30]. Fujii et al. [22] reported that outbreaks of scombroid fish poisoning were caused by ingestion of fi-ozen-thawed fish and its products, even when the viable count of histamine-forming bacteria was low. The L-histidine decarboxylation activity of halophilic histamine-forming bacteria was highest at the beginning of the stationary phase of the growth and gradually decreased as the stationary phase proceeded [31]. This fact might explain the very low content of histamine in the commercial sauces analyzed in this study as shown in Table 2. All the commercial sauces analyzed, particularly those made in Japan showed a very low histamine content. The Japanese food industries are extra careful when it comes to hygiene in foods and food products, which might also explain the inhibited growth of microorganisms in the samples during fish sauce production. The Korean sauce contained a little higher amount of histamine compared with other samples analyzed, but it was still safe from human hazards as set by the U.S. FDA, [32]. AU.S. regulatory limit is set for histamine for tuna but not for other fish. The level which constitutes a human health hazard, is set at 50 mg/100 g tuna and is known as "hazard action level" [33]. Also, a "defect action level" has been established for tuna and is set at 10 mg/lOOg tuna when signs of decomposition are present.

Table 2 Histamine contents of commercial fish sauces Samples'"

mg/mL^ MeaniS.E.

Patis (Philippines)

A B C D E Nampla (Thailand) Nampla (Japan) Fish sauce (Korea) Shottsuru (Japan) Anaerobically fermented (Japan)

0.04 0.02 0.10 0.07 0.03 0.43 0.37 1.38

±0.01 ±0.01 ±0.02 ± 0.03 ±0.01 ±0.02 ±0.01 ±0.02

nd nd

""Values are average of two replicates. ''All samples were obtained from their country of origin. nd: not detected. Figure 1 shows the behavior pattern of histamine in the supernatant of fresh fish added with 30, 20, 10 and 5% salt during fermentation. Results indicate that histamine is hardly formed in the control sample (30%), but as the concentration of sah used decreased, the histamine content increased. The fish body deteriorated faster in the low salt concentration (10% and 5%) than in the higher concentrations (30% and 20%). These findings suggest that salt inhibits the growth of microorganisms that could enhance the formation of histamine during fermentation. In all samples, the content of histamine decreased from around the third or fourth day of fermentation and continued to decrease as fermentation progressed.

643 0.3• : Fish + 30% Salt 0.25 H F • &

•

• : Fish + 20% Salt A : Fish+ 10% Salt

0.2

• : Fish + 5% Salt

E

*

*

—

^

c o 0.15 01 i+-•

c 0)

o c

0.1

o

o 0.05-1

I I I I I I 1I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

5

10

15

20

25

30

35

40

45 50 (Days)

Figure 1. Histamine in fish sauce added with different concentrations of salt.

14

16 (Days)

Figure 2. Histamine in a fish sauce added histidine. Fish were lefl; to spoil for one day before use. 2% histidine was used. Salt was 99% NaCl.

644 Figure 2 shows the behavior pattern of histamine during the 16 days incubation. In the spoiled fish (control), histamine started to increase on the second day but gradually and continuously decreased fi'om the third day and until the end of the experiment. In the absence of salt, fish added with histidine showed a sharp increase in histamine on the second day and continued to increase until it reached its peak on the fourth day but started to descend following the same pattern in control sample. In the histidine and salt added sample, although the histamine was a little lower than the histidine added sample but without salt, a decrease in the content of histamine was also observed. In the mixture of fish and salt without any added, histidine, the change in the amount of histamine was not significant. The same decreasing tendency in histamine was also observed in the only fish sample (control spoiled) and decomposed linearly with the progress of incubation time. Among the samples analyzed, the histidine added sample without salt showed the highest production of histamine. It appeared that histidine added tofi"eshfishhad no significant effect on the formation of histamine but when added to spoiled fish, it enhanced the histamine formation. In short, histamine concentration increased at the initial stage of fermentation but decreased linearly with the progress in incubation time. Though this experiment was carried out in a short time, the very low concentration of histamine in fish sauces fermented for a long time (as commercial fish sauces) might support this behavior of histamine during fermentation. Figure 3 shows the remaining soluble histidine in the fish mixture after 16 days incubation. There was no change in the amount of soluble histidine in the fresh fish salt mixture added with histidine during the 16 days incubation It seems that histidine could be added to fish without undergoing degradation or change provided that growth of bacteria is inhibited, and in this study, the presence of salt in high concentrations and the condition offish before incubation were important. However, in the histidine added fish mixture without salt, the added histidine was almost retained a day after addition but abruptly decreased on the third day and continuously decreased until hardly detected on day 16. Although the relation between the decrease in histidine and the increase in histamine was not statistically determined, it could be that histidine was microbially decarboxylated to form histamine. The total amount of amines in fish and histidine added fish mixtures increased with incubation time. In the presence of salt, amines remained at a low level and remained constant throughout the incubation (Figure 4). This result seems to follow the pattern for the formation of histamine where salt inhibited the formation of histamine and addition of histidine to salted freshfishdid not increase the histamine in the mixture but when added to spoiled fish with or without salt, histamine content rose to a high level and the increase was parallel to the incubation time.

4.

SUMMARY

Addition of histidine to freshfishdid not increase the amount of histamine formed, contrary to that observed for spoiled fish; in both cases, histamine decreased as fermentation progressed. The amount of soluble histidine decreased with the progression of incubation time. The state offi'eshnessoffish greatly influenced the formation of histamine during the fermentation process in the manufacture offish sauce. The very high concentration of salt also inhibited the growth of microorganisms that could dccarboxylate histidine to form histamine. Furthermore, good hygiene had a great influence on the formation of histamine. The very low concentration of histamine in commercial sauces might be explained by the appearance, decrease and disappearance of histamine during fermentation.

645

• • A •

: Fish : Fjsh+Histidine : Fish+Salt : Fish+Salt+Hlstidine

=^=r12

14

16 (Days)

Figure 3. Histidine remained in a histidine addedfishsauce. Fish were left to spoil for one day before use. 2% histidine was used. 1.6-r

I.4J

J ^

• • A •

: Fish : Fish+Histidine : Fish+Salt : Fish+Salt+Histidine

H

I 0.6 H O 0.4 J 0.2 H

I

I

I

I

I

I

I

'

'

'

I

I

I

I

I

10

12

I

I -j—1

I

I

14

I

16 (Days)

Figure 4. Amines formed in a fish sauce added histidine. Fish were left to spoil for one day before use. 2% histidine was added.

646 5.

REFERENCES

1 2 3 4 5 6

J.A. Maga, CRC Crit. Rev. Food Sci. Nutr.,10 (1978) 373. S.L. Rice and P.E. Koehler, J. Milk Food Technol., 39, (1976)166. W. Lovenberg, J. Agric. Food Chem., 22 (1974) 23. D.M. Kuhn and W. Lovenberg, Lancet, 1 (1982) 879. P. Antila, Kieler Milchwirtschaftliche Forschungsberichte, 35 (1983) 373, S.L. Taylor and S.S. Sumner, Seafood Quality Determination, Proceedings of an International Symposium Coordinated by the University of Alaska Sea Grant College Program, D.E. Kramer and J. Liston (eds.), Elsevier Science Publishers B. V. Amsterdam, 1986. M.H. Merson, W.B. Baine, E.J. Gangaros and R. Swanson, J. Am. Med. Assoc, 228 (1974) 1268. T. Kawabata, K Ishizuka and T. Miura, Bull. Jap. Soc. Sci. Fish., 21 (1955) 335. S.H. Arnold and W.D. Brown, Adv. Food Res., 24 (1978) 113. M. Kimata and A. Kawai, Mem. Res. Inst. Food Sci. Kyoto University, 6 (1953) 1. S.L. Taylor, CRC Crit. Rev. Toxicol., 17 (1986) 91. M. Kimata, In Fish as Foods, G. Borgstorm (ed.). Vol. 1, 329, 1985. T. Kawabata, K Ishizuka, T. Miura and T. Sazaki, Nippon Suisan Gakkashi, 22 (1956) 41. J.E. Stratton, R.W. Hutkins and S.L. Taylor, J. Food Protec, 54 (1991) 460. M. Hayashi, J. Pharmacol. Soc. Jap., 74 (195 4) 1148. O. Arunlakshana, J.L. Mongar and H.O. Schild, J. Physiol, 123 (1956) 32. J.L. Parrot and G. Nicot, Pharmacology of histamine. Absorption de I'histamine par I'appareil digestif. In Handbook of Experimental Pharmacology, M. Rochae Silva (ed.). Vol. 18, Past I, Springer-Verlag, New York p. 148, 1966. R. Lafont, Proceedings of Indo-Pacific Fish Councol, 15* Meeting, Bangkok, Thailand Section II and II, p. 163, 1955. N. Sanceda, T. Kurata and N. Arakawa, J. Food Sci., 61 (1996) 220. D. Fardiaz and P. Markakis, J. Food Sci., 44 (1979) 1562. E. Cousin and B. Noyer, Rev. Med. Franc. dExtreme-Orient (Hanoi), 22 (1944) 82. T. Fujii, K., Kurihara and M. Okuzumi, J. Food. Protec, 57 (1994) 611. T. Sato, T. Fujii, T. Masuda and M. Okuzumi, Fisheries Science, 60 (1994) 299. M. Okuzumi, A. Hiraishi, T. Kobayashi and T. Fujii, Inter. J. System. Bacteriol., (1994) 631. M. Hayashi, J. Food Hyg. Soc. Japan., 11 (1970) 429. M. Ohashi, E. Nomura, M. Suzuki, M. Otsuka, 0. Adachi and N. Arakawa, J. Food Sci., 59(1994)1. NEC. Nee User's Guide: Basics, Ver. 3.0 Ed. Nee Micro. Corp., Japan, p. 69, 1983. C.K Murray, G. Hobbs, RG. and Gibcrt, J. Hygiene, 88 (1982) 215. K.D.H. Chin and P.E. Koehler, J. Food Sci., 48 (1983)1826. T. Sato, B. Kimura and T. Fujii, J. Food Hyg. Soc. Jap., 36 (1995) 22. K. Kurihara, Y. Wagatuma, T. Fujii and M. Okuzumi, Nippon Suisan Gakkaishi., 59 (1993) 1401. S.N. Whetstone, FDA's seafood regulatory program. International Canned Tuna Workshop, Songkia, Thailand, June 7, 1993. S.L. Taylor, Histamine Poisoning Associated with Fish, Cheese, and Other Foods, Monograph, World Health Organization, Washington, DC, 1985.

7 8 9 10 11 12 13 14 15 16 17

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

647

Effect of crystallization time on composition of butter oil in acetone F. M. Fouad\ O. A. Mamer\ F. SaurioP and F. ShahidP. ^The Biomedical Mass Spectrometry Unit, McGill University, 1130 Pine Avenue West, Montreal, Quebec, H3A 1A3, Canada ^Department of Chemistry, McGill University, Montreal, Quebec, H3A 2K6, Canada ^Department of Biochemistry, Memorial University of Newfoundland, St. Johns, Newfoundland, AlB 3X9, Canada

Abstract Kinetics ofthermal modification ofbutter oil in acetone at a constant temperature was studied. Anhydrous butter oil was stirred in 50% acetone by weight at room temperature in order to remove insoluble residues (SQ), mainly high molecular weight-saturated lipids. The resultant lipids in acetone were further subjected to cooling at 0°C for 10,20 and 50 min and the corresponding solid fractions (Sj, S2 and S3, respectively) were collected. The remaining liquid lipid (L) together with solid fractions Sj, S2 and S3 were characterized for their fatty acids and triacylglycerol (TAG) profiles. Results indicated that crystallization ofbutter oil in acetone at low temperatures may produce less saturated products, similar to those obtained via supercritical fluid extraction using CO2. The L and Sfractionswere found to contain the same TAGs, but in different proportions. Profiles of triacylglycerols and fatty acids of these lipid fractions were compared with corresponding results for butter lipidfractionatedby supercritical carbon dioxide (SC-CO2) or a dual process involving SC-CO2 of temperature controlled partial crystallization (TCPC) harvested liquid fraction of neat butter oil at 17°C(L. 17).

1.

mXRODUCTION

High contents of cholesterol and saturated long chain fatty acids in animal fat and butter have been associated with coronary heart disease (CHD). Accordingly, partially hydrogenated vegetable oils (margarine) were introduced as a nutritional substitute [1]. Economics of changing the consumption pattern ofbutter and margarine was costly in general to dairy industry and particularly to dairy farmers. Consequently, as early as the 1940's several investigators tried to develop procedures to improve the acceptability ofbutter via temperature controlled partial crystallization (TCPC) ofbutter oil. TCPC ofbutter oil was carried out to produce fractions having a fatty acid distribution that would yield lipids with physical characteristics more suitable for food and industrial applications [2-8]. In view of earlier results pertaining to synthetic or natural manipulation ofbutter oil to yield fractions with different spectra of triacylglycerols, fatty acids and accordingly physical properties.

648 we were prompted to examine TCPC of butter oil solutions in acetone at various time intervals at low temperatures. A simple one-step TCPC of anhydrous butter oil failed to yield lipid fractions that were characteristically different in their chemical composition and physical properties [2, 5]. The differences in triacylglycerol and fatty acid analyses between solid (S) and liquid (L) fractions of butter oil resembled those brought about by seasonal variations [2]. Crystallization of anhydrous butter oil is rather complex [3, 4] because of the problem oi crystal packing associated with the large number of triacylglycerols resulting from in-vivo substitution of at least 37 different fatty acids on the glycerol backbone [9]. Furthermore, it is possible that the unsaturated and polyunsaturated chains pack within the same layer and that saturated chains pack within unsaturated layers. The phase diagram of simple triacylglycerol binary systems, tristearin (SSS) and a mixed chain triacylglycerol, stearodipalmitin (PS? or SPP), best illustrates the intricacy ofthe crystallization pattern of butter oil and reflects the complexity of the interaction between triacylglycerols with similar structures. The SSS-PSP system has an eutectic mixture melting at 63.9°C at about 65 mole % PSP. On the other hand, the mole fraction of SPP in SSS at the eutectic temperature is about 27% [10,11], even though it differs only in the position ofthe stearic acid moiety. Therefore, temperature dependent co-nucleation of various triacylglycerols of butter lipids yields solid and liquidfractionswith the same spectrum oftriacylglycerols and fatty acids, but in different proportions [2] rather than fractions having different triacylglycerol and fatty acid compositions [5 ]. As a result, laboratory scale TCPC of butter oil failed to yield fractions with significantly different chemical and physical properties, namely melting range. Accordingly, co-nucleation occuring at various temperatures, rate of cooling, agitation and filtration is expected to minimize the spectral differences of chemical distribution of triacylglycerols and fatty acids and accordingly the physical properties of isolated lipidfractions[2, 7, 12, 13]. This contrasts with earlier conclusions that butter oil can befractionallycrystallized in a one-step process to yield products, which were markedly different in their physical and chemical properties [5] to warrant their commercialization as a healthy alternative to hydrogenated vegetable oils. Thus one-step TCPC may not be suitable for industrial food applications [5]. Therefore, it is the intent of this paper to examine TCPC of butter oil in acetone at various time intervals at low temperatures in order to produce lipid fractions with different characteristics. As previously reported [3], acetone was used to induce perturbation ofthe crystalline packing of various triacylglycerols of butter oil, which is expected to influence crystallization behavior of butter oil. However other solvents such as ethanol or petroleum ether could also be used. The harvested lipid fractions at various time intervals are expected to have a distinctly different triacylglycerol and fatty acid profiles which would reflect on their physical properties compared to TCPC of neat butter oil or butter oil solutions in organic solvents.

2.

EXPERIMENTAL

The effects of continuous lipid depletion at 0°C according to soLibiii^y aiid molecular weight on the profiles of triacylglycerols and fatty acids of isolated lipid solids at various time intervals from a solution of butter oil in acetone was examined. In general, butter was melted and kept at 60°C until complete separation of oily butter lipid and v^ater layers. Separated butter oil was dried over anhydrous sodium sulfate and then manipulated as described below. For efiicient separation ofcrystallized lipidfractions,Kenag milkfilters(Kenag Inc., Ashland, OH) were used which allowed complete separation of lipid crystals from mother liquor within 5 to 15 min. For fatty acid and

649 traicylglycerol analyses, fused silica capillary columns coated with SP-2340 or DB-5 stationary phase were used, respectively. Anhydrous butter oil was mixed with acetone (1:1, w/w) and stirred at room temperature to remove an insoluble residue, SQ. Filtrate was kept at 0°C where precipitated solid lipid fractions, Sj, Sj and S3 at 10, 20 and 50 min respectively were separated by filtration. A liquid lipid L was obtained from the final mother liquor by acetone evaporation. For the purpose of comparison with earlier results [2-9], butter oil was mehed at 60°C, treated with anhydrous sodium sulfate and fihered under vacuum (Whatman # 1 filter paper). The resulting filtrate was stored under nitrogen at -20°C until use. Molten neat butter oil, warmed to 60°C and separated into liquid and solid fractions at temperatures 29, 25, 21 and 17°C, without agitation [2]. The crystallized triacylglycerols were harvested using Kenag milk filters (Kenag Inc. Ashland OH) which allowed complete separation of crystals from the mother liquor within 5 to 15 min. The solid or liquid fractions obtained were used both as a reference material to compare butter lipids fractionated by supercritical carbon dioxide (SC-CO2) or crystallization at various time intervals from acetone solution at low temperature and as starting material, e.g., L.17 fraction, for further SC-CO2 fractionation. All samples were kept at -20°C until analyzed. Solid (S) and liquid (L)fractionswere designated according to the temperature at which they were isolated, e.g. S.17orL.17. SC-CO2 Extraction of Anhydrous Butter Oil In all experiments, research grade CO2 (99.995% pure, Medigas, St. Laurent, Quebec) was used in a Newport SC-CO2 apparatus (Newport Scientific Inc., Jessup, MD) with an extraction vessel of 0.85 L capacity maintained at 35°C. The separation vessel was kept at 61.2 atm (900 psi) and 30°C throughout each experiment. The pressure in the extraction vessel was set at 136 atm (2000 psi) and maintained constant while extraction of anhydrous butter oil samples was carried out for a period of 14hr. At 2hr intervals, the solubilized fraction was removed from the separation vessel without interruption of the run. The mass remaining in the extraction vessel at the end of the experiment was collected and analyzed. In a second set of experiments, the thermally fractionated L.17 lipids were subjected to SC-CO2 and isolated fractions were analyzed for their triacylglycerol and fatty acid profiles. 3.

RESULTS AND DISCUSSION

Upon stirring butter oil at room temperature in acetone (1:1, w/w) the remaining insoluble lipid residue, S^ was collected and found to be mostly composed ofhigh-molecular-weight saturated lipids. Perturbation of the crystalline packing of butter oil triacylglycerols by the combined effects of removing high molecular weight lipids and addition of acetone forced precipitation of lipids according to their molecular weights and solubilities (Table 1). Fraction SQ contained 40% less of C24-C36 and 50% more C44-C54 triacylglycerols than butter oil. Unsaturated and low-molecularweight triacylglycerols are expected to be more readily soluble in polar organic solvents. Such differences will be a function ofthe proportions oflow melting triacylglycerols extracted by acetone treatment, the amount of acetone used, and number of extractions employed. This effect decreased with progression of cry stallization time as similar changes in the C3 4-C3 6 and C44-C54 triacylglycerol content of Si, S2 and S3 (collected at 10,20 and 50 mins respectively at OT) were found to be 11% and -6%; +12% and -9%; and +14% and -11%, respectively. Most interesting is the increase (+27%), (+24%) and (+17%) in C36-C38 triacylglycerols in fractions Si, S2 and S3 with respect to butter oil, respectively while the C40-C42 lipids remained almost

650 Table 1 Kinetics of medication of butter oil in acetone solution at 0°C ACN 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54

Butter Oil 0.32 0.52 0.78 1.89 2.64 6.95 12.35 12.25 10.74 7.45 6.95 6.51 7.64 8.42 5.49 1.49

So

SI

S2

S3

L

0.02 0.11 0.52 1.25 1.77 3.68 7.98 7.60 7.04 7.08 9.36 12.06 13.18 12.48 6.56 1.32

0.05 0.14 0.27 0.66 1.43 4.96 15.18 15.99 10.77 7.59 6.35 5.34 6.33 9.84 5.41 1.07

0.05 0.27 0.72 1.28 2.94 7.68 15.48 15.07 10.82 7.44 6.11 5.91 7.59 7.26 4.93 1.37

0.15 0.37 0.75 1.65 2.87 8.21 15.15 13.62 10.91 6.86 5.62 6.31 7.98 6.24 4.96 1.29

0.27 0.88 1.77 3.14 5.94 8.76 10.98 14.59 11.42 8.81 5.71 5.45 4.23 4.28 4.47 1.29

ACN, Acyl carbon number; SQ, white powdered lipid; SI, S2 and S3, solid butter lipids harvested at 10, 20 and 50 min at 0°C respectively; and L, low molecular weight residual liquid lipid.

unchanged. The change in the content of C44-C54 (+50%, -6%, -9% and -11%) in SQ, SJ, SJ and S3 respectively is most likely due to the initial substantial depletion ofthese triacylglycerols contained in SQ fraction and their progressive depletion in Si and Sj. The opposite trend was observed for C24-C36 as these low molecular weight triacylglycerols are presumably more soluble in acetone. Similarly, proportions of medium molecular weight triacylglycerols C40-C42 in Sj, S2 and S3 remained almost unaffected (Table 1). It is interesting to note a 58% increase in C24-C34 and a 30% decrease in C44-C54 in the residual liquid fraction 1. Nevertheless, the proportions of C36C42 in fraction 1 remained almost unaffected as compared to butter oil. This observation is the likely basis for the observed low mehing temperature of3°C for the fraction 1. The same data (Table 1) shows that the triacylglycerol profile of SQ is similar to values reported earlier for solid fractions isolated by TCPC of butter oil at 29-2 TC and thoroughly washed with acetone to remove most ofthe low-molecular-weight triacylglycerols. Acetone-extracted low-melting triacylglycerols left behind a white lipid powder which showed a differential scanning calorimetry (DSC)-thermogram with one single sharp melting peak at 53°C and was characterized by one sharp peak on size exclusion HPLC. By comparison with parent butter oil, this white lipid was enriched 1.2 to 2.4 fold in C44-C52 triacylglycerols, 0.2 to 0.4 fold in C34-C40 and contained almost no C22-C32 triacylglycerols. The short chainfatty acidsC4:0-C10:0andc/5-unsaturatedfatty acids C14:l-C18:3 were reduced 90 and 70%, respectively, whereas medium-chain (C12:0-C15:0) and long chain (C16:0-C20:0) saturated fatty acids were both increased approximately 1.5 fold. A similar, but less pronounced, trend was observed when the S.25 and S.21 TCPC fractions were treated with

651 acetone. On the other hand, fraction 1 contained C24-C54 profiles very similar to that of butter oil extracted for 12 hr at 35°C and 136 atm (SC-CO2). Under constant pressure and temperature, SC-CO2 extraction of butter oil (203 g) removed a nearly constant percentage ofthe lipid with time; that is 15.6, 14.7, 13.4, 12.3, 11.5, 10.2 and 9.0 g of butter oil lipids were extracted at 2 hr interval over a period of 14 h. The initial extract contained larger proportions ofthe more volatile and lower molecular weight triacylglycerols [4, 15] and later fractions showed increasing proportions ofthe higher molecular weight triacylglycerols. The 2 hr extract contained approximately 2 times as much C22-C38 triacylglycerols as butter oil whereas the 12 hr extract contained about 1.5 times that amount. The residue, after 14 hr extraction, contained only -59% ofthe low-molecular-weight triacylglycerols and was enriched approximately 1.4 times as compared to that of butter oil in the C44-C54 triacylglycerols. In comparison, the 2 hr and the 12 hr extracts contained approximately 54 and 77% ofthe C40-C54 triacylglycerols ofthe butter oil, respectively. The 2 hr extracts, richer in low-molecular-weight triacylglycerols, showed lower melting range (10-12°C) and had altered DSC profiles with decreased proportions ofhigh melting range that corresponds to high-molecular-weight triacylglycerols, relative to butter oil. The C26-C34 triacylglycerols were increased from 100 to 600% with respect to their proportions in butter oil and the C42-C54 compounds decreased from 20 to 80%. However, these large increases in the low-molecular-wei^t triacylglycerols must be viewed in the context of their low relative proportions in butter oil (C26-C34 constituted - 9.8% ofthe triacylglycerol analysis). The extraction efficiency for C26-C34 is also reflected in the fatty acid data for 12 hr extracted sample, but to a lesser extent since most ofthe low molecular weight (C26 and C28) triacylglycerols had been removed by that time. The fatty acid profiles for the 2 hr and 12 hr extracts and for the residue also reflected the trend observed for triacylglycerol profiles as higher proportions of C4:0-C14:0 fatty acids were found in the 2 hr extract as compared to butter oil (butyrate showing an increase of 165%). However, the residue showed lower proportions of these acids than those present in butter oil. Conversely, the extracts showed lower proportions ofthe longer chain acids (CI 8:0, C20:0) than the starting material, while the residue contained higher proportions of these acids (exceptforC18:3 and C20:0 which wereslightly lower). The intermediate C 14:1, CI 6:0 and CI 6:1 acids were present in approximately the same proportions in both extracts, the residue and the butter oil. In the 2 hr extracted sample, the content of unsaturated cis- and trans-C\^ acids decreased by 38 and 35%, respectively, while that ofthe polyunsaturated CI 8:2 and CI 8:3 acids showed smaller decreases. The unsaturated CI8:0 acids were present in slightly higher proportions in the residue, relative to butter oil. Interestingly, the residual triacylglycerol profile (57%) after 14 hr extraction of butter oil corresponded to the S.29 fraction obtained in 10% yield with 4 hr TCPC of butter oil. The content of cholesterol in the lipid extracts was essentially constant at about 325 mg per lOOg extract, corresponding closely to its concentration in the starting butter oil [4, 14]. The potential of a dual-process treatment involving SC-CO2 and TCPC ofbutter oil to produce fractions with greater distinctness was examined. The C22-C38 triacylglycerols, which were increased in the L. 17 fraction by 25% relative to the starting material, were further concentrated by 82% after 2 hr SC-CO2 extraction at 35°C and 136 atm, and comprised 70% ofthe total weight oftriacylglycerols. There were corresponding decreases of 53% in the SC-CO2 extract ofthe C40C54 components, which were decreased by -12% in L. 17, compared to butter oil. Anticipated increases in the proportions of C4:0-C14:0 fatty acids were found, but those for C14:1 and C16:0 were essentially unchanged from their butter oil values. Other than the large increase in the butyrate residue, the fatty acid profile for this extract was virtually identical to that obtained for 2 hr extracted butter oil, but showed less ofthe CI 8 and C20 acids. The 2 hr extract of L. 17 (which represented -7% ofthe total butter oil) melted over a low range of 6 to 8 °C and showed a single HPLC peak,

652 eluting at 22.2 min, indicative of a fraction largely composed of low-molecular-weight triacylglycerols. Thus, TCPC of butter oil in organic solvents at low temperature produced, in a shorter period of time, a liquidfractionwhose profile was similar to that of SC-CO212 hr extract. A close examination of the gas chromatograms offractionsL and So,using an RTX-65tg column, revealed that similar triacylglycerols with a given acyl carbon number (ACN) have widely different distribution patterns in butter oil, SQ and Lfractions(Table 2, Figure 1). This is indicative of the different solubilities in organic solvents and resulting crystallization behavior of components with a given ACN but with different geometries and/or chemical structures. This observation supports a previous report ofthe widely varying crystallization behavior ofbinary mixtures of geometrically different triacylglycerols [11].

Table 2 Quantitative Distribution of triacyglycerols of individual lipids in ACN pattern of TCPC butterfractionsin acetone solution at 0°C L Butter _So ACN ACN Butter _So L 6.36 6.21 33.86 62.30 1 6.31 1 61.67 2 5.82 6.45 4.69 2 43.40 17.33 17.08 C:38 3 23.49 24.97 8.33 C:46 3 8.61 6.49 11.34 4 5.00 4.65 10.10 4 6.38 2.06 5.19 5 21.47 22.52 5.81 5 8.17 4.66* 4.51* 6 37.92 35.04 60.42* 1 34.41 55.63 11.35 1 15.13 20.91 4.31* C:48 2 45.23 24.34 43.68 2 9.53 11.97 8.29 3 10.89 6.32 20.56 3 17.16 17.19 2.73 4 9.49 5.38* 16.72* C:40 4 16.64 14.93 30.00 1 7.09 Nil Nil 5 5.63 5.62 3.09 2 1.65 Nil Nil 6 14.01 11.16 21.31 3 15.25 40.58 1.82 7 11.63 11.26 18.10 C:50 4 43.99 38.19 16.40 8 10.27 6.96 4.07 5 15.70 9.33 51.60 1 37.12 52.17 4.08 6 5.58 3.66 5.49 2 14.37 4.90 14.86 7 3.68 4.18 7.86 3 19.64 5.56 23.69 8 7.25 4.06 10.16* C:42 4 6.86 15.10 10.93 1 9.37 20.39 Nil 5 2.91 4.21 3.00 2 34.40 40.84 3.67 6 14.84 2.1 8.76 C:52 3 40.93 22.11 79.63 3.21* 7.15 4 3.1 2.63 Nil 7 4.28 62.14 Nil 5 12.20 6.04 16.70 1 28.64 13.51 56.20 1 7.65 21.32 Nil 2 6.21 C:44 3 26.08 6.69 13.22 C:54 2 23.60 38.56 54.52 4 10.00 5.61 30.58 3 49.36 32.72 45.48 3.8 4 19.39 7.41 Nil Nil 5 24.00 6 5.07 7.35 Nil For symbols see footnotes to Table 1, TCPC, temperature controlled partial crystallization. *In this ACN new lipid peaks appeared with concentrations ranging from 2-8% (see Figure 1).

653 Table 3 Distribution of identified triacyglycerols in butter and its TCPC fractions fi-om acetone at 0°C. Lipid

PPP

PPS

PPO

PSO

POO

sso oos

000

Butter So L

2.49 7.33 0.48

1.28 5.07 0.08

3.69 4.77 0.70

1.89 2.68 0.16

2.45 1.45 3.56

0.35 0.51

0.29 0.10

0.74 0.43 0.70

Table 4 Distribution of identified triacylglycerols in vegetable oils and animal body fat from TCPC fractions from acetone at 0°C Lipid

POO

000

Olive

17.39

21.31

Com

PPP

PPS

PPO

PSO

SSO

OOS

28.87

BBF

27.58

7.26

1.15

1.22

8.02

6.57

2.13

6.83

BKF

18.76

10.59

0.84

4.07

9.95

15.15

1.19

7.23

LBF

20.92

4.02

2.16

2.77

9.24

10.72

4.50

7.81

HBF 18.53 1.98 0.82 3.38 8.44 24.64 3.12 2.36 BBF, beef body fat; and BKF, beef kidney fat, LBF, lamb body fat; HBF, hog body fat. Olive oil contained 4.47 POP, 2.74% PLP, 8.86% PLO, 10.51% OLO and 2.80% OOL triacylglycerols. Com oil contained 7.51%, LLL, 4.70% PLL, 5.62% PLO, 22.88% OLO and 17.28% OOL triacylglycerols. P, palmitic; S, stearic; and O, oleic acid. Identified lipids of butter oil fractions TCPC from acetone solutions at 0°C, animal fat and vegetable oils.

Some triacylglycerols ofbutter oil and its fractions were identified by comparison with authentic standards. The distribution ofthese identified triacylglycerols among crystallizing fractions collected at various time intervals from acetone solution ofbutter oil followed the same trend; a general increase of - 7 0 % and a decrease of --57% in fractions SQ and L with respect to butter oil, respectively (Table 2). Except for POO which decreased in SQ and increased in L and OOS that decreased in Soand remained unchanged in L, generally the proportions of other identified lipids were enriched in SQ and reduced in L (Table 3). For example, PPS increased by 296%o in SQ and decreased by 94% in L while SSO increased by 46% and 0 0 0 decreased by 66% in So and were undetectable in L. Considering the variable relationships ofthese lipids within the SQ and L fractions and the parent butter oil, it may be concluded that minor chemical changes in a constituent of a

654 mixture of triacylglycerols leads to substantially altered crystallization behavior. This conclusion is supported by earlier published data [10, 11]. Reflecting on the observation that: (i) only 0 0 0 is common to all lipids (Table 4) and POO is common to olive oil and animal lipids, and (ii) the POP proportion of 4.5% in olive oil is approximately half of that of geometrically different PPO in the animal lipids (Table 4), it may be speculated that vegetable oils, butter oil and various animal adipose tissue lipids may have a common ancestoral origin. Fast atom bombardment (F. A.B.) mass spectral analysis, in a matrix of m-nitrobenzyl alcohol and with added NaCl, showed that samples L, SQ and a fraction precipitated at 4°C from an acetone wash of butter oil, were composed of the same triacylglycerols but in different proportions. The observation of mass spectral fragments indicative of the existence of triacylglycerols containing odd-chain fatty acids is also of interest (Figure 2). The results of ^^C-NMR analyses lends further support to this conclusion (Figure 3).

4.

REFERENCES

1 2

S. Stender, J. Dyerberg, G. Holmer, L. Ovesen and B. Sandstrom, Clin. Sci. 88 (1995) 375. F.M. Fouad, F.R. van de Voort, M.D. Marshall and P.O. Farrell, J. Am. Oil Chem. Soc. 67 (1990)981. F.M. Fouad, F.R. van de Voort, W.D. Marshall and P.G. Farrell, J. Food Lipids 1 (1993) 119. F.M.Fouad,F.R. van de Voort, W.D. MarshallandP.G. Farrell, J. Food Lipids 1(1993) 195. V.A. Amer, D.B. Kupranyez, and B.E. Baker, J. Am. Oil Chem. Soc. 62 (1985) 1551. J.E. Schaap and G.A.M. Rutten, Neth. Milk Dairy J. 30 (1976) 197. J.M. deMan, Can. Inst. Food Technol. J.l (1968) 90. R.G. Black, Aust. J. Dairy Technol. 30 (1975) 153. S. Patton and R.G. Jensen (R.T. Holman, ed), Progess in the Chemistry of Fats and Other Lipids, Oxford: Pergmon Press Ltd., 1975 p. 163. D.M. Small, J. Lipid Res. 25 (1984) 1490. M. OUivon and R. Perronn, Chem. Phys. Lipids 25 (1979) 395. J. Makhlouf, J. Ami, A. Boudreau, P. Verret and M.R. Sahasrabudhe, Can. Inst. Food Sci. Technol. J. 20 (1987) 236. E. Deffense, Fette Wiss. Technol. 133 (1987) 3. A. Shishikura, K. Fujmoto, T. Kaneda, K. Arai and S. Saito, Agric. Biol. Chem. 50 (1986) 1209.

3 4 5 6 7 8 9 10 11 12 13 14

655

Figure 1. GLC of butter oil and its TCPC fractions SQ and L fractions from acetone at 0°C using an Rtx-65TG column.

656

ijgg

3 2 7 , 1367 1

I

I

I

ijJjilijAllJTl 360

350

460

4^0

Figure 2. F. A.B. mass spectra in m-nitrobenzyl alcohol of (I) S^, (II) low molecular weight butter oil fraction precipitated from acetone mother liquor at 4°C and (III) L.

e

Le

1

u u

—j J

i

Le

U

u

h

—

is

fo

"j 0

[^

fs"

Ls

("oinie)

u

r-

is

1

u u

t

r-

u u u

r^

f^

js H B

A

m

^ ^

(woiniq)

rx n I ro 90 ro c-o o

1 001

009

J

Le

—1

u u u u rt2

1-

M (d

i««

H 0

1U

r-

Z3 J — 3

OOC

(•aofinw)

00^

657

oB eta

o

I op

I 'o S o

I

c o o

c 2 H-l

eta

1 5 U

Z3

o pi o

s

O

c o

0)

2

E

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

659

Antimicrobial effect of volatile oils ofgarlic and horse-radish Gy. Patkai, J. Monspart-Senyi and J. Barta Department for Canning Technology, University for Horticulture and Food Industry, P.O. Box H'1052 Budapest Pf53, Hungary ABSTRACT Both the seasoning and antimicrobial effect of garhc (Allium sativum) and horse-radish (Armoracia lapathifolid) was investigated in salad-dressings and ketchup. The test microorganims were: Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. The organoleptically optimal concentration of garlic oil made by extraction was high enough to ensure microbiological stability of ketchup. However, the concentration of oil of horse-radish needed for microbial stability was sufficiently high to become organoleptically undesirable. 1. INTRODUCTION Volatile oils of spices have an antimicrobial effect in addition to their stimulating effect on digestion and appetit. Theoretically they could be used as natural food preservatives, but the concentration needed for microbiological stability is generally associated with an intolerable taste. Nevertheless, these oils may have an important role combined with other food preserving treatments. The authors investigated simultaneously the seasoning and antimicrobial effect of volatile oils of garlic and of horse-radish. The aim of this work was to determine the optimal concentration for improving microbial stability, while at the same time adding desirable flavor and seasoning to salad-dressings and to tomato ketchup. 2. INVESTIGATION METHODS. - Separation of garlic oil by extraction with ethyl-ether and by steam distillation 900 g of cleaned and pulped garlic were extracted in four steps with 1800 cm^ (600+600+400+200 cm^) ethyl-ether. After separation of the liquid and solid phases by decantation the ethyl-ether was perfectly removed by hot-air stream, so only the pure garlic oil-remained for sensory and microbiological investigations. During this process the temperature of the extractant was about O^C because of the intensiv evaporation. The steam-distillation of the volatile components was performed from the mixture of 500 g garlic pulpe and 1000 cm^ water. (Temperature: 100°C; duration: 70-80 min.) The first 100 cm^ of the distillate were gathered, the aqueous and oily phases were separated, and the oily phase was used for investigations. - The parameters of the separation of the volatile components of horse-radish by extraction with ethyl-ether and by steam distillation were the same as above mentioned separation parameters of garlic oil.

660 Determination of diallyl-disulphide and diallyl- monosulfide concentration in garlic preparate by gas-chromatography Type of the gas-chromatograph used: CHROM 4; detector with flame ionization; spiral column with 200 cm diameter,filledwith 3% OV 17 Chromasorb WHP 100-200. Diallydisulphide and diallyl-monosulfide, the degradation products of alHcin served as standards for the quantitative measurement of garlic-oil components. Sensory analysis: Samples with different concentrations of seasoning oils were compared and ranked on the basis of the summed up quality points (taste, aroma, general impression) given by the panel The sum of the ranking numbers was evalueted on the basis of the Kramer test. Figure 1. and 2. are showing the resuh of the comparison of the summed up quality points on the basis of statistical evaluation. Investigations of the antimicrobial effect were performed by the method of agar-diffusion , by plate-counte and by the thermostat-test. The number of the mesophyl-aerob cells determined by counting of the "most probable numbpr" of the cells (MPN technic, temperature: 37°C, culture medium: nutrient broth, MERCK 5443) The antimicrobial effect of the seasoning oils was detected by the agar-difiiision method. Holes were boren in the agar-agar culture medium inoculated with the test-microbes, and 0,1 cm^ of the antimicrobial substance was given into the holes. The antimicrobial effect of the substance used can be evaluated on the basis of the diameter of the steril (transparent) circles surrounding the holes after incubation of the plate. Following culture media were used: 1 Test microbe 1 Staph, aures Lactobacillus brevis Sach. cerevisial 1 PenicillOiri oxalicui^

Culture medium Baird Parker agar (Merck 5406) MRS agar (Merck 10660) OGY agar (Merck 10877) OGYagar (Merck 10877)

Incubation temperature 1

3TC 30°C 25T: 25T

661 3. RESULTS 5.1.Results concerning the garlic preparates Tomato ketchup was seasoned with gariic oil made by extraction with diethyl-ether, while garlic oil made by steam distillation was used for seasoning of an acidic cucumber-salad dressing. Results of the sensory analysis are shown on Figure 1.

Tomato ketchup

Salad dressing

N u m b e r oT s a m pies

Figure 1. Results of the sensory analysis of the seasoning effect of garlic oil preparates * limit for significantly worse sample (1? point); sample (10 points)

limit for significantly better

Concentration of diallyl-disulfide plus diallyl-monosultide in the samples: 1. 0.35ul/100g; 2. 0.70ul/100g; 3. 1.40 ul/100 g (present in form of allicin;) 4. 0.35ul/100g; 5. 0.70ul/100g; 6. l.lOul/lOOg The garlic preparation contained 0.5%(v/v) diallyl-disulfide and 0,2%(v/v) diallylmonosulfide, both in form of allicin. Sensory analysis indicated that a concentration of 0.1 % (v/w) of the extracted gariic preparation ensured a significantly better seasoning effect in ketchup than did the lower or the higher level. In the case of the dressed cucumber-salad, the destilled preparation was used. 30 % higher concentration was needed for optimal seasoning, because this preparation contained only allicin degradation-products diallyldisulfide and diallyl-monosulfide instead of allicin as a result of the distillation process utilized. Allicin is degraded to diallyl-disulfide and to diallyl-monosulfide in hours at room temperature in aqueous solution and it is degraded in weeks when in an oily extract. Though diallyl-disulfide and diallyl-monosulfide also have a pharmacological effect, their sensory and microbiological effect is much lower, than that of allicin in the fi-esh garlic preparation. (VOIGT, etal 1986).

662 Results of the microbiological investigations are shown in the Table 1.

Table 1. ANTIMICROBIAL EFFECT OF VOLATILE COMPONENTS OF GARLIC Volatile component

Garlie extract DMS allicin D0S allicin D k S allicin DDS aUicin "Knoblauch Olmazerat** DMS+DDS DMS+DDS DMS+DDS Distilled garlic oil DMS+DDS "Fluka" standard DMS+DDS DMS+DDS DMS+DDS DMS+DDS

Cone. (Hl cm ^)

Test microorganism Lactobacillus brevis

Saccharomyces cerevisiae

Penicillicum oxalicum

Aspergillus niger

Pseudomonas aeruginosa

0.02 0.05 2.00 5.00

+++ +++ +++ +++

+++ +++ +++ +++

+++ +++ +++ +++

/ / / /

/ / / /

0.02+0.12 0.05+0.3 0.1+0.6

-

-

-

.

-

/ / / /

+ + ++ +++

/ / / /

+ + ++ +++

+ ++

160+330

20+60 40+120 200+300 400+600

DMS: diallyl-monosulflde / no investigations + slight antimicrobial effect DDS: diallyl-disulfide - no antimicrobial effect + + expressed antimicrobial effect +++ strong antimicrobial effect The data are indicating a strong antimicrobial effect of the garlic preparation made by extraction with ethyl-ether against the tested microbial phylii. Similar effect was observed in the case of the pharmaceutical preparation "Knoblauch Olmazerat" and in that of the steamdistilled product but only at a much higher concentration. 3.2. Seasoning and antimicrobial effect of the volatile components of horse-radish Horse-radish preparation made by steam distillation was used for the seasoning of mayonnaise. The volatile oil of horse-radish contains about 20% sulphur containing organic

663 compounds; the main component is sinigrin, which can be enzymatically hydrolysed to allylisotiocyanate (KARWOWSKA et al., 1977). The watery and oily phase of the distillate were examined separately. Sensory and microbiological tests were carried out with both fresh and stored preparation (Storage time:30 days, storage temperature: +4^ C). Results of the sensory analysis are shown on the Figure 2.

Figure 2. Results of the sensory analysis of mayonnais seasoned with oil of horse • radisch ^ limit for significantly worse sample (18 points) "^^ limit for significantly better sample (10 points) Volatile oil concentration in the samples: 1. 0.6 % (w/w); 2. 1.0 % (w/w); 3. 1.3 % (w/w) According to the data of the sensory analysis, the mayonnaise sample containing 1% (w/w) distilled horse-radish oil, was significantly better, and the one containing 1.3% (w/w) horseradish oil was significantly worse than all the other samples. The antimicrobial effect of the aqueous and organic (oily) phases of the horse-radish distillate was investigated using the method of agar-difiusion. Investigations were repeted after storage for 30 day 4^0.

664 The results are shown on the Table 2.

Table 2. Antimicrobial effect of distilled horse-radish preparations Preparate

Storage time (days)

Storage temperature CC)

Tested microbes

Aspergil- Saccharonu lus niger cerevisiae Distilled watery phase Distilled oily phase Distilled watery phase Distilled oily phase Extract 1% cc. Extract 100% cc. ControU

30

2

++

Escherichia coli

Bacillus cereus

+

+

++

++

++

+++

Pseudomo- Lactobacilnas lus brevis aeruginosa + 1

++

1

4

2

+

+

+

4

2

+++

++

+++

4-

++

1

0

1

/

-

/

/

/

-

0

1

/

+++

/

/

/

+++

/

1

-

-

-

-

-

/

/ no investigations - no afttimicrobial effect

1

+ slight antimicrobial effect DMS: diallyl-monosuMde + + expressed antimicrobial effect DDS: diallyl-disulfide + + + strong antimicrobial effect

A strong antimicrobial effect of the undiluted oily phase was shown. Steam-distillation and cooled storage did not diminish this antimicrobial effect. 4. SUMMARY The seasoning and antimicrobial effects of different garlic preparations (made by extraction with ethyl-ether at low temperature or separated by steam distillation at 100^ C) and a pharmazeutical product "Knoblauch Olmazerat, incapsuled" were compared. Tomato ketchup contmning 0.1%(v/w) extracted garlic oil was shown to be an excellent seasoning. Furthermore, this concentration was high enough, to ensure microbial stability of the product. This garlic preparation contained 0.5% (v/v) diallyl-disulfide and 0.2%(v/v) diallylmonosulfide in the original allicin form because of the very low processing temperature. Steam distilled garlic oil and "Knoblauch Olmazerat" had no effect when used at the same level as ethyl-ether extracted garHc oil because their allicin content was almost completely degradted to diallyl-disulfide and diallyl- monosulfide. Sensory analysis and microbiological investigations performed with steam-distilled horse-radish preparation indicated that the mayonnaise sample, containing 1 % (w/w) horse-radish oil became significantly the best sensory qualification. However this concentration was uneffective against microbial growth. Distilled, undiluted horse-radish oil proved to have a strong antimicrobial effect. Seasoning

665 and antimicrobial effect of horse-radish oil was not diminishing after distillation or low temperature storage. 5. REFERENCES 1 K. Karwowska and B. Tokarska, Prace Institutow i Laboratoriow Badowczych Przemyslu spozywczego, 27 (1977) 7-12. 2 M. Voigt and E. Wolf, Dtsch. Apotheker Zeitung, 126 (1986) 591-593.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

667

Changes in the flavour of monoterpenes during their autoxidation under storage conditions J. Pokomy, F. Pudil, J. Volfovd and H. Valentovd Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technickd 5, CZ-16628 Prague 6, Czechia

Abstract Limonene and linalool were oxidized at 40 and 60°C at limited access of oxygen, and their oxidative changes were determined by gas chromatography - mass spectroscopy (GC/MS). The respective oxides belonged to important oxidation products. The changes were compared with those of sensory odour profile. Citrus odour notes slowly disappeared, while the intensity of woody, acidic and heavy odour notes increased. Heavy note reminded higher esters, tropical fruits and flowers. Dihydropyridine antioxidants acted as inhibitors of medium activity; their presence slightly influenced the composition of oxidized products and the sensory character.

1. INTRODUCTION Monoterpenes are important components of many essential oils, particularly citrus oils, which consists of 80-95% monoterpenes, mainly limonene. Limonene is easily oxidized via free radicals, which are converted into a mixture of six unstable hydroperoxides by reaction with oxygen [1], in a manner similar to the structurally related carvomenthene [2], a-terpineol and a-pinene [3,4]. Similar reaction proceeds under intensive ultraviolet irradiation even in absence of photosensibilizers [5-7]; they are oxidized into hydroperoxides, however, their composition is different. Carvone and carveol [5] and 1,2-limonene oxide [1] belong to important oxidation products that deteriorate the sensory character. Then* precursors are two mesomeric free radicals formed on the 6th carbon atom of limonene [8]. In addition to 1,2epoxide, the 8,9-isomer is produced. The epoxides and hydroperoxides are converted into the respective hydroxylic derivatives [8]. The relationship between the changes of the chemical composition of terpenes undergoing autoxidization and changes of their sensory character were the object of this study.

2. MATERIALS AND CHEMICALS Monoterpenes, (S)-(-)-limonene (96%), (±)-linalool (97%) and various reference substances, were produced by Aldrich. Dihydropyridine antioxidants, Diludine (2,6-dimethyl3,5-6/5-ethoxycarbonyl-l,4-dihydropyridine) and OSI 7284 (2,6-dimethyl-3,5-6wbutoxycarbonyl-l,4-dihydropyridine), were synthetized at the Institute of Organic Synthesis, Riga, Latvia.

668 3. ANALYTICAL METHODS 3.1. Solid phase microextraction (SPME) Volatiles were adsorbed on a 65 |im Carbowax-divinylbenzene fiber for a manual holder (Supelco, USA). The extraction time was 10 nun at 40°C, the desorption 2 min at 220^*0; the cleaning time 30 min at 220°C.

3.2. Gas liquid chromatography (GLC) The GC8000 Series Fisons gas chromatograph was equipped with a headspace autosampler HS800 and a 60 m x 0.32 mm Supelcowax 10 (layer thickness 0.25 mm) capillary column (Supelco, USA). Column temperature was programmed from 50°C (2 min), heating rate 2°C/min to 220''C (30 min). The injector temperature was 220°C, the flame ionization detector (FID) temperature 250°C; helium carrier gas pressure was 100 kPa, and the input/split ratio 1:25. Retention indices were calculated using a mixture of ^t-alkanes as reference substances. For the GC-mass spectrometry (GC/MS) analysis, the MSD8000 mass spectrometer was used; the ionizing energy was 70 eV. Internal standards were used for the calculation of absolute amounts of components (A2-decane). 33, Sensory analysis Sensory analysis was performed according to the international standard [9] in a test room provided with six standardized test booths [10]. The assessor panel consisted of 12 selected and trained persons [11] with at least 6 months experience in sensory profiling of. A 100 mg sample was placed into a 250 mL wide-neck ground-glass bottle, and the odour intensity was evaluated by sniffing. For the analysis of stabilized samples, 100 ^L of 1% methanol solution of the antioxidant was added, and the solvent evaporated. The sample was then added, the bottle closed, and shaken. Odour acceptability was determined using an unstructured graphical scale: straight lines 100 mm long [13] (0% = rather bad, 100% = excellent). The sensory profile [12] consisting of 24-36 descriptors (see the spider-web diagrams in Figure 2), was evaluated using unstructured graphical scales, i. e. straight lines 100 mm long [13] (0% = imperceptible, 100% = very strong). Two samples were served per session. A total of 24 responses were used for the calculation of means. The standard deviation of means varied between 2-6% of the scale. 4. OXTOATION PROCEDURE A portion of 100 mg of a terpenic substance was placed into a 10 mL glass vial, and 5 mg of A2-decane (internal standard) were added. The vial was sealed and the mixture aged at 40^C in a thermostat. Volumes of 100 |LIL vapor phase were injected into the GC injector using a gas-tight Hamilton syringe. For sampling into the GC-MS, the solid phase microextraction (SPME) sampling technique was used. In experiments with stabilized samples, 100 mL of a 1 % methanol solution was added, the solvent was evaporated, the sample of terpenes was

669 added, the vial sealed, and the antioxidant dissolved by shaking, stored at 40 or 60''C, respectively.

5. RESULTS AND DISCUSSION 5.1. Oxidation of limonene Limonene was oxidized both at 40 and 60°C in the presence of insufficient air in the headspace for complete oxidation, simulating the actual conditions in packed flavoured foods and beverages. Pure limonene was added at the beginning of storage, but after several hours at 40°C, several major oxidation products were formed simultaneously; at least 52 components were detected after 20 hours of storage. An example of limonene oxidized at 60^C for 20 hours is shown in Figure 1, and the list of substances identified in the oxidized mixture is shown in Table 1.

too

4

•

7

75

50-

f 25

1 1

<

1 tl

i«

11

It

iliililijl ^j 10

19 I t

L

1

. L lllilL

20

Figure 1. GC profile of limonene oxidized at 60°C for 20 h

II

It i i

1

LiijLilJIJ 70 TdbM CalA.]

670 Table 1 Identification of reaction products of limonene oxidation Peak Retention Retention Identified No. time [min] index compounds 1 6.57 822 acetone 2 7.50 890 methanol 3 8.31 933 ethanol 4 10.05 1000 «-decane 5 19.91 1209 limonene 6 21.93 1249 1,3,8-p-menthatriene 7 23.79 1277 p-cymene 8 34.85 1448 cw-limonene oxide 9 35.66 1462 /raw^-limonene oxide 10 46.46 1616 menthadienol 11 47.63 1626 ascaridol 12 49.09 1639 2,8-menthadien-l-ol 13 50.13 1647 citral 14 53.02 1670 carvone 15 58.85 1833 cw-carveol 16 59.69 1848 p-cymenol 17 60.58 1863 /rcms-carveol 18 67.77 1990 menthadienol 19 68.23 1999 7-hydoxylimonene Peak numbers refer to Figure 1; n-decane was used as inner standard (retention index RI 1000). Hydroperoxides, the primary reaction products, were destroyed under conditions of GC separation so that only tiie more stable oxidation products were detected, such as cis- and /raMs-limonene oxides, carvone, acetone, citral, cis- and /raAis-6,8-menthadien-2-ols (carveols), 2,8-menthadien-l-ol, l,8-menthadien-7-ol (7-hydroxy limonene), /7-cymenol and ascaridol (a cyclic peroxide, which is relatively stable). During the interval studied, limonene remained the most prominent compound present; the reason was that the availability of oxygen in the sealed sample was low so that only small part of the original amount could be oxidized. Numerous compounds were formed in the beginning of oxidation (most likely produced by decomposition of hydroperoxides), but were decomposed during further storage. Other compounds, such as cw-limonene oxide and menthadienol, were formed in larger amounts only after more than 60 hours of oxidation, probably being only tertiary reaction products. Examples are given m Table 2.

671 Table 2 Time development of some limonene reaction products during storage at 60°C Time [h] Acetone 1,3,8-p-Men- cw-Limonene Carvone cw-Carveol thatriene oxide 0.88 0.26 0.92 25.14 1.29 2.0 3.29 2.01 1.12 1.68 19.10 10.5 3.79 4.95 16.44 1.62 2.56 19.5 4.11 5.34 3.23 13.15 2.77 28.0 3.49 5.40 11.16 3.67 3.03 34.5 3.16 2.90 3.09 5.66 9.67 41.0 3.31 5.01 7.69 7.88 3.31 47.5 3.42 11.38 3.69 6.07 3.15 56.0 2.49 11.35 2.65 7.38 1.18 60.5 3.84 12.73 0.69 11.76 65.0 4.85 5.73 7.84 18.24 0.73 15.86 67.0 13.66 27.59 0.71 23.42 17.99 69.0 Peak areas are expressed in % total peak area Sensory profiles obtained at defined times of oxidation are shown in Figure 2; relative values are given as they are more suitable for comparison. The lemon odour note decreased. heavy-^ pungentx

V

fresh^-^..^^ menthol y—-^

lemon juice ^ I

uamnn peel

i \ ^ ^'\

/^3K!L2\

XK^^Si-.^^ orange peel

^^^^^^^^^^

terpenic"^

/

f

woody^ / anise^

X orange juice

\

\

—

1

fruity

^floral

A ^acidic ^spicy

* « — 0 hours

>-o-48 hours

-o—161 hours

—e—232 hours

~^—66 hours 1

Figure 2. Sensory profile of stored limonene while woody, acidic and heavy odour notes became stronger during storage. Some ratios of odour intensities are given in Table 3. Odour notes reminding higher esters (10-14 carbon atoms), tropical fruits and tropical flowers are denoted as heavy (the term is used in the fragrance industry).

672 Table 3 Effect of storage time of oxidizing limonene on the ratio of intensities of some odor notes in the sensory profile Storage time Ratio woodyiorange acidic:lemon heavyifresh woody:lemon 0.35 0.54 0.35 0 0.53 0.67 0.50 48 0.42 0.69 0.77 0.49 66 0.52 0.53 1.05 1.18 0.70 2.62 161 1.71 3.29 232 3.25 3.83 Defmitions of odour notes: woody = associated with oak lactones; lemon = associated with lemon juice; acidic = associated with acetic acid; heavy = associated with odour of higher esters (10-14 carbon atoms). Tropical fruits or tropical flowers; fresh = associated with meadow flowers of temperate climate, with low molecular weight esters (3-5 carbon atoms). 5.2. Oxidation of linalool Linalool was oxidized under the same conditions as limonene, but, because of its lower stability, results were obtained at 40°C. An example of the GC separation of the reaction mixture obtained after 700 h at 40°C is shown in Figure 3, and the identification of some prominent peaks is shown inTable 4. Contrary to limonene, linalool was

Figure 3. GC profile of oxidizing linalool stored at 40**C for 700 hours

673 Table 4 Identification of compounds in stored oxidizing linalool Peak Retention Retention Compound No. time [min] index identified acetaldehyde 709 5.68 1 acetone 824 6.49 2 7.34 crotonaldehyde 887 3 methyl vinyl ketone 933 8.21 4 pentanone (probably 2-pentanone) 953 8.64 5 pentenone (probably 4-penten-2-one) 1038 11.13 6 methyl pentenone (probably 4-methyl-3-penten-2-one) 1131 15.02 7 2,3-metiiyldihydro-4-methylfiiran 1203 18.98 8 3-hydroxy-3-methyl-2-butanone 1284 23.67 9 hydroxypropanone 1289 24.05 10 hydroxybutanone 1304 25.09 11 a furan derivative 1404 31.52 12 32.34 a pyran derivative 1418 13 34.07 14 1445 m-linalool oxide (furan derivative) acetic acid 1459 34.91 15 1474 35.93 16 /ra«s-linalool oxide (fiiran derivative) 3,7-dimethyl-l-octen-3-ol 1537 39.98 17 linalool 1553 40.94 18 43.29 19 4,4-dimethyl-2-butenolide 1588 hotrienol 1608 45.01 20 48.78 21 1641 5-ethenyldihydro-5-methyl-2(3H)-furanone 5-ethyldihydro-5-methyl-2(3H)-fiiranone 1661 51.62 22 cu-linaiool oxide (pyran derivative) 1672 52.67 23 /ra/u-linalool oxide (pyrane derivative) 1683 54.09 24 3,7-dimethyl-l ,5-octadien-3,7-diol 1939 64.42 25 8-hydroxylinalool 2103 73.95 26 Peaks correspond to those in Figure 3; «-decane was used as the internal standard (RI = 1000) decomposed at a uniformly constant reaction rate during the storage. It was decomposed not only by oxidation but also by other reactions, such as retroaldolization. Among the most prominent reaction products were four isomeric linalool oxides were identified as cisr- and /ra«5-5-ethenyl-tetrahydro-a,a,5-trimethyl-c/.s-furanmethanols and cis- and trans-Sethyltetrahydro-2,2,6-trimethyl-pyran-3-ols. Several aliphatic products were also identified. Linalool oxides of the pyran type (contrary to those of thefiirantype) do not increase in their intensitiesfiromthe beginning of reaction, but rather increase as an exponential fimction of time (Table 5).

674 Table 5 Changes of relative intensities of linalool oxides during the oxidative storage Time[h] cw-Linalool trans-Lmalool cw-Linalool tranS'Lmalool oxide (fiiran) oxide (pyran) oxide (pyran) oxide (fiiran) 0.0022 0.0014 0.00008 0.00008 17 0.00064 0.0402 0.0369 161 0.00061 0.00115 0.0657 234 0.0727 0.00105 0.1065 0.00229 330 0.1266 0.00191 0.1565 424 0.1925 0.00416 0.00329 0.00711 0.2303 519 0.2856 0.00569 687 0.03299 0.5245 0.6293 0.02717 Peak have structure as given in Table 4; relative intensities are expressed on the basis of linalool =100 5.3. Effect of dihydropyridine antioxidants on the oxidation of limonene and linalool Dihydropyridine antioxidants inhibited the oxidation of limonene, but also affected the rate of formation of various products at different degrees, e.g. they inhibited the formation of carvone (Figure 4), but enhanced the formation of limonen oxidesfi-omlimonene (Figure 5). On the contrary, they inhibited the formation of linalool oxide firom linalool (Figure 6). The sensory profile of limonene was influenced by the presence of 1,4-dihydropyridines, both Diludine and OSI7284 (Figure 7).

+ 0.2

8 a* 0.15

JS 0.1

4-0.06

50

100

150

250

Exposure time [h]

Figure 4. Effect of antioxidants on the formation of carvone in autoxidizing limonene

675

120 100 I

-Rosemary extract -Oiludine -OSI 7284 -pure

I I 100

150

Exposure time [h|

Figure 5. Effect of antioxidants on the fonnation of limonene oxides in autoxidizing limonene

0.035 -Rosemary extract

0.03 -I

-Oiludine -OSI 7284 -pure

0.025

< 0.02 0.015 0.01 •{

0.005

0

100

200

300

400

500

600

Exposure time [h]

Figure 6. Effect of antioxidants on the formation of linalool oxides in autoxidizing linalool

676

lemon juioe heavy^

I

lamonoaal

\ 50/pungentx

fresh /—

V

/\

menthol \——"^

terpente^

^ \

/

^^^^jof

>5^Wi V

/

/

ifC

x

I'Sf

woody^

/

'

\

-o-Diludlne

^^Sta^--^ orange peel

^

\

•nise -•-Limonene pure (fresh]

xoiangejulce

^

—

^

'"^

^floral

^acidic spicy -ili-OSI7284

-S-Rosemary

Figure 7, Sensory profiles of limonene oxidized at 60°C for 222h

6. SUMMARY During storage of flavoured foods, terpenes are oxidized even in presence of low content of oxygen. Limonene and linalool, typical components of citrus oils, were used as model substrates. Nonoxidative reactions of terpenes and secondary reactions of oxidation products proceed, especially, after exhaustion of oxygen. Intensities of typical citrus flavour notes decrease and off-odours imparted by oxidation products increase, particularly woody, heavy and acidic notes. Antioxidants inhibit the flavour deterioration, rosemary extract being more active than 1,4-dihydropyridines. The compositions of stored limonene and linalool are affected differently by antioxidants used, which influences the respective sensory profiles. The effect of the particular antioxidant on flavour changes should ne taken into account when they are applied to foods and beverages.

?• ACKNOWLEDGEMENTS The project was supported by COPERNICUS CIPA-CT94/0121; Prof G. Dubursfromthe Institute of Organic Synthesis, Riga, Latvia, prepared the samples of dihydropyridine antioxidants.

7. REFERENCES 1 S. Anandaraman and G. A. Reineccius, Food Technol, 40 (1986) 11, 88. 2 P. Schieberle, W. Maier, J. Firl and W. Grosch, J. High Resolution Chromatog., 10 (1987) 588.

677 3 C. S. Foote, S. Wheeler and W. Ando, Tetrahedron Lett., 46 (1965) 4111. 4 G. O. Schenck, K. Golhiick, G. Buchwald, S. Schroeter and G. Ohloff, Liebigs Ann. Chem.,674(1964)93. 5 L. Lawrence, J. R. Buckholz and H. Daun, J. Food Sci., 43 (1978) 535. 6 Schieberle and W. Grosch, Z. Lebensm.-Unters.-Forsch., 189 (1989) 26.Cannona, J. P. 7 P. Carmona, J. Bellanato and A. Hidalgo, Ann. Technol. Agr., 25 (1976) 159. 8 G. O. Schenck, O.-A. NeumilUer, G. Ohloff and S. Schroeter, Liebigs Ann. Chem., 687 (1965) 26. 9 ISO 6658: Sensory analysis - Methodology - General guidance. ISO, Geneva, 1985. 10 ISO 8589: Sensory analysis - General guidance for the design of test rooms. ISO, Geneva, 1988. 11 ISO 8586: Sensory analysis - General guidance for the selection, training andmonitoring of assessors. ISO, Geneva, 1989. 12 ISO 6564: Sensory analysis - Flavour profile. ISO, Geneva, 1985. ISO 4121: Sensory analysis - Grading of food products by methods using scale categories. ISO, Geneva, 1988.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

679

Effect of rosemary and 1,4-dihydropyridines on oxidative and flavour changes of bergamot oil F. Pudil, J. Volfovd, V. Janda, H. Valentovd and J. Pokomy Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technicka5,CZ-166 28 Prague 6, Czechia

Abstract Bergamot oil was oxidized in the temperature range of 40-60°C at restricted oxygen levels. The course of oxidation was investigated by gas chromatography (GLC) and gas chromatogaphy with mass spectrometric detection (GC/MS). Flavour acceptabilities and sensory profiles were determined at the same time. The sensory acceptability decreases on storage, and sensory profiles change, too. Limonene oxides and carvone, which were identified as main oxidation products, have particular sensory profiles. An addition of rosemary extracts inhibits the oxidation, but it influences the ratios of various components of stored oil. Therefore, it changes moderately the sensory profile. The sensory profile changes are more pronounced, when 1,4-dihydropyridine antioxidants are used. Both oxidative and sensory changes depend on the structure of dihydropyridines, too.

1. INTRODUCTION Citrus oils are widely used as flavorings for food and cosmetic products. Bergamot oil, producedfi'ombitter orange {Citrus aurantium subsp. Bergamie Risso et Poiteau Engler) were applied for various foods and beverages, such as fiuit juices [1], soft drinks [2], liqueurs [3], firuit jams [4],flavouredtea [5] and even flying oil [6]. The disadvantage of bergamot oil is its high content of monoterpenes [7], particularly limonene, linalyl acetate and linalool, which decrease resistance against oxidation under storage. Black tea, flavoured with bergamot oil, was stable only for two months at room temperature [8]. At 50 or 60°C, the peroxide value of 300 mval/kg was soon reached which caused deterioration of flavour quality [9]. Stored samples of bergamot oil have high peroxide values [10], unless they have been stabilized with antioxidants; they are oxylabile even in cold orfi^ozenstorage [11]. The peroxide value was found a good indicator of sensory quality of orange oil, but tiie gas-chromatographic profile was preferable [12]. More than 160 components were identified in aged bergamot oil, using GC/MS [13]. Oxidation mechanisms of terpenic hydrocarbons and their oxygenated compounds were discussed [14]. Oxidation products modify the flavour of essential oils. Therefore, we studied the relationship between the chemical composition and the sensory profile of stored bergamot oil, and the effect of antioxidants on the product.

680 2. MATERIALS AND CHEMICALS Bergamot oil (free of antioxidants, without deterpenation) was supplied by Sigma (UK), (S)(-)-limonene (96 % purity) and (±)-linalool (97 % purity) by Aldrich (Gemany). Rosemary extract was produced by extracting 100 g of fresh dried rosemary leaves (grown in Poland, in 1995) four times with 1 L acetone, macerating overnight at room temperature of 22°C, combining the filtrates, removing the solvent by distillation, and drying on a boiling water bath. The main active components were identified as camosic acid and camosol. Diludine (2,6-dimethyl-3,5-diethoxycarbonyl-l,4-dihydropyridine) of 99 % purity and OSI 7284 (2,6dimethyl-3,5-dibutoxycarbonyH,4-dihydropyridine) of 98 % purity were prepared in the Institute of Organic Syntheis (Director: Prof Dr. G. Duburs) in Riga, Latvia.

3. EXPEMMENTAL PROCEDURES 3.1. Oxidation of bergamot oil A 100 fiL portion of bergamot oil was placed into a 10 mL vial containing an internal standard (5 mg of «-decane). The vial was sealed and conditioned at 40°C (in some experiments, at 60'^C) in a thermostat. A volume of 0.1 mL of vapour phase was injected by gas-tight Hamilton syringe mto the gas- chromatograph (using a headspace autosampler). Alternatively, the solid phase microextraction (SPME) sampling technique was used. To study the antioxidant efficiency, 1 mg of the antioxidant dissolved in 100 mL methanol, was added, and the solvent evaporated. Essential oil was added afterwards, the vial was sealed, and thoroughly shaken. 3.2. Sotid phase microextraction (SPME) A 65 pm Carbowax-divinylbenzene fiber for a manual holder (Supelco, USA) was used for extracting volatiles for GC/MS. The fiber was inserted into the vial containing the stored sample (see Section 3.1). Extraction time was 10 min at 40°C and desorption time 2 min at 220°C. The fiber was cleaned for 30 min At the same temperature. Details of the apparatus are given in another chapter in these proceedings[16]. 3.3. Gas chromatography (GLC) and GLC with mass spectrometric detection (GC/MS) The gas chromatograph GC8000 (Fisons Instruments) was equipped with an autosampler HS800 (injector temperature 220°C) and a 60 m x 0.32 mm column, coated with Supelcowax 10 (fibn thickness 0.25 mm) was used. The column temperature was programmed from 50*^0 (2 min isothermally), heating rate 2°C/min to 220°C (isothermal for 30 min) Tnc fl^me ionization detector (FID) was used (temperature 250*^0). The c^^T?er gas was helium at an initial pressure 100 kPa. The input/split ratio 1:25. Retention indices were calculated using a mixture of «-alkanes as reference substances, as well as for calculation of peak areas. A MSD8000 mass spectrometer was used for GC/MS; the ionizing energy was 70 eV. Pure standards (Aldrich, purified by GLC) were used for identification of mass spectra.

3.4. Sensory analysis Conditions for the sensory analysis were in agreement with the international standard ISO [17]; the test room was equipped with six standardized test booths [18]. The panel of assessors consisted of selected, trained and monitored persons [19] with experience in sensory profiling of at least six months. The amount of 100 mg of sample was placed into a 250-mL wide-neck

681 ground-glass bottle, and left at least 2-3 hours to equilibriate. The odour intensity was evaluated by sniffing. For the analysis of stabilized samples, 100 jiL of 1% methanolic solution were added, the solvent evaporated, the sample was added, the bottle thoroughly shaken, and left for 2-3 h. For the hedonic rating, the odour acceptability was determined using an unstructured graphical scale - straight lines 100 mm long [19] (0% = rather bad; 100% = excellent). The sensory profile [20] consisted of 24-36 descriptors (they are shown in the respective figures in spider-web diagrams); unstructured graphical scales 100 mm long were used (0% = imperceptible; 100% = very strong). Two samples were served at a session in random order. The total of 24 responses were used for the calculation of means. The standard deviation of the means varied between 2-6% of the scale.

4. RESULTS AND DISCUSSION 4.1. Composition of bergamot oil Bergamot oil was analyzed by GC/MS, and a chromatographic profile is shown in Figure 1. The sample was moderately oxidized (20 h at 40°C) in order to better determine the peak position of some oxidation products. The chemical structure of identified compounds was determined by comparison of mass spectra and retention indices with those of authentic substances; they are listed in Table 1. Linalool, linalyl acetate and limonene were present as major terpenic components, in agreement with the literature [7, 22, 24], and myrcene, cis- and /ran^-ocimene, and p-cymene were found in smaller amounts. Caryophyllene was the only sesquiterpene detected.

3.5] 21

3.0J

S

141 1<

4

If

r

2.5

|| ii

f

• 1.5-1

3

1

iMuJLuLjLL^U 0,5-]

1 11

1

14 1

11 1 1

t

i.oj 1

1

1

17 1

h

k

JLJ 30

0 1

^

40

Time [min] Figure 1. Chromatographic profile of bergamot oil after storage at 40°C for 20 h. Numbers above each peak refer to the compound shown in Table 1.

682 Table 1 List of identified substances in the sample of very slightly oxidized bergamot oil (20 h at 40°C, corresponding to Figure 1) Retention Retention Identified Peak substance index time [min] No. acetone 840 6.53 1 7.54 methanol 2 910 946 8.23 3 ethanol 16.79 4 1174 p-myrcene limonene 1209 18.65 5 1246 20.83 6 c»5-ocimene 1263 21.85 7 trans-ocimeiae 1281 23.01 8 /j-cymene c/f-linalool oxide 1453 34.04 9 1467 34.95 10 acetic acid 35.91 11 1481 /rans-linalool oxide 1536 39.38 12 3,7-dimethyl-1 -octen-3-ol 1540 39.62 13 y-terpineol 1561 40.97 14 linalool 1571 41.68 15 iinalyl acetate 1594 43.15 16 sesquiterpene (probably »o-caryophyllene) 1648 49.36 17 citral 1654 50.13 18 a-terpineol 1668 51.98 19 neryl acetate 1675 52.67 20 carvone 1681 53.78 21 geranyl acetate 1975 66.47 22 caryophyllene oxide

4.2. Oxidation of bergamot oil Bergamot oil was oxidized under simulated storage conditions of flavored food products, i. e. under limited access of oxygen at 40°C and in thin film of about 1 mm. Examples of chromatographic profiles are shown in Figure 2 where a sample taken near the beginning of oxidation is compared with a sample stored for about a month, when oxygen has been consumed and various secondary (non-oxidative) reactions proceeded. Various oxidation reaction proceeded in the beginning of storage, but when oxygen in the headspace has been exhausted, various secondary reactions followed. Changes at 60°C were very sunilar, therefore, they are not presented here. Limonene was gradually destroyed firom the start of storage and the course followed the zero order kinetics until complete exhaustion of oxygen. Data for several other compounds are given in Table 2. Myrcene was oxidized from the beginning, too. On contrary, the degradation of/?-cymene followed after a distinct lag period. The contents of a- and y-terpineols and 3,7dimethyH-octen-3-ol mcreased during llie storage. The two ocimenes reached their maximum in the beginning of storage. Acetone, a typical oxidation product formed by decomposition of a hydroperoxide, was produced very rapidly, reaching a maximum after 40 hours. Both cisand /ran^-linalool oxides were formed slowly, and attained their maxima when oxygen was already consumed.

683 Table 2 Changes of some compounds during the storage of bergamot oil at 40'^C 428 h 39 h Peak No. 739 h 354 h 185 h 612 h 4.38 4.16 5.22 6.88 14.60 1 6.48 31.33 4 16.75 46.89 107.45 148.63 20.52 5.34 9.21 7.69 6.65 6 6.53 8.47 16.62 18.15 27.59 7 9.50 11.46 27.08 40.23 179.57 216.48 288.96 8 6.18 6.36 6.05 5.46 4.26 9 6.07 5.87 5.65 3.01 5.73 4.60 11 1.74 0.55 12 1.82 1.61 1.31 0.91 1.27 1.69 1.12 0.50 1.35 0.69 13 7.54 6.25 4.42 3.50 2.40 18 6.03 Note: Peak numbers are the same as in Table 1. Numbers refer to FID peak area. 4.3. Effect of rosemary extract on the degradation of bergamot oU Rosemary extract, an efficient natural antioxidant used for the stabilization of fats and oils [25, 26], was found active in stabilizing limonene and linalool under storage conditions at 40°C. No changes or very small changes were observed in most components for the first 400 hours of storage. The activity was similar to that observed in Citrus hystrix oil [16]. Changes of the composition of bergamot oil stabilized with rosemary extract during the storage at oxygen are partially shown in Table 3. The contents of acetone, 3,7-dimethyl-locten-3-ol, /7-cymene, P-myrcene, a- and y-terpineols changed similarly during the storage as in non-stabilized oil. The degradation oicis- and /ra^w-ocimenes was moderately inhibited by rosemary extract. Both cis- and /ra/w-linalool oxides were formed in inhibited samples at the same rate at the beginning as in the control containing no antioxidants, however, they were decomposed at a faster rate at later stages of storage (after 400 h) if rosemary extract was present. Table 3 Changes of some components of bergamot oils stored with rosemary extract at 40°C Peak No. 41 h 187ji 356 h 543 h 713 h 1 20.91 2.39 1.34 5.10 1.60 58.78 120.74 57.77 4 31.89 24.26 8.04 7.80 8.24 8.82 7.98 6 30.96 22.04 29.36 17.02 13.83 7 96.29 90.01 175.51 21.67 2.16 8 4.71 5.06 4.95 3.87 2.28 9 3.77 4.01 4.07 3.65 2.33 11 0.79 1.28 0.92 1.95 1.56 12 0.64 0.69 1.11 1.81 1.23 13 4.91 3.07 3.54 7.59 5.55 18 The structure and numbers of peaks are the same as in Table 1.

Since bergamot oil is a complicated mixture of terpenes, the sensory changes that occur with storage are difficult to evaluate and compare. Therefore, the effect of rosemary extract

684 was studied using purified limonene and linaiooi. An example of limonene is shown in Figure 3 (changes occurring in stored linaiooi were very sunilar). Degradation of citrus notes was very pronounced in the non-stabilized sample, but it was slightly smaller in the stabilized sample. In presence of rosemary extract, the formation of woody, acidic and heavy odour notes were suppressed in stored bergamot oil; on contrary, the formation of spice and fresh odour notes was stimulated. The hedonic rating decreased by 28% of the scale in case of unstabilized sample and by 14% only in case of stabilized sample, respectively. 70-

60-

SO

a e

<0

0S 30

., 1

1

20-

10-

-1 t

• 1

0-

LJLL-

IJJi

JI II

^

^ •.

.-

t

Time [min] Figure 2. Chromatographic profiles of oxidized bergamot oil, A - 40 hours at 40*^0, B - 740 hours at 40°C.

lemon juice heavy ^ pungent ^

V

terpenic^

y 1

*7

> orange juice

/\^^^} r 7 i \ y ^ \ ^ ^ , . ^ - ^ orange peel

^^^^"^^^^

menthol ^—^-^ '

I _ , _ _ _ ^ lemon peel

'Z^^^^^i^^^^^^^^^^'*^~^~~~' ^ ' ^

/

i

^

\

^floral -o-O hours

woody

f anise

\

acidic spicy

- o — rosemary ~-^r- dliudlne - • ^ OSI7284

Figure 3. Sensory profiles of limonene stored with and without rosemary extract

685 4.4. Effect of 1,4-dihydropyridine antioxidants on the degradation of bergamot oil Dihydropyridine derivatives are used for the stabilization of feeds and phannaceutical preparations such as carotene supplements [27, 28]. In terpenes they were found less efficient as inhibitors of oxidation, similarly as in pure terpenes [29], but they influenced the formation of various oxidation and other degradation products. Changes of several important components are summarized in Table 4 for oil stabilized with Diludine and in Table 5 for oil stabilized with OSI 7284, respectively.

Table 4 Changes of some components during the storage of bergamot oil at 40°C in presence of Diludine 717 h 547 h 358 h 189 h 43 h Peak No. 15.71 15.40 15.55 21.76 27.25 1 29.34 142.14 14.17 53.72 125.20 4 6.77 4.32 8.60 6.52 7.70 6 13.41 7 7.56 14.90 23.25 25.43 21.30 361.06 525.62 266.62 S 9.71 12.67 10.36 9 6.03 4.00 2.86 9.25 11.33 9.72 5.45 11 1.11 0.60 1.00 1.57 0.85 12 0.57 0.66 1.67 1.11 0.87 13 5.24 5.89 0.35 2.69 4.12 18 Compounds and numbers of peaks are the same as in Table 1.

Table 5 Changes of some components during the storage of bergamot oil stabilized with OSI 7284 Peak No. 45 h 207 h 375 h 548 h 721 h 1 40.28 18.01 11.16 6.07 4.05 4 217.14 154.47 70.95 29.77 18.55 12.87 6 10.76 9.43 8.32 6.04 7 45.91 10.47 35.87 24.18 15.56 282.24 8 207.30 53.46 334.63 1.89 6.64 9 6.05 6.95 5.80 4.22 11 4.73 5.46 6.14 5.51 4.17 12 0.98 1.11 1.25 1.70 1.47 0.93 13 1.03 1.25 1.00 1.10 3.82 4.44 18 5.20 6.06 6.12 Compounds and numbers of peaks are the same as in Table 1. The destruction of myrcene was efficiently inhibited in presence of OSI 7284, but no effect was observed with Diludine. The maximum contents of cis- and trans—linalool oxides were higher than in absence of antioxidants or in presence of rosemary extract. The maximum was particularly high, when Diludine has been added. The content of p-cymene changed similarly. The maxima of cis- and /ran^-ocimenes were much higher in oil stabilized with OSI 7284 than m oil stabilized with Diludine. During 300-400 h of storage the concentrations of a- and

686 y-terpineols were almost the same in all samples studied. During further storage their content decreased in presence of dihydropyridines, especially Diludine, while it continued to increase in non-stabilized oil or in oil stabilized with rosemary extract. Similar differences were observed for 3,7-dimethyl-l-octen-3-ol. The formation of acetone was also stimulated by dihydropyridine derivatives. These differences could be explained by presence of a nitrogen group in dihydropyridines. Their weakly basic character may influence not only the course of hydroperoxide decomposition, but the course of non-oxidative degradative changes such as retroaldolization. From thesedata, it is evident that changes in sensory profiles of stored bergamot oil are not due only to the inhibition of primary oxidation reactions, but also depend on the structure of antioxidant added.

5. ACKNOWLEDGEMENTS Supported by the research grants Copernicus CIPA-CT94/0111 and OK 175 ( M S M T CR). The rosemary extract was prepared and characterized by Dr. J, Korczak, Agricultural Univeristy, PoznapPoland. The dihydropyridine antioxidants were preprared and characterized by prof. Dr. G. Duburs and co-workers. Institute of Organic Synthesis, Riga, Latvia.

6. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

16 17

E. Benk, Mineralbrunnen, 34 (1984) 410. C. Arena, Ind. Bevande, 10 (1980) 209. E. Benk, Alkohol-Ind., 98 (1985) 278. E. Benk, Ind. Obst- GemOseverwertung, 70 (1985) 358. H. P.Neukom, D. J. Neier and D. Blum, Mitt. Geb. Lebensm.-Unters. Hyg., 84 (1993)337. W. J. Begemann and P. D. Harkes, EP No. 0191 519 Al (1986). P. E. Shaw, J. Agr. Food Chem., 27 (1979) 246. N. Dhanaraj, C. P. Natarajan and R. Seshadri, J. Sci. Food Agr., 37 (1986) 185. J. Pokomy, D. Wiewi6ricova, E. Nfiiec, H. S^ankova and D. Curda, Sb. V§CHTPrazeE,34(1972)5. A. DiGiacomo, F. Bovalo and M. Caliciuri, Essenze, Deriv. Agrumeri, 45 (1975)110. P. Rovesti, Essenze, Deriv. Agrumari, 45 (1975) 100. J. Pokomy, D. Curda, L. Jirdskova, D. Wiewiorkovd and G. Janidek, Sb. V§CHTPrazeE,34(1972)21. G. Mazza, J. Chromatogr., 362 (1986) 87. G. Mazza, Essenze, Deriv. Agrumari, 57 (1987) 5. J. Pokomy, F. Pudil, K. Ulmannovd and E. Ficov^ in: Food Flavors: Generation, Analysis and Process Influence (G. Charalambous, ed.). Barking, Elsevier Science, 1995, p 815. F. Pudil, H. Vijaya, V. Janda, J. Volfova, H. Valentovd and J. Pokomy, These Proceedings, p. ISO 6658: Sensory analysis - Methodology - General Guidance. ISO, Geneva, 1985.

687 18 19 20 21 22 23 24 25 26 27 28

ISO 8589: Sensory analysis - General guidance for the design of test rooms. ISO, Geneva, 1988. ISO 8586: Sensory analysis - General guidance for the selection, training and monitoring of assessors. ISO, Geneva, 1989. ISO 6564: Sensory analysis - Flavour profile. ISO, Geneva, 1985. ISO 4121: Sensory analysis - Grading of food products by methods using scale categories. ISO, Geneva, 1988. L. Mondello, P. Dugo, K. D. Bartle, G. Dugo and A. Cotroneo, Flavour Fragrance J., 10 (1995) 33. R. Huet, Fruits, 46 (1991) 501, 551, 671. N. Nakatani, Nippon Nogeikagaku Kaishi, 62 (1987) 170. J. Pokorny, H. T. T. Nguyen and J. Korczak, Nahrung, 41 (1997) 176 L. Koui^imska, J. Pokorny and G. Tirzitis, Nahrung, 37 (1993) 91. L. Farnikova, J. Pokorny, G.Tirzitis, Potrav. Vedy, 13(1995) 225. J. Pokorny, F. Pudil, J. Volfova and H. Valentova, These Proceedings, p.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

689

Effect of a - t o c o p h e r o l ( v i t a m i n E) o n t h e r e t e n t i o n of e s s e n t i a l oil, color a n d t e x t u r e of Chios m a s t i c r e s i n d u r i n g s t o r a g e . D. Papanicolaou, M. Melanitou and K. Katsaboxakis National Agricultural Research Foundation, Institute of Technology Agricultural Products, 1 Sof Venizelou, Lycovrissi, 141 23, Greece

of

Abstract Vitamin E was added to Chios mastic resin packed in laminated packages. Product hardness, color and essential oil concentration were measured during storage for 450 days at 25 and 37°C. It was found that although sample weight remained constant, essential oil decreased gradually during storage as determined by clevenger distillation. This indicates that essential oil was transformed into other, nonvolatile, products. The presence of vitamin E retarded the essential oil loss significantly at both storage temperatures in comparison to control samples. Vitamin E retarded resin hardening and also Umited changes in color in comparison to control samples.

1. INTRODUCTION

The Chios mastic, is extracted from a tree, Pistacia lentiscus var. chia, which grows spontaneously on the Greek island of Chios. It is used primarily as a natural chewing gum and its essential oil, obtained by steam distillation, is used for the preparation of Hquors, as a flavor agent in food products (sweets, icecreams), in cosmetics e.t.c. The major problems of mastic resin during storage are the loss of essential oils and the simultaneous increase of its hardness which results in a product of inferior quality and of low commercial value (1). Previous studies suggest that the mastic gum could be considered as a system of a resin and a solvent and that the loss of the later by evaporation is the principal cause for the hardening of the product (2, 3). However, the effect of evaporation on the texture has not been studied. Furthermore, mechanisms other than evaporation have not been investigated whether they are involved in the loss of essential oil and/or the increase in hardness of the raw material. The known positive effect of antioxidants on oil preservation led the authors to study the effect of the addition of vitamin E on the stabiUty of the essential oil and the mastic texture during storage. Color measurement is a quahty parameter of the product and changes in color were also expected to be influenced by the addition of vitamin E.

690 Specifically, the main objective of this research were to study the effect of evaporation and of vitamin E (a-tocopherol), a natural antioxidant, on the loss of essential oil, the increase of hardness and the changes in color during storage.

2. MATERIALS AND METHODS The mastic was harvested according to the method developed recently (4). The essential oil concentration was four times higher than that of the traditionally harvested product and was semi-fluid in texture. In order to remove foreign impurities from the raw material, the harvested resin was slightly heated to liquefy it and then it was passed through a centrifugation filter. The filtered mastic was divided in two lots; Vitamin E (a-tocopherol) was added to the first at a concentration of 0.02% (w/w) and thoroughly mixed so that it was well distributed into the mass of the resin. No addition was made to the second lot. Samples of 5 g from each lot were packed in heat sealed plastic laminated bags, so that the loss of essential oils by evaporation was prevented. The packages were stored at two different temperatures: 25°C and 37°C. In addition, samples were stored at 37°C in uncovered petri dishes in order to study the loss of essential oil by evaporation through the surface of the samples. The weight loss of the samples, the texture, the color and the retention of essential oil were determined during storage. The retention of essential oils was determined by clevenger distillation. The texture (hardness) was measured by means of a Food Technology Corporation apparatus using a probe of 5 mm diameter which was submerged to a depth of 2 cm and with a speed of 5 cm/sec. The sample was placed on a hive of 7 mm diameter. The color of the samples was measured with a Minolta portable color difference meter.

3. RESULTS AND DISCUSSION

3.1. The influence of vitamin E in the loss of essential oil. Figure 1 presents weight loss and percent loss of essential oil during storage of mastic samples at 37°C in uncovered petri dishes. As shown in this figure, although the sample weight remained practically stable during storage, a loss of about 70 % of essential oil occurred within the first 60 days. This loss reached about 90 % after 90 days and increased sUghtly until the end of the storage period. This loss of essential oil as determined by clevenger distillation is higher than that which could be explained by the loss of weight of the samples (as a result of

691

0 0 -1

90

WEIGHT OF SAMPLE T LOO (% LOSS) •

ESSENTIAL OILS (% LOSS)

80 70

t=37 ^C

60 50

-p

40

-•-ess.oil

30

-^—weight 1

0.50

20 10 0 i, 0 0 0

or—^^—Q—1

50

100

1

1

150 200 TIME (DAYS)

250

1

—

°—i 0.00 300

F i ^ r e 1. Essential oil and weight loss of Chios mastic resin stored at 37 °C in uncovered petri dishes. % Loss= (Essential oil at time 0 - Essential oil at time T) X 100 Essential oil at time 0

evaporation mechanisms). This impUes that mechanisms other than evaporation contribute to the decrease of essential oil. This loss of essential oil is also demonstrated in figure 2 where the samples of mastic resin were stored at 25 and 37°C in sealed laminated packages preventing any weight loss by evaporation. As shown in this figure, the essential oil loss reached about 80 % at both temperatures within the first 100 days and above 90 % at the end of storage period (450 days). The addition of vitamin E limited the loss of essential oil, particularly for samples stored at 25°C. It was remarkable that at 25°C the presence of vitamin E reduced by half the loss of essential oil within the first 100 days. The level then remained almost constant even after 450 days of storage. The decrease in the essential oil content of mastic suggests that some of the components were transformed into nonvolatile substances which were no longer recoverable by clevenger distillation. The mechanism of that transformation could be polymerization and/or oxidation of those substances. That could also explain the increase in hardness of the resin as well as the modification of the chemical composition of the essential oil taking place throughout the storage of the product, the most important being the decrease in the myrcene content and the increase in a-pinene (5). Vitamin E reduces the rate of loss of essential oil. This indicates that vitamin E blocks the mechanism of polymerization and/or oxidation.

692

100 90

ESSENTIAL OILS (% LOSS)

80 70 60 50 + 40 30 20 10 0 200 300 TIME (DAYS)

500

F i ^ r e 2. Effect of a-tocopherol on essential oil loss of Chios mastic resin stored at 25 and 37 °C in sealed laminated packages

3.2. The influence of vitamin E in the texture (hardness) of mastic during storage. As mentioned above, the progressive hardening of mastic during storage is the most serious problem in the quality and the commercial value of mastic resin. As can be observed in figure 3, the addition of vitamin E retarded this undesirable change in texture significantly for 60 days at both temperatures (25 and 37 °C) in comparison with the control samples. The presence of vitamin E minimized also the negative effect of the high temperature (37°C) for 40 days while a higher increase rate of hardening was observed thereafter. This was probably due to the destruction of vitamin E at this temperature. The increase in hardness of the mastic during storage was attributed to the polymerization reactions of the constitutents of the resin (3). It seems, therefore, that vitamin E acts protectively by blocking or retarding this mechanism of polymerization and/or oxidation, as in the case of the essential oil.

693

160 140 120 100 80 + 60 40 20

25 oC+vitE 25 oC 37 oC+vitE 37 oC

HARDNESS (Kg)

30 TIME (DAYS)

Figure 3. Effect of a-tocopherol on hardness of Chios mastic resin stored at 25 and 37 °C in sealed laminated packages.

3.3. The influence of vitamin E on the color of mastic during storage. Figure 4 presents changes in L value and b/a ratio of color values measured with a Minolta Colorimeter during storage. A gradual loss of L value which represents the lightness can be observed in all samples with a higher loss rate at 25 °C than at 37 °C. The addition of vitamin E did not influence significantly these changes in L value. L VALUE

b/a VALUE

^-25oC B—37oC+vitE e—37oC L VALUE •—25oC+vitE •-25oC A—37oC+vitE

100

150

200

250

300

•—37oC

TIME (DAYS)

Figure 4. Effect of a-tocopherol on color of Chios mastic resin stored at 25 and 37 °C in sealed laminated packages. An increase in b/a ratio indicates the development of a yellowish color in the product which is an undesirable alteration. As shown in figure 4, the addition of

694 vitamin E retarded this increase in comparison to controls at both temperatures. The alteration of color is observed even in the partial absence of oxygen as the samples used for measurements were stored in laminated packages of aluminum foil in the absence of atmospheric oxygen.

4. R E F E R E N C E S 1. M. Melanitou, D. Papanicolaou, K. Katsaboxakis and K. Stamoula. Comparison of some physicochemical characteristics between solid and fluid Chios mastic resin. Dev. Fd. Sci. 37B, Proceedings of the 8th International Flavor Conference, Cos, Greece, July 1994, 1937. 2. M. Codounis. Application du froid au conditionnement et a la conservation du mastic de Chios. Rev. Gen. Froid (1979) 11, 657. 3. A. Kehayoglou, G. Doxastakis, V. Kiosseoglou and M. Mikedis. Compressional properties of Chios Mastiche (Pistacia lentiscus var. Chia) (1993). In: Food Flavors Ingredients and Composition Ed. G. Charalambous Elsevier Publishers p.429. 4. D. Papanicolaou, M. Melanitou, K. Katsaboxakis, D. Bogis and K. Stamoula. A new method for harvesting of Chios mastic resin in fluid form. Dev. Fd. Sci. 37A, Proceedings of the 8th International Flavor Conference, Cos, Greece, July 1994,311. 5. D. Papanicolaou, M. Melanitou and K. Katsaboxakis. Changes in chemical composition of the essential oil of Chios mastic resin from Pistacia lentiscus var. Chia tree during solidification and storage. Dev. Fd. Sci. 37A, Proceedings of the 8th International Flavor Conference, Cos, Greece, July 1994, 303.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

695

Dietary oil and endogenous antioxidants in hyperlipemia: Uric acid T. R. Watkins, D. K. Kooyenga and M. L. Bierenbaum K. L. Jordan Heart Research Foundation 48 Plymouth St., Montclair, NJ 07042 USA

Abstract Accumulating evidence has shown that limited serum antioxidant reserves are an important indicator of risk of cardiovascular disease. Would depletion of exogenous antioxidants (e. g., serum vitamins), by oxidant stress, also lead to depletion of endogenous antioxidants? Serum antioxidants in hyperlipemic subjects eating selfselected diets, with dietary addenda of canola oil or milled flax seed, were measured. Dietary stress of polyunsaturated oil, here canola, depleted serum vitamins and urate, too.

1. INTRODUCTION Life is a fire. Oxygen sustains life. It serves as the terminal electron acceptor in normal oxidation in mammalian life as we know it. No oxygen, no life. Though some organisms use other acceptors, e. g., the sulfur bacteria use sulfur, life as we know it uses oxygen. However, if we increase the oxygen concentration above certain narrow limits, an organism's growth may cease altogether. In the plant kingdom. Hardy [1] has shown that the soya plant exposed to excess oxygen will suffer growth and yield retardation. The yields fall as ribulose-1,5bisphosphate carboxylase is inhibited, poisoned by oxygen. And mammals? If the laboratory rat is exposed to an elevated oxygen atmosphere of 70%, it will begin to suffer pulmonary edema within a few days and die soon thereafter. The premature human infant incubated in an elevated oxygen atmosphere begins to adapt with decreased vascularization of the retina. Once outside of the incubator, the eye adapts by increased vascularization and the retina becomes damaged, the retinopathy leading eventually to blindness [2]. Oddly, oxygen, vital for life, may be toxic under certain conditions. Some evidence exists that, even in a healthy body, normal metabolism generates toxic oxygen species. Is oxygen our deadliest friend? This question has been raised about the Damoclesian nature of oxygen. How can oxygen be so toxic? Gershman and Gilbert [3] noticed that the death rates associated with elevated oxygen tensions were much greater than expected in several microorganisms and small mammals. Hence, they posited that the toxicity observed is induced by radical forms of oxygen. Since superoxide anion was a reduced form of oxygen and possibly implicated, their observation was

696 extended and the role of oxygen as superoxide has been styled 'the superoxide theory of disease' by McCord & Fridovich [4]. If this is true, then one should be able to measure the presence of oxygen radicals, and such levels should be elevated in 'disease states'. Any condition leading to elevated free radicals in the tissues ought to lead to enhanced risk of disease. For example, prolonged exposure to smoke, whether from cigarette, cigar, auto, factory or other source (such as some medicinals, like doxorubicin), /. e., sources of exogenous free radicals, ought to lead to elevated risk [5]. Life itself leads to accumulation of free radicals, via leakage in the electron transport chain, redox cycling and uncorrected errors. A fire consists of a source of electrons, an electron acceptor, yielding byproducts (water, if oxygen is the electron acceptor), other byproducts and heat. So does the fire of life. Biochemically speaking, life is a fire sustained by a source of combustible material, /. ^., food, a reservoir of electrons; air, a source of oxygen to take up the liberated electrons; and heat captured to do useful work, or lost to the environs, and waste, such as water. Hence, we need a constant supply of food for its electrons, and air-containing oxygen—as the terminal electron acceptor to sustain the vital fire. Let us consider the step-wise reduction of oxygen. The reduction of oxygen Oxygen is reduced in four steps. In the first step, diatomic oxygen takes up one electron, thereby becoming superoxide anion. Next, this is reduced to hydrogen peroxide. This is followed by reduction to hydroxyl free radical and hydroxide ion. See Figure L (The dot indicates the unpaired, free electron.) Here one sees several toxic species of

Figure 1. The reduction of oxygen to water. O2 + e -

0 2 - * + 2H^ + e H2O2 + e - ^

UQ.

Superoxide formation

-^ 0 2 - * ^

H2O2

HO* +

OH-

+ e- -* OH-

Superoxide to Hydrogen peroxide Peroxide reduction to hydroxyl Hydroxyl to hydroxide

2 0 H - + 2H+ -> 2H2O O,

+ 4H^ + 4e-

->

H,0

the essential oxygen molecule: superoxide, hydrogen peroxide, and hydroxyl radical. These species react aggressively with living tissues, leading to tissue damage and disease, yet the body has a defense system. Both enzymatic and non-enzymatic protective systems function to prevent tissue damage. Enzymes like superoxide dismutase, catalase and glutathione peroxidase offer local protection against certain of these highly reactive

697 radicals. In other cases, the tissue relies on facile electron donors, styled antioxidants, to stop the local burnt tissue injury from enlarging. These include ascorbic acid, tocopherols, tocotrienols, glutathione and other phenols. Fatty acids, proteins, carbohydrates and nucleic acid bases represent good sources of readily oxidizable substrate. Tappel [6] has shown the importance of antioxidants in preventing unchecked chain reactions perpetuated by free radicals in the case of the fatty acid. In Figure 2, L represents a polyunsaturated fatty acid. In the presence of an available electron donor, an 'antioxidant', indicated AH, the chain can be terminated. By intercepting the free radical LOO-, the antioxidant, AH, can prevent further radical generation, i. e., radically slow the chain. Further, the antioxidant free radical. A- is much less reactive with fatty acids. Evidence has been published showing that various other peroxidized forms exist [7].

Figure 2. Free radical formation of a polyunsaturated fatty acid. LH -* L» + H«

H abstraction

L» + O2 ^

Combination with oxygen

LOO*

LOO* + LH ^

LOOH + L*

Perpetuation

LOO* + AH ^

LOOH + A*

Chain termination

Under normal conditions, the level of oxygen free radicals is balanced by antioxidant defenses. In circumstances of stress, such as a disease state, the balance can be readily tipped in favor of further tissue damage and toxicity by the radicals. Clearly, one would expect that consumption of a diet providing inadequate amounts of antioxidant vitamins, particularly vitamins C and E, and sulfhydryls, such as cysteine, as well as metals like selenium, the latter two cofactors of glutathione peroxidase, and riboflavin, essential for glutathione reductase, and possibly protein, could lead to unchecked tissue damage and disease. Further, conditions leading to chronic inflammatory disease states, such as conditions leading to elevations of xanthine oxidase, superoxide dismutase, or phagocytes activated in inflammatory disease, would be expected to impose a burden of oxidative stress [8]. Consider the phagocyte as an example. Neutrophil + foreign particle (on cell surface)

-*

02~»

[NADPH oxidase].

At a site of local injury, NADPH oxidase can generate superoxide as a defense against local injury. This superoxide radical then wreaks havoc on the local tissue. Though it does not enter the cell directly, it can be converted into hydrogen peroxide which readily enters the cell (as illustrated in Figure 1). Once in contact with iron or copper, it

698 produces highly toxic OH* radical. The superoxide may also react with NO* to yield peroxynitrite, ONOO". It may also break down to yield OH*. Various forms of oxygen based radical damage have been measured. Thiobarbituric acid reactive species (TEARS) have been widely reported. Though not specific to lipid substrates, these have been widely reported. Radicals other than oxygen based ones may be important in disease; nitrogen based radicals have received some attention. Amino acids having been H* abstracted have been studied extensively by Davies [9]. Peripheral neural damage by oxygen has received considerable attention in the condition of diabetes, being measured as a decreased level of reduced glutathione, & increased conjugated dienes and hydroperoxides [10]. Oxygen damaged nucleic acid has also been studied, as 8-OH guanine and 8-OH adenine [11,12]. The -OH radical has been associated with metastasis of breast cancer. [13]. Tissue elevations of these compounds have been linked to carcinogenesis, e. g., breast and prostate cancer [14]. The accumulation of these compounds has also been associated with pre-mature aging [15]. In measuring states of elevated oxygen radical species, then, one would also expect to be able to measure decreased levels of reducing species, more specifically, some of the antioxidant vitamin species. The major antioxidants measured in mammalian species have been ascorbic acid, the phenolic sort, like a- and y-tocopherol. Any other important ones would be expected to be first cousins of phenols, or possibly quinone-type molecules with appropriate redox potentials. The normally metabolizing mammalian cell has to deal with toxic oxygen species. As electrons are shuttled from food to oxygen, the terminal electron acceptor, some leakage occurs in the electron transport chain. Some estimates place the magnitude at 3%, or possibly somewhat higher. This could amount to nearly two kg of oxygen radicals per year per person. These radicals must be reduced in order to protect the tissue from cross-linking and other chain related damage events. The lipid ferried in the LDL lipoprotein particle may be a culprit in the etiology of heart disease. Does dietary supplementation with antioxidants raise tissue antioxidant levels (serum, in this instance)? We have shown [16] that as the dietary vitamin E level was raised, subsequently, the serum level also rose in the laboratory rat. Also, we have seen that serum vitamin E level reflects dietary intake: supplementation of the human diet with vitamin E boosts the serum level. See Figure 3. Does the accumulation of oxygen damaged lipid in the blood and the associated decreased antioxidant reserve result in increased levels of risk factors of cardiovascular disease? In a study from our laboratory [18], the fatty acid hydroperoxide level was shown to vary inversely with the serum vitamin E concentration in hyperlipemics. The tendency of the platelet to aggregate, to form a thrombus rises in step with the serum peroxide level, and malonaldehyde, typically. Additionally, should these subjects be supplemented with 800 lU/d of the vitamin, their platelet aggregability, a functional indicator of thrombogenic risk, returned to normal values. If the risk factors faithfully represent actual morbidity, one would expect to find some data corroborating it. Population data has been published. In an eight-center epidemiologic study of morbidity and mortality of cardiovascular disease in which more than 32,000 subjects were studied. Gey [20] has reported that the risk of the disease correlated inversely with the serum concentration of vitamin E,

699 Figure 3. Serum vitamin E level as a function of dietary intake: human subjects.* Dietary Intake, lU/d Serum level, range, mg/dL None (Elgin study)

<0.2

10

0.2 - 1

400

1 - 2.5

800

2.5 - 4.0

2000 4.0 - 5.5 * Data compiled from a series of studies. See [17, 18, 19]. a-tocopherol. Further, the serum level of this lipid-soluble, antioxidant vitamin was a more powerful predictor of risk than any of the traditional risk factors, serum cholesterol, blood pressure elevation or cigarette habit. Angina has been similarly correlated with serum vitamin E concentration by Riemersma [21]. In a more recent supplementation trial, Stephens ['Cambridge study', 22] has shown that non-fatal cardiovascular events decreased about 75% after subjects received daily vitamin E. Further, antioxidant levels have been correlated with stroke disease. Aspirin inhibits cyclooxygenase, thereby limiting PGE2 production, a powerful agonist of platelet aggregation, like vitamin E [23]. Aspirin, a powerful anti-platelet factor, has been reported to decrease the incidence of stroke in several recent studies, such as the Swedish aspirin study [24], using 75 mg/d. Verlangieri and Bush [25] reported that Rhesus monkeys, in a 36-month vitamin E supplementation trial, showed decreased incidence of carotid stenosis compared with controls. In a study from our laboratory, tocopherols supplementation (including a- and y-tocopherols and tocotrienols) of persons who have suffered a transient ischemic attack (TIA), monocular blindness of less than 24 hours duration, or a minor stroke, resulted in improved-decreased-serum peroxide levels within six months. This improvement persisted for more than two years [26]. Supplementation also resulted in significantly improved carotid artery blood flow, regression of the carotid artery stenosis, a 'roto rooter' job in the stenosed carotid artery [27]. Have toxic oxygen radicals been associated with other diseases? Several have been evaluated. These include: ionizing radiation induced tissue injury; selenium deficiency; inborn defects of antioxidant enzymes; impaired intestinal fat absorption leading to vitamin E deficiency; retinopathy of prematurity; copper overload injury (Wilson's disease); iron overload disease (hemachromatosis); cataract induced by ultraviolet radiation; other conditions related to smoke or elevated oxygen levels. In addition to cardiovascular disease, other diseases tend to increase with oxidative stress, such as rheumatoid arthritis, inflammatory disease (like Crohn's and ulcerative colitis); shock and traumatic injury to the spinal cord or brain. In these conditions, the trauma or stress often results in elevated levels of xanthine oxidase, generating more superoxide, and later more free iron, leading to hydroxyl radical production. Ames, et al [15], have reported oxidized guanine and adenine, which, when present at sufficient levels, lead to altered DNA templates. In actively dividing cells, these will lead to transcriptional errors.

700

Eventually, they result in mutations. He and his group have shown that in male cigarette smokers, the urinary level of modified purines, such as 8-OH-guanine, correlate with the number of cigarettes smoked per day. Also, above a certain level of smoke exposure, the testicular tissue cannot be purged of the oxidatively modified bases, resulting in by sperm non-virulence. The LDL hypothesis of the initial injury leading to heart disease has been proposed by Steinberg [28]. According to this hypothesis, an LDL particle laden with peroxidized lipid can be recognized by a special receptor in the intima of the artery. Once recognized, it will be taken out of the circulation and left in the subendothelial space. Association of macrophage cells with this debris accumulating in the subendothelial space eventually becomes transformed into immobile foam cells. As these living fat droplets accumulate, they tend to occlude the lumen. The injury site may later become calcified. Atherothrombotic risk rises.

2. PURPOSE and EXPERIMENTAL In altering one's dietary fat pattern from more- to less-saturated fatty acids, to decrease serum cholesterol levels, do antioxidant levels change? If altered fat is used as a strategy to alter the serum cholesterol levels, then antioxidant levels may change. In general, if a polyunsaturated fatty acid rich diet were fed, one would expect cholesterol levels to decrease. The antioxidant vitamins inhibit platelet aggregation, a concern among persons with hyperlipemia [18]. The antioxidant vitamin levels would be expected to decrease, since peroxide and other oxidizers oxidize the tocopherol antioxidants, as during processing. Strong oxidizers have routinely been used to bleach vegetable oil. Feeding diets rich in polyunsaturated fatty acids have resulted in increased incidence of neoplasms. Dayton reported [29] the success of com oil in modulating cholesterol levels. Within ten years, however, he reported an increased incidence of tumor disease in his remaining cohort. When he reported this, others denied his conclusion [30]; they did not wish to accept the results. Could diets rich in polyunsaturated fat deplete antioxidant reserves? We tested the effect by feeding canola oil, a commonly eaten polyunsaturated fat, to hypercholesterolemic subjects. It would be expected to lower serum cholesterol levels, and possibly antioxidants. If canola results in depletion of serum antioxidants, then feeding a flax seed addendum, a source of more highly unsaturated fat, rich in (0-3 fatty acids, might lead to further depletion of serum antioxidants. Would either oil temper platelet hyper-aggregability? Uric acid has a redox potential more negative than some of the antioxidant vitamins, therefore, it might also be used up when subjects ate diets rich in polyunsaturated fat. By feeding canola, rich in 0-6 lipids, and flax seed, rich in (0-3 lipids, light may be shed upon these questions. At the appointed time, fasting serum was drawn into Vacutainer® serum separator tubes, allowed to clot, centrifuged and then analyzed immediately or stored frozen under nitrogen and assayed within a month of drawing. Standard laboratory assays for lipids, antioxidant vitamins and peroxidation products have been described [18,31]. Standards were obtained from Sigma Chemical Co. (St. Louis, MO) or Roche (Basel, Switzerland).

701 3. RESULTS Feeding human subjects commercially available, processed canola oil (30 g/d) modulated serum lipids and resulted in depleted antioxidant reserves (31). Table 1 shows the serum lipid values of a human feeding study in which hyperlipemic subjects (N = 36) were fed a daily allotment of canola oil for eight weeks. Though no significant change occurred in total cholesterol, the LDL fraction decreased significantly, from 173 to 160, p < 0.05. The HDL cholesterol fraction did not change. The triglyceride levels also remained stable. Serum vitamin levels changed, particularly the antioxidant vitamins E and j8carotene. Table 2. Retinol levels remained steady. The oil soluble antioxidants, however, decreased markedly, when the body was stressed with the daily addendum of an ounce of canola oil, and no extra antioxidants. Hence, in a subsequent study, we began to define the relation between toxic, serum peroxides and serum antioxidant reserves in hyperlipemic subjects (N = 16). As peroxide levels rose, antioxidant vitamin levels declined [18]. Further, the supplementary antioxidant a-tocopherol supplement did not result in any improvement in serum lipids in these hyperlipemic subjects. In addition, the serum level of uric acid decreased in concert with the antioxidant vitamins. Table 3. Throughout this study subjects were monitored for general health status using routine tests. The indices suggested that the subjects were indeed healthy, with respect to renal and liver function. Table 4. In a separate part of the study, we attempted to define the relation between serum antioxidant reserves and peroxide levels. Would the hypercholesterolemia these hypercholesterolemic subjects suffered improve with antioxidant supplementation? Based upon an earlier supplementation trial in which diabetics were supplemented with 2000 I. U. of vitamin E daily, we did not expect them to decline [18]. They did not. The hyperlipemics had elevated serum peroxide levels. In an effort to detoxify their elevated peroxide levels we gave them a-tocopherol antioxidant. As their lipids remained elevated, we turned to flax seed, a seed rich in polyunsaturated fatty acid, providing more than twice as much n-3 fatty acid (a-linolenic acid) as canola oil, besides soluble fiber, both potentially hypolipemic [32-35], Table 5. Table 1. Serum cholesterol levels in subjects given canola oil daily for 8 weeks. Fraction Base End Cholesterol

254 ±12

248 ±12

LDL-cholesterol

173±9.0*

160±10

HDL-cholesterol

47±4.8

51±5.0

Triglycerides 214 ±28 226 ±32 Data as mg/dL; * p < 0.05. Reprinted from M. L. Bierenbaum, et aL, J. Am. Coll. Nutr. 10 (1991) 230.

702 Table 2. Serum vitamin levels during canola oil supplementation for 8 weeks. Vitamin Base End P A,Atg/dL

52.4±3.3

54.3±2.4

n. s.

E, mg/dL

1.62±0.1

1.36±0,06

0.005

j8-Carotene, /tg/dL 27.4±4.0 14.9±1.8 0.005 Reprinted from M. L. Bierenbaum, et aL, J. Am. Coll. Nutr. 10 (1991) 230.

Table 3. Serum uric acid levels with canola oil supplementation for 8 weeks. Analyte Base End p Uric acid, mg/dL

6.4±0.35

6.1±0.34

0.025

Would a daily supplement of 30 g of flax seed meal incorporated into bread modulate the serum cholesterol levels down toward normal? Data had showed that it could [34]. Half of the subjects (N = 20) ate the supplement for six weeks. This period was followed by a four-week washout period and then six weeks eating the wheat bread as a control, or vice-versa. The serum lipids decreased significantly [35], Table 6. During the study serum peroxides and vitamins were monitored. Data showed that lipid hydroperoxides (indicated as LOPS) decreased in both arms of the study, the decrement being much greater during the flax periods. Table 7. Both vitamin C

Table 4. Health indices of subjects in the 8-week canola study. Index Status BUN*

No change

GOT

No change

GPT * BUN: blood urea N; CJCJT: y-gl^ transferase;

No change GPT: glu-pyr transferase.

and j3-carotene increased significantly during flax feeding. Vitamin E tended to improve. With flax feeding, though we expected to see antioxidant vitamin levels decrease, flax

Table 5. Fatty acid profile of canola and flax seed lipids, weight percent. Fat Saturated Monounsat. Diunsat. Triunsat. (o) 3) Canola

6

58

26

10

Flax 11 22 14 53 Reprinted from : U. S. D. A. Food Composition Handbook/VY103, Washington, D. C.

703

derived antioxidants spared some antioxidant vitamins, as seen in the serum data. The serum data for ascorbic acid and uric acid varied in concert in this feeding study. As tissue ascorbate levels decreased, urate levels also decreased. This relation, too, was consistent with urate functioning in concert with other antioxidants, in this case ascorbate.

Table 6. Serum lipids of subjects eating a daily allotment of flax seed bread, mmol/L. Fraction Wheat, base Wheat, end Flax, base Flax, end Cholesterol

6.0±0.02*

5.7±0.2

5.8±0.2**

5.4±0.2**

LDL-chol.

4.2±0.2*

3.9±0.2

4.2±0.1**

3.4±0.1**

HDL-chol.

1.1±0.1

1.2±0.1

1.0±0.1

1.1±0.1

0.2+0.03 0.17±0.02 0.16+0.03 0.15±0.02 Triglyceride * p < 0.05; ** p < 0.01. Reprinted from: T. Watkins, et aL, G. Charalambous (ed.) Food Flavors: Generation, Analysis, Process Influence, Elsevier, Amsterdam, 1995, p. 653. Table 7. Serum peroxides and vitamins during flax feeding for six weeks, /xmol/L. Wheat, base Substance Wheat, end Flax, base Flax, end LOPS

4.0±0.28*

1.44±0.17

5.3±0.59*

1.5±0.21

Vitamin C

86.3±9.1

85.7±5.7

74.4±4.0*

87.4±6.8

j8-carotene

0.65±0.11

0.47±0.1

0.63±0.05*

1.01±0.1

45.7±3.9 56.0±10.4 62.9±9.3** 43.0±5.3 Vitamin E p < 0.001; ** p < 0.02. Reprinted from: T. Watkins, et aL, G. Charalambous (ed.) Food Flavors: Generation, Analysis and Process Influence, Elsevier, Amsterdam, 1995, p. 653, 654.

Tables. Serum uric acid during flax seed meal feeding for six weeks. mg/dL. Analyte Wheat, base Wheat, end Flax, base Flax, end Uric acid 6.05 ±0.41 5.84±0.50 5.59±0.48 5.56±0.66

Table 9. Health indices measured during flax supplementation. Index Wheat, base Wheat, end Flax, base

Flax, end

18.4±2.2

17.0±2.3

19.0±1.8

33.3±8.0 35.2±7.8 GGT Data units: BUN, mg/dL; GGT: U/L.

26.2±8.1

30.1±9.3

BUN

19.1±1.7

704 The uric acid data remained stable during flax feeding, Table 8. As the antioxidant components in flax spared body reserves of antioxidant vitamins during flax feeding, uric acid apparently was not needed as an antioxidant reserve. During flax feeding, other indices indicated that the subjects remained healthy. Table 9.

4. SUMMARY We may draw several conclusions from these data. First, canola oil feeding attenuated serum cholesterol levels, as did flax seed. The more potent flax effect was especially marked in the LDL fraction. Second, antioxidant levels in tissue vary directly with dietary input, as in the case of tocopherol. Third, serum antioxidant levels varied inversely with dietary unsaturation load and antioxidant input. The canola oil addendum depleted antioxidants. Fourth, depletion of urate followed depletion of antioxidants such as vitamin E and j8-carotene, during the canola study. In contrast, the flax diet spared urate, in spite of higher unsaturation levels in the flax than the canola study. Sixth, when the antioxidant ascorbate was depleted from tissue stores, urate was also depleted, showing that these water-soluble antioxidants worked in concert (data plot not shown). In summary, we have seen from two studies that as increased polyunsaturated fat intake consumes antioxidant vitamin reserves, the body begins to use other available reducing equivalents. Uric acid, another potential reducer, acts as a source of reducing equivalents. Other compounds, such as flavonoids, may function similarly. The trail of our recent work has been leading to evaluation of more of them, such as those in wine.

5. REFERENCES 1 2 3 4

5 6 7 8 9 10 11 12 534.

B. Quebedeaux, U. Havelka, K. Livak and R. W. F. Hardy, Plant Phys. 56 (1975) 761. L. Johnston, D. Schaffer, D. and T. R. Boggs, Am. J. Clin. Nutr. 27 (1973) 1158. D. L. Gilbert (ed.). Oxygen and living processes. Springer, New York, 1981. J. McCord, M. Crapo and I. Fridovich., In: A. M. Michelson, J. M. McCord and I. Fridovich (eds.). Superoxide and Superoxide Dismutases. Academic Press, New York, 1977. J. M. C. Gutteridge and B. Halliwell. Free Radicals in Biology and Medicine, 2d ed. Clarendon Press, Oxford, 1989. A. L. Tappel, Fed. Proc. 32 (1973) 1870. R. Yamauchi, T. Matsui, Y. Satake, et al Lipids 24 (1989) 204. B. M. Babior, New Engl. J. Med. 298 (1978) 645, 721. K. J. A. Davies, J. Biol. Chem. 262 (1987) 9895. K. K. Nickander, J. D. Schmelzer, D. A. Rohwer, et al J. Neuro. Sci. 126 (1994) 6. R. A. Floyd, FASEB J. 4 (1990) 2587. S. Loft, A. Astrup, B. Buemann and H. Enghusen Poulsen, FASEB J. 8 (1994)

705

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

D. C. Malins, N. L. Polissa and S. J. Gunselman, Proc. Nat'l. Acad. Sci. 93 (1996) 2557. D. C. Malins, N. L. Polissa and S. J. Gunselman, Proc. Nat'l. Acad. Sci. 94 (1997) 259. B. N. Ames, M. Shigenaga T. Hagen, Proc. Nat'l. Acad. Sci. 90 (1993) 7915. T. R. Watkins, P. Lenz, A. Gapor, et al, Lipids 28 (1993) 1113. M. K. Horwit, Am. J. Clin. Nutr. 8 (1960) 408. M. L. Bierenbaum, R. Reichstein, H. N. Bhagavan, et aL Biochem. J. Int'l. 28 (1992) 57. M. L. Bierenbaum, F. J. Noonan, L. J. Machlin, et al Nutr. Repts. Int'l. 31 (1985) 1171. G. F. Gey, P. Puska, P. Jordan, et al Am. J. Clin. Nutr. 53(1991) 326S. R. A. Riemersma, D. A. Wood, C. A. Macintyre, et al Lancet 337 (1991) 1. N. G. Stephens, A. Parsons, P. M. Schofield, et al Lancet 347 (1996) 781. F. Violi, D. Pratico, A. Ghiselli, et al Atherosclerosis 82 (1990) 247. M. Britton, C. Helmers and K. Samuleson, Stroke 18 (1987) 325. A. J. Verlangieri and M. J. Bush, J. Am. Coll. Nutr. 11 (1992) 130. A. C. Tomeo, M. Geller, Watkins, T. R, et al Lipids 30 (1995) 1179. D. K. Kooyenga, M. Geller, T. R. Watkins, et al Asia Pacific J. Clin. Nutr. 6 (1997) 72. D. Steinberg, S. Parthasurathy, T. E. Carew, et al New Engl. J. Med. 320 (1989) 915. S. Dayton, Circulation (Suppl.) July, 1969. F. Ederer, P. Leren, O. Turpeinen, et al Lancet 2 (1971) 203. M. L. Bierenbaum, R. Reichstein, Wm. McGinnis, et al J. Am. Coll. Nutrition. 10 (1991) 228. H. O. Bang and J. Dyerberg, Adv. Nutr. Res. 3 (1980) 1. T. Terano, A. Hirai, T. Hamazaki, et al Atherosclerosis 46 (1983) 321. M. L. Bierenbaum, R. Reichstein, T. Watkins, J. Am. Coll. Nutr. 12 (1993) 501. T. R. Watkins, A. C. Tomeo, M. Struck, et al In: G. Charalambous (ed.). Food Flavors: Generation, Analysis and Process Influence, New York, Elsevier (1995) 649.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

707

Changes in Citrus hystrix oil during autooxidation F. Pudil, H. Wijaya*, V. Janda, J. Volfova, H. Valentova and J. Pokomy

Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technicka 5, CZ-166 28 Prague 6, Czech Republic

^Faculty of Agricultural Technology, Bogor Agricultural University, P.O. Box 220, Bogor 16002, Indonesia

Abstract The essential oil from Citrus hystrix is an interesting new raw material for the food and cosmetic industry. The composition of oil was analyzed by capillary gas chromatography. Changes due to autooxidation were studied at 40*^C and 60°C using a headspace autosampler. Volatiles were identified by GC-MS after solid-phase microextraction (SPME) sampling using a Carbowax coated fiber. Changes in the sensory profile of autooxidized oil were determined imder conditions specified by ISO standards, using unstructured graphical scales. Influences of a dihydropyridine antioxidant, Diludine, and of rosemary extracts on the course of oxidation of Citrus hystrix essential oil were determined; correlations between sensory and chromatographic data were calculated.

1. INTRODUCTION Citrus hystrix is a citrus plant which is widely used in various foods and beverages in Indonesia ("jeruk purut") and other Asian countries as a source of natural flavor. The leaves of Citrus hystrix^ called "som makrut" in Thailand and "suwangi limau" or "purut limau" in Malaysia, are used to give unique oriental flavour to soups, curries, and many other cookies and cakes. The flavor is similar to that of citrus flavours, but it is harsher, reminding the panelists of citrus leaves. This flavor source is ahnost unknown in the European countries. Investigations on Citrus hystrix oil composition have been akeady reported by several authors [1-7]. It was reported that the oil contained citronellal, citronellol and nerol. A recent study by Sato et al. [2] has confirmed citronellal as a principal component of the oil. Furthermore, the authors foimd that citronellal in the oil is present in the L-form. Discussion

708 on the oil constituents was also published in a review paper by Lawrence [3]. Processing methods to produce Citrus hystrix leaves flavor were compared and evaluated by Wijaya [4]. The earlier study on the Citrus hystrix oil composition was published by Pudil et al. [7].

2. MATERIALS AND CHEMICALS Citrus hystrix leaves were obtained from a local market in Kramat Jati, Jakarta. Indonesia.The oil was prepared from Citrus hystrix leaves by steam distillation at atmospheric pressure for 2 hours using a pilot plant modified oil separator trap. No terpene removal was made. Two samples were analyzed: Oil A, prepared in 1995 and oil B, prepared in 1996. The mixture C was a synthetic mixture containing only citronellal, citronellol and limonene in the ratio contained in Oil A. Rosemary extract was prepared by extracting dry rosemary leaves with acetone; the 1,4dihydropyridine derivatives, Diludine (2,6-dimethyl-3,5-diethoxycarbonyl-l,4-dihydropyridine, and OSI 7284 (2,6-dimethyl-3,5-dibutoxycarbonyl-l,4-dihydropyridine) were prepared in the Institute of Organic Synthesis (Director Prof. Dr. G. Duburs) in Riga, Latvia.

3. EXPERIMENTAL PROCEDURES 3.1. Oxidation of the Citrus hystrix oil A 0.1 mL portion of pure Citrus hystrix oil was placed m a 10 mL vial. An internal standard of 5 mg of n-decane was also added. The vial was sealed and conditioned at 40°C in a thermostat. A 0.1 mL sample of vapor phase was injected via a gas-tight Hamilton syringe into the gas chromatograph (using a headspace autosampler). Alternatively, the SPME sampling technique was used. To study the antioxidant efficiency, 1 mg of the antioxidant (1,4-dihydropyridines or rosemary extract) was added. 3.2. Gas liquid chromatography GLC analyses of oxidized Citrus hystrix oil were carried out in a the GC 8000 series gas chromatograph (Fisons Instruments) equipped with a flame ionization detector and a 60 m x 0.32 mm Supelcowax 10 capillary column fihn thickness (Supelco, USA). The column temperature was programmed from 50°C (constant for 2 min), at a rate 2°C/min to 220°C (and then at 220°C for 30 min). The injector temperature was 220°C and the detector temperature was 250°C. The carrier gas (helium) pressure was kept at 100 kPa. The input split ratio was 1 : 25. To determine retention indices, a mixture of n-alkanes was injected with the sample of Citrus hystrix oil.

709 3.3. Gas liquid chromatography - mass spectrometry (GLC-MS) GLC-MS analyses of oxidized Citrus hystrix oil were carried out on a MSD 800 mass spectrometer with a GC 8000 series gas chromatograph (Fisons Instruments). The energy of ionizing electron was 70 eV. The capillary column and the temperature program were as above. The compounds were identified on the basis of Aeir mass spectra (NIST library in the MassLab software package; Fisons) and retention data of pure standards.

I

3.4. Solid phase microextraction (SPME) A 0.065mm Carbowax - Divinylbenzene fiber for a manual holder (Supelco, USA) was used for extracting volatiles; the fiber was inserted into the vial (Figure 1). The extraction time was 10 min and the temperature was 40*^0. The time of desorption was 2 min at 220°C. At the same temperature the fiber was cleaned for 30 min.

Figure 1. The detail of SPME sampling 3.5. Sensory analysis The sensory analysis was performed according to international standard [8] in a test room provided with six standardized test booths [9]. The panel of 12 assessors consisted of selected and trained persons [10] with experience in the sensory profiling (2-4 sessions a week, 4 samples each) of at least 6 months. The sensory profile consisted of 24 -36 descriptors, and the intensities of partial flavor notes were rated by unstructured graphical scales [11]. Two samples were served at a time, consisting of the tested product supported on a piece of cotton, placed in a 500-mL ground-stoppered bottle. The absolute odour intensity was evaluated by sniffing, and rated using linear graphical scales.

3.6. Sociological study Each person evaluated the Citrus hystrix oil by sensory profiling and was familiar with the flavour, was served on a special sheet containing questions concerning the optimimi application of oil. Some application were preprinted (the same as shown in Table 2), and the respondents could add any other comments Aey judged as useful. The results were expressed in % of total responses.

710 4. RESULTS AND DISCUSSION

Citrus hystrix oil was slightly oxidized and shows peaks of oxidation products in addition to natural components (Figure 2). The chemical composition was similar to that published earlier [7] especially the major peaks (Table 1); only the content of citronellal was lower than reported in the literature. During autoxidation under simulated storage conditions, the content of citronellal decreased rapidly, and followed first order reaction kmetics (Figure 3A). For the GLC/MS analysis of oxidized oil, the device shown in Figure 1 was applied. No great difference was observed between the degradation of non-stabilized and stabilized samples. The formation of oxidation and other reaction products was affected by antioxidants. The formation of acetone was efficiently inhibited by tiie rosemary extract (Figure 3B), but both 1,4-dihydropyridines were ineffective. The same was observed in the case of linalool oxides formation (in this case, Diludine was found slightly better than OSI 7284), especially after long reaction times. In case of the formation of p-mentha-l,4(8)-diene and p-menth-8-en-3-ol, the 1,4dihydropyridine derivatives showed some moderate antioxidant activity as well. The last two compounds are typical intermediate derivatives, which are formed relatively rapidly, and decompose during the oxidation. Sensory profiles of two samples of Citrus hystrix oils are shown in Figure 4. The essential features are the same, but nevertheless, certain differences existed as their chemical composition was different. In both oils, citrus odour notes prevailed in the profile. The sensory profile of three main terpenic constituents are shown in Figure 5. They had a similar character as the essential oil, and the calculated contributions of citronellal, linalool and citronellol to the profile of oil A have been calculated; the results are compared with the three components under Mixture C. There were differences between the model mixture and the natural oil, showing that minor components markedly affected the sensory profile. Nevertheless, a significant correlation existed between the model mixture C and die natural material. The correlation coefficient was very high in case of oil A (r = 0.9451), but was lower in case of oil B (r = 0.7154); it should be considered, however, that the model mixture shnulated the composition of oil A. The correlation between the sensory profiles of the two oils and the profile of citronellal were very high (r = 0.9617 and 0.7905, respectively). Odor acceptabilities were determined as well, using unstructured graphical scales (0 % = very agreeable; 100 % = bad); they were rated 29 and 47 % in case of oil A and B, respectively, which is considered a favorable rating, especially in oil A.

711 During autoxidation under simulated storage conditions, the content of citronellal decreased rapidly, and followed first order reaction kinetics (Figure 3A). For the GLC/MS analysis of oxidized oil, the device shown in Figure 1 was applied. No great difference was observed between the degradation of non-stabilized and stabilized samples. The formation of oxidation and other reaction products was affected by antioxidants. The formation of acetone was efficiently inhibited by die rosemary extract (Figure 3B), but both 1,4-dihydropyridines were ineffective. The same was observed in the case of linalool oxides formation (in this case, Diludine was found slightly better than OSI7284), especially after long reaction times. In case of the formation of p-mentha-l,4(8)-diene and p-menth-8-en-3-ol, the 1,4dihydropyridine derivatives showed some moderate antioxidant activity as well. The last two compounds are typical intermediate derivatives, which are formed relatively rapidly, and decompose during the oxidation. Sensory profiles of two samples of Citrus hystrix oils are shown in Figure 4. The essential features are the same, but nevertheless, certain differences existed as their chemical composition was different. In both oils, citrus odour notes prevailed in the profile. The sensory profile of three main terpenic constituents are shown in Figure 5. They had a similar character as the essential oil, and the calculated contributions of citronellal, linalool and citronellol to the profile of oil A have been calculated; the results are compared with the three components under Mixture C. There were differences between the model mixture and the natural oil, showing that minor components markedly affected the sensory profile. Nevertheless, a significant correlation existed between the model mixture C and the natural material. The correlation coefficient was very high in case of oil A (r = 0.9451), but was lower in case of oil B (r = 0.7154); it should be considered, however, that the model mixture simulated the composition of oil A. The correlation between the sensory profiles of the two oils and the profile of citronellal were very high (r = 0.9617 and 0.7905, respectively). Odor acceptabilities were determined as well, using unstructured graphical scales (0 % = very agreeable; 100 % = bad); they were rated 29 and 47 % in case of oil A and B, respectively, which is considered a favorable rating, especially in oil A.

712

o

1 8

I O o

I I •I 1 ^^

s " §2 CO

C

O H

'^ -

O

e

Q> Jo o

^

CO

8

.11

713 Table 1 The main identified components of slightly oxidized Citrus hystrix oil Figure 1), Peak No. RT(min) RI Identification Note acetone 825 6.63 1 7.61 2 methanol 891 ethanol 934 8.37 3 chloroform 9.79 4 air contamination 989 n-decane 10.01 5 internal standard 1000 10.83 6 1023 a-pinene 1028 11.00 7 a -phellandrene 1107 14.27 8 P-pmene 1123 14.94 9 P -phellandrene 1166 17.16 10 p -myrcene a -terpinene 1182 17.99 11 limonene 1198 19.05 12 c/5-ocimene 1240 21.25 13 1255 21.85 14 y-terpinene ^an5-ocimene 1257 22.29 15 p-mentha-1.4(8)-diene 1274 23.46 16 3-hydroxycyclopentanone 1342 27.69 17 4-methyl-(2-methyl1356 28.53 18 1 -propenyl)-tetrahydropyrane 2,2,6-trimethylcyclohexanone 1361 28.84 19 octahydro-3A-methyl-cw-2H1371 29.61 20 inden-2-one cw-linalool oxide 1446 34.51 21 /ra/w-linalool oxide 1475 36.39 22 citronellal 1485 37.15 23 ? 2-cubebene sesquiterpene, M==204 1528 39.79 24 linalool 1552 41.40 25 ? p-menth-8-en-3-ol 1568 42.42 26 7 p-menth-8-en-3-ol 1575 42.89 27 ? caryophyllene sesquiterpene, M=204 1590 43.86 28 terpinen-4-ol 1599 44.43 29 citronellyl formate 1617 46.33 30 citronellyl acetate 1636 48.61 31 geranyl acetate 1681 54.33 32 citronellol 1684 54.76 33 ? nerolidol sesquiterpene, M=222 1840 34 59.23

714

B 30 . -pure - Diiudine -Rosemary extractj -OSI7284

—X—pure -HB— Diiudine —A—Rosemary extract —9—0317284

25 S 20 S. 15 o

1 105 .

500

ir>r « u 4—a—a 0

1000 time(h)

« * A . ^ m—u u m '-* 500 1000 time(h)

• /A

1 1500

C 1.2 ^

1 S 0.8 (0 2£

g.0 8 9

0.2 -

^OJS—

^s^

—A

0 -1 ) C

500

A—nA

_A_ A

*

,^, 1000 time (h)

1500

A A A

500

1000 time (h)

-pure - Diiudine -Rosemary extract] -OSI7284

A-A

-pure - Diiudine - Rosemary extract] -0317284

—JK—pure —Q— Diiudine —^—Rosemary extract —9—0317284

•f 0.4 -

pure Diiudine Rosemary extractj 031 7284

A A A

500 time(h) 100°

1500

Figure 3. Changes of selected components during autooxidation of Citrus hystrix oil under conditions described above, A - citronellal, B - acetone, C, D - cis, /ra«5-linalooloxides, E p-mentha-l,4(8)-diene, F - p-menth-8-en-3-oL

715

O

1 "S

i 9>

o

.is

^

c O

CM

Ep 3 O

O

i

716

1

8 3^

0 D) Q. C

0

\

0

•MM*

0 Q. 0 C

1 1

o

i1

© -J^^

^ 1

CI -|-9fl^^ f k.

£ CO 0 >f /

(0 (0

t» ^ ^

J x^

—' >

(c 0 c

2

0 QL

i

L

(0

J? O LL

wr\

pi

c ^

/

o

1

CD

(D L13 ^^ X ^

1

5

o

1 o

3 o

"0

c I

«4-'

1/5 £

o «

il

C o

^ o

i: .2

I

U

2

Q.

b

J5

LJ

c

CO

o o

0 \

CO

>^ "^

•0

o o o

" ^

\

\ 0

Q.

c o *-*

b

1

o ^

« 3

O

5.S

^

»r> .SI' d)

717

Areas of possible applications of this oil are shown in Table 2. In Indonesia, Citrus hystrix oil is generally considered as suitable for the application in foods, but the best application field according to the answers given by our sensory panel was assigned to cosmetic products in case of oil A, but some food products could be successfully flavoured as well. The reverse rank was foimd in the case of oil B. The application would thus depend on the actual composition of the material.

Table 2 Possible applications oi Citrus hystrix oils (% of total responses). Application Perfumes Cosmetic creams Toothpastes Fruit beverages Liqueurs Candy

Oil A 48 52 47 39 36 37

OilB 29 33 31 41 35 40

5. CONCLUSION 1. Essential oil from Citrus hystrix is shnilar to other citrus oils, consisting mainly of terpenes and related compounds. 2. The odor character is influenced by main terpenic components, but minor compounds also affect its sensory profile. 3. The area of application depends on the actual chemical composition and thus the odour profile of the sample. 4. During oxidation, the odour character is affected marginally, and typical oxidation products of monoterpenes represent main oxidation products. 5. Antioxidants, such as rosemary extract or 1,4-dihydropyridines, not only inhibit oxidation of Citrus hystrix oil, but also influence the composition of oxidation products.

6. REFERENCES 1 E. Gildmeister and F. HofBnann, Die Atherischen Ole, Band V, Akademie Verlag, Berlin (1959). 2 A. Sato, K Asano and T. Sato, J. Essent. Oil Res. 2 (1990) 179. 3 B. M. Lawrence, Perfumer <& Flavorist 18 (1993) 43. 4 C, H. Wijaya, Oriental Natural Flavor: Liquid and Spray-Dried Flavor of "Jeruk Purut" {Citrus Hystrix DC) Leaves. In: Food Flavors: Generation, Analysis and Process Influence. Ed.: G. Charalambous, p. 235, Elsevier (1995).

718 5 B. M. Lawrence, J. W. Hogg, S. J. Terhune and V. Podimuang, Phytochemistry 10 (1971) 1404. 6 C. Moreuil and R. Huet, Fruits 28 (1973) 703. 7 F. Pudil, H. Wijaya and V. Janda, Chemical Composition of Citrus Hystrix Oil. In: XVIII th International Symposium on Capillary Chromatography. Eds.: P. Sandra and G. Devos, Vol. II, 20.5. - 24.5.1996, Riva del Garda, p. 1027 (1996). 8 ISO 6658: Sensory analysis - Methodology - General Guiddance, ISO, Geneva, 1985. 9 ISO 8589: Sensory analysis - General guidance for the design of test rooms. ISO, Geneva, 1988. 10 ISO 8586: Sensory analysis - General guidance for the selection, training and monitoring of assessors. ISO, Geneva, 1989. 11 ISO 6564: Sensory analysis - Flavour profile. ISO, Geneva, 1985. 12 ISO 4121: Sensory analysis - Grading of food products by methods using scale categories. ISO, Geneva, 1988.

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

719

Studies cm the develapment of a qmcd^ test for piedictiiis the sorption properties of refillable polycarbonate bottles P,G. Demertzis^ and R. Franz^ ^Laboratory of Food Chemistry, Department of Chemistry, University of loannina, GR-45110 loannina, Greece ^Fraunhofer - Institute for Food Technology and Packaging, Giggenhauserstr. 35, D-85354 Freising, Germany

Abstract Quick tests are recently proposed in the literature as alternatives to the large scale contamination studies performed to assess the quality and safety-in-use of recycled and reused plastic beverage bottles (Demertzis et al. Packaging Technology and Science, in press; Nielsen et al. Food Additives and Contaminants, in press). The work reported here has m e a s u r e d the interaction of small plastic specimens (strips) in place of bottles with selected mixtures of surrogate contaminants to model the great number of chemicals that could in principal contaminate returned bottles because of consumer misuse. Results showed that significant amounts of chemicals can be sorbed into the plastic material if mis-used to establish a re-migration potential in the bottle material after refilling. Results were also compared with tests using actual polycarbonate bottles.

1.

INTRODUCTION

Polycarbonates offer a unique combination of properties not encountered with any other thermoplastics. The main features are excellent mechanical properties which remain constant over an unusually broad t e m p e r a t u r e range, low absorption of liquids, resistance to weather, chemical resistance, total inertness to food components and transparency. These properties have allowed them, in spite of their high cost, to p e n e t r a t e several packaging applications such as plates for ovenable dinners and refillable bottles for water, soft drinks and milk [1-3]. Recently, in certain European countries, a system for washing and refilling polyethylene terephthalate (PET) and polycarbonate (PC) bottles used for soft drinks and other food packaging applications has been introduced. Although the result of this approach is a reduction in the production of new plastic bottles, serious contamination problems might arise if safeguards are not provided. If, for instance, these bottles are used for storage of any household chemicals, such as detergents, photographic liquids, pesticides etc., before being returned to the loop, they might become contaminated by substances

720 which, unless removed during the washing process, could damage the quality and/or the safety of the product filled subsequently. Many studies, some of them highly time-consuming, have been made for determining the transfer of pollutants into foods [4-121. The potential public health risks in connection with the use of PET refillable bottles as a consequence of possible mis-use have also been reviewed and summarised recently [13]. From the results of all mis-use studies carried out so far and from probability considerations, it is generally concluded that returnable PET bottles can be safely reused, provided that specific food manufacturing procedures and inspection systems are required to eliminate abused bottles. Although this approach can be recommended because it is thorough and tests the actual re-use system in practice, it does have the disadvantage that many hundreds of bottles must be contaminated, large quantities of toxic substances are used, and these are by necessity introduced into a commercial washing and bottling plant which must be set-aside for the trials and subsequently will need decontamination until the process is judged acceptable. In order to test refillable bottles for inertness it is, therefore, desirable to establish a practical, cost efficient and relatively quick standard test procedure that could also be carried out in the surveillance laboratories. To this end, a quick inertness test procedure for multi-use PET bottles has been recently proposed, based on the sorption behavior of surrogate contaminants into bottle wall strips [14-15]. The principle of the intended work was to measure the uptake of four sets of model contaminants onto PC bottle strips and the subsequent remigration of these chemicals in food simulants and to test if the sorption into PC strips exposed to contaminants by immersion is comparable to sorption into whole bottles exposed single-sided by filling. This correlation would allow for an estimation, from the measured sorption values for the strips, of t h e remigration of these chemicals from a bottle contaminated under the same conditions as the strips.

2.

EXPERIMENTAL

2.1. Materials Returnable PC bottles were supplied by Continental PET Europe, France. The four sets of model contaminants used are listed in table 1. All chemicals were of analytical grade (i.e. purity > 99%).

2.2. Methods 2.2.1. Preparation of contamination liquids from sets A-D. - Set A : Mixture of equal weight parts of each of the contaminants (without dilution). - Set B : After mixing equal weight parts of the contaminants a 8 % dilution of the mixture with PEG-400 was made. - Set C : After mixing equal weight parts of the contaminants a 6 % dilution of the mixture with PEG-400 was made.

721 - Set D : The mixture of equal weight parts of the contaminants was 12 % diluted with PEG-400. Table 1 Model contaminants (mol. weight) applied Set A alcohol-type compounds

SetB ester/ketone-type compounds

SetC hydrocarbon-type compounds

Ethylene glycol (62) Phenol (94) n-Hexanol (102) 2-Phenylethanol (122) Menthol (156) 1,2-Decanediol (174)

Ethyl acetate (88) Cyclohexanone (100) iso-Amyl acetate (130) Benzophenone (182) Linalyl acetate (196) Methyl stearate (299)

Toluene (92) n-Heptane (100) p-Xylene (106) Limonene (136) Phenyl cyclohexane (218) Phenyl decane (218)

SetD chlorinated (strongly interactive) compounds Chlorobenzene (112) 1,14-Trichloroethane (133)

2.2.2. Contamination of bottle wall strips and whole bottles. A number of strips having dimensions of approx. 1.1 x 6.0 cm were cut from the middle part of the PC bottle walls. Strips were placed each in 20 ml screw-cup glass vials containing enough volume (10 ml) of contamination liquid to contact the whole strip area. The vials were then stored horizontally at 40 oC for 14 days. The whole PC bottles were filled with glass beads of 4 mm diameter and the contamination liquid was added, so that it just exceeded the bead's level. The bottles were then closed with plastic caps and placed at 40 ^C for 14 days. 2.2.3. Work up and washing of bottles and strips. After the contamination phase, the bottles were emptied and rinsed 4-5 times with tap water and one time with ethanol. They were then washed by filling with 80 ^C hot 1.5 % NaOH solution and keeping this temperature for 10 min. After washing, the bottles were emptied and rinsed again several times until pH was neutral. Contaminated strips were removed from the vials and briefly immersed three times in ethanol (for approx. 5 seconds each time) to remove contaminants from the surface. They were then wiped clean using soft wipe paper to remove excess ethanol from the surface. Strips cleaned in this way were either going directly into the sorpt ion/re-migration determination or further washed with 1.5% NaOH (as the bottles) and then going into the remigration process.

722

2.2.4. Determination of sorption of contaminants into the strips. The contaminated strips (after washing and cleaning) were weighed. The edges of each strip were cut off (approx. 1 mm around the whole strip was removed) to eliminate any edge sorption in the determination and the strips were re-weighed. The strips were then cut into small pieces and transferred into 10 ml vials for extraction. 1 ml of CH2CI2 and the appropriate amount of the internal standard solution were added. The vials were stored for 24 hours at 40 oC. The swollen PC material was extracted with 4 ml isopropanol for further 24 hours at 60 ^C under periodic agitation. The samples were then stored for 3 hours at 20 ^C to allow for re-precipitation of the dissolved polymeric material. Aliquots of the extracts were cleaned by filtering through disposable regenerated cellulose filters having a pore size of 0.2 pm. The filtered extracts were GC-analysed with calibration against uncontaminated strip extractions spiked with known amounts of analytes using the internal standard method. Triplicate determinations were performed for each case. The concentrations of contaminants found in the extracts were expressed in mg/dm2 PC. 2.2.5. Gas chromatographic analysis. The analytical GC methodology applied has been described in details in a previous paper [14].

3 . RESULTS AND DISCUSSION 3.1. Test strategy The main disadvantage of any chemical mis-use testing of refillable plastic bottles is the impossibility to mimic the real- life situation (which cannot be defined). Consequently, common practice is to select "real life relevant" misuse chemicals which means representative or model contaminants. The question of which conditions the misuser really applies cannot be answered. This remains another field of probability considerations. Any chemical misuse testing has to use agreed upon test protocols (which by the way is common sense and practice for any migration testing of food contact articles). Consequently, there is a need for establishing a conventional method for reproducible inertness testing of refillable bottle materials. Hence, model contaminants and well defined contamination conditions should be used to determine in a reproducible and comparable way the interactivity of a given PC material. Another more realistic conclusion is the possibility of using small plastic specimens (bottle wall strips) in place of bottles and comparing the results using actual bottles. Such a test scheme would allow work to be done with smaller amounts of chemicals and solvents compared to use of the whole bottle and could give relatively quick answers to the question of the uptake of chemicals by the PC material and the potential for re-migration after refilling (provided that a relatively short contamination phase can be applied). The contamination conditions should be reproducibly constant (standard contamination conditions) to allow for real comparative testing. The use of mixtures of model contaminants having different chemical

723 structures (which can be tested in one analytical gas chromatographic run) instead of individually selected compounds may be criticised using the following argument : To justify working with mixtures of model contaminants, one must demonstrate how such mixture-derived values correlate with singlecompound contamination results. This, however, represents a critical argument with return-character because all known artificial and unknown real-life contamination have been carried out irreproducibly at different conditions and concentrations. For instance, at a high c o n t a m i n a n t concentration, incompatibility with the test material might be visually observable due to a swelling effect. On the other hand, the same contaminant can diffuse into the bottle wall without visually changing it when applied at lower and therefore less aggressive concentrations. The relevant question arising here is : What is the borderline concentration which does not lead to swelling of the polymer? This situation is critical and cannot easily be recognised by visual inspection. It should be borne in mind that at such a borderline concentration (before reaching a plasticising effect) the diffusion characteristics of the individual compounds are not dramatically influenced by each other which justifies application of mixtures of model contaminants at the right concentration. Furthermore, in real life mis-use with mixtures is much more likely than with single compounds. From this the use of model contaminants is justified, however, at such a concentration which leaves the polymer visually intact. The model contaminants used in the present study were selected for different considerations : variation of chemical structures/polarities, variation of molecular weights, comparison of aromatic versus non-aromatic structures, consideration of strongly interactive compounds, consideration of surrogates proposed by FDA and simple analysis of all model contaminants using a single method (GC/FID). Following these considerations three sets (A, B and C) of six compounds each and a mixture of two stongly interactive (with polymers) chlorinated hydrocarbons have been selected. Each set was measured by a single GC run using flame ionisation detection. Set A was used undiluted, e.g. contamination liquids were prepared by mixing of equal weight parts of each of the contaminants. Sets B, C and D were diluted by mixing the original contamination liquids (prepared as above) with suitable amounts of polyethylene glycol having an average molecular weight of 400 (PEG 400). This dilution was performed to reduce aggressiveness of the compounds of sets B, C and D because these compounds are sorbed to a relatively high degree into the PC matrix causing swelling of the material. Another point of interest was that the edges of the contaminated strips were removed (cut off) before extraction and quantification of the sorbed compounds. The removal of the edge material by cutting 1 mm from each edge was performed to ensure that the amount of sorbed contaminants determined was coming only from such types of surface areas which are accessible to contaminants on the body of the actual bottles. 3 . 2 . Sorption - re-migratioii measurements Due to the thickness of the polymer samples (550 pm), equilibrium in sorption was not reached during the period of exposure to contaminants. This means that the sorbed amounts should be proportional to the exposed area rather than

724

^

^N ^

\

\

'N ^

[3uip/6iu] pe)ej6jujaj JO paqjosqe sseiu

3

O

=6 £

C\J_

9

0)

2

f 1"o 0)

"c ^

"o c (0 0) •^

C

0)

s •^ ^

o a E o

i

0

o

<

CO

CJ

fi
v

ia ^ ^ ^

§

T3

a o CO

4-»

^

1 C/3

S

> a o T3

CO

o

&

0) C/3

4-3

< 4-3

(D

4^ C/3

a

4J •r-*

eu

i3 w U

[3UJP/B1U] paieiBjiuaj JO paqjosqe sseiu

725

g

o

fe

O

CO

O

P3

726

Y^^=^^=V=^=V=^ [3Ujp/6ui] paiejBiujaj jo paqjosqe sseiu

'u CD

I I 2i

CO

o

o CO

OH

CO

CO

E u

[3iup/6uj] pa}ej6|UJej JO paqjosqe sseiu

c a> c o a E o o O 0) (J)

727

'u CD

I I

g

fe £

g O

CO

0)

CO

a; CO

PH

o

728 to the mass of the polymer and therefore the sorption values are expressed here on an area basis (mg/dm^ PC). This enables a direct comparison of results for strips exposed by total immersion with results for bottles exposed single-sided. Concerning the analytical aspects of our measurements, the repeatability of the sorption - remigration measurements was excellent with a relative standard deviation of less than 5% between triplicate exposures. S i m p l e extraction of PC samples with dichloromethane over 24 hours was very effective and a second extraction yielded no more than an extra 0.5%. The results obtained for sorption and remigration experiments with strips for each of the four sets of model contaminants (A-D) are graphically summarised in Figs. 1-4. The corresponding results with whole bottles are presented in Figs. 5-8. It is observed that due to the relatively low diffusivity of PC material for organic compounds, only a small fraction of the total amount available for sorption actually enters the polymer matrix. However, compounds having high chemical affinity for the polymer (for example, phenol of Set A, ethyl acetate of Set B, toluene of Set C and chlorobenzene of Set D) are sorbed in significantly larger amounts than other contaminants which are more or less incompatible with PC (for example, propylene glycol and menthol of Set A and n-heptane of Set C). Another observation made in these studies was that compounds with aromatic structures (for example : phenol, toluene, chlorobenzene) w e r e generally sorbed in larger amounts than the other compounds. This could be attributed to the planar aromatic ring structure which allows for b e t t e r penetration into the polymer matrix. The comparison of single-sided (actual bottles) and double-sided (strips) contamination was to establish if PC strips immersed in the surrogate contaminants exhibited the same sorption behaviour as PC whole bottles filled with the contaminants. It is observed that samples subjected to single-sided (bottles) and double-sided (strips) exposure exhibited similar sorption levels on an area basis for the majority of substances. Our results are in agreement with those obtained by Nielsen et al [151 for refillable PET bottle material. It can be concluded that, with the recommended precautions regarding the removal of cut edges before sorption (or re-migration) tests, the behaviour of PC bottles towards contaminants can be predicted by experiments using strips and this should facilitate the development and testing of materials and processing.

4. CONCLUSIONS AND PROSPECTS This study has shown that chemicals can be sorbed into refillable PC bottles mis-used to various extents, depending on the chemical nature and molecular weight of the chemical compound. Furthermore, the results have shown that due to sorption into the bottle wall, a re-migration potential can be generated for subsequent transfer of pollutants into the foodstuff after refilling if not removed by washing and passing the electronic/ optical inspection system of a refill line. Generally, immersion of strips in contaminant mixtures satisfactorily simulates contamination of intact whole bottles. These results, along with the findings from other research teams working within our collaborative project, have provided the foundation for a subsequent EU-funded project to develop

k i u p / 6 i u ] U0|jBj6iLU-Gy

729

730

[3Uip/6ui] uo!}ej6jui-3y

U CD U

CO

•B

o

LO

.^ ft

CO

CO

'B T3 CD

CO

O

o

g CD

CO

g CO

I u a;

CD

[ZUip/Bui] uojiBjBjui-aa

0)

c >.5? i^

731

03

u u CO

m

a i3 CQ T^

w

U

o

g

o 4:1

S

O

4^ 4-3

a^ 0)

Xi 4^

o c o -J

C

cu c o

E o o

o CO

o

i U

fe

732

[^uip/Bui] uoj)ejBjLU-abi

c 0)

c o Q. E o o

o

(J)

^ CD U cd CO

05

O

s O

g

.fci

CD U

CD CO

g CO

cd

'u

I o U

fo

733

reference materials and test methods for the inertness of refillable PET bottle material.

5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

G.L. Robertson, Food Packaging: Principles and Practise, Marcel Dekker, Inc., New York, 1993. W.A. Jenkins and J.P. Harrington, Packaging Foods with Plastics, Technomic Publishing Co., Inc., Lancaster, PA, 1991. J. Stepek, V. Duchacek, D. Curda, J. Horacek and M. Sipek, Polymers as Materials for Packaging, Ellis Horwood Ltd, Chichester, 1987. F.W. Bodyfelt, M.E. Morgan, R.A. Scanlon and D.D. Bills, J. Milk Food Technol., 39 (1976) 481. J.D. Lundsberg, F.W. Bodyfelt and M.E. Morgan, J. Food Protect., 40 (1977) 772. J.M. Casaway, J. Food Protect., 41 (1978) 851. J.M. Casaway, J. Food Protect., 41 (1978) 965. M. D. Cassiday, R.J. Streu, R.L. Wence and P.T. Delassus, J. Plast. Film Sheeting, 6 (1990) 268. R.S. Khinavara and T.M. Aminabhavi, J. Appl. Polym. Sci., 45 (1992) 1107. T.H. Begley and H.C. Hollifield, Food Technol., 47 (1993) 109. T.J. Nielsen, J. Food Sci., 59 (1994) 227. V.J. Feron, J. Jetten, N.De Kruijf and F. van den Berg, Food Addit. Contam., 11 (1994) 571. R. Franz, M. Huber and O.-G. Piringer, Food Addit. Contam., 11 (1994) 479. P.G. Demertzis, F. Johansson, C. Lievens and R. Franz, Packaging Technol. Sci., 1997, in press. T. Nielsen, A.P. Damant and L. Castle, Food Addit. Contam., 1997, in press.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

735

Recycling old polymers in bi-layer bottles. Effect of the volume of the solid food on the contaminant transfer. I.D. Rosca^ and J.M. Vergnaud^ ^Dept. of Chemical Engineering., University Politeknica, Bucarest, Romania. ^Lab. Materials and Chemical Engineering., Faculty of Sciences, University of St-Etienne, 23, Dr. P. Michelon, 42023 St-Etienne, France. Abstract Recycling old polymers as new bi-layers bottles may be of value, if a virgin polymer layer is placed between the recycled polymer and the food. In the case of solid food, the process of contamination by the recycled polymer is controlled by radial diffusion through the bottle and the food. The effect of the volume of the bottle is considered, as it plays an important role. In fact, an increase in the volume of the bottle is associated with a decrease in the area of the bottlefood interface per unit volume of food. The profiles of concentration of contaminant through the bottle and food are determined, as weU as the kinetics of transfer, and the concentration-time histories in various places of the food. 1. INTRODUCTION Recycling waste polymers is becoming of greater importance for various reasons which may be ecological, economical or even political. Different procedures are utilised, using either combustion for energy (1), or pyrolysis for recovering new monomers. Another method based on reusing old packages made of polymers as new food packages, has also been considered. However a problem arises with this last method, as potential contaminants may be introduced into the reused polymer during its first contact with food (2-4). It is well known that when a polymer is in contact with a liquid, the liquid enters this polymer, sometimes permitting a release of some additives of the polymer. This double transfer is controlled by diffusion (5, 6). In order to prevent food from contamination by the recycled polymer, bi-layer packages consisting of the reused polymer and a virgin polymer layer are made, the virgin polymer being in contact with the food (7-10). The virgin polymer layer plays the role of a functional barrier, and the question which arises is how to determine the period of time over which the food is protected. The process of contamination is controlled by diffusion of the contaminant through the bi-layer package (11) and either convection into the liquid food (12) or diffusion through the solid food (13, 14). This problem can be resolved either by tedious and highly time consuming experiments (4, 16) or by calculation (11-14). The mathematical treatment is feasible when the diffusivity is constant, especially in the following two cases : when the coefficient of convection is finite and the volume of liquid infinite, or when the coefficient of convection is infinite and the volume of liquid is finite (6, 17). An attempt to mix the analytical solutions obtained in this two cases is inappropriate (18). In the more general case, when the volume of food is finite as is the coefficient of convective transfer, numerical models must be built (6, 11-14), These models take into account all the known facts, e.g., the double transfer into and out of the polymer (5,6) the diffusion through the polymer and convection into the liquid food (12), and the diffusion through the polymer and the solid food (13,14). The first purpose in this paper is to show that numerical models can be built in the case of the radial diffusion of the contaminant through the bottle and the solid food (13,14), when the bilayer package is made of both a recycled and virgin layer.

736 The other objective is to determine precisely the effect of two parameters of great interest: i). The relative thickness of the recycled and virgin layers of the package by keeping the thickness of the bottle constant. This ratio of these thicknesses is varied within a wide range, from the recycled polymer alone to a rather large thicknesses of the virgin polymer : ii). The volume of the bottle, and thus the ratio of the volume of food per unit area in contact with the bottle, which ratio increases with the volume of food. 2. THEORETICAL 2.1. Assumptions The following assumptions are made in order to define the process precisely : i) - The diffusion of the contaminant is radial through the bottle and the food, ii) - The bottle is made of two layers, the recycled and the virgin layer, the virgin layer being in contact with the food. iii) - Initially, the concentration of contaminant is uniform through the recycled polymer, iv) - Perfect contact exists between the bottle and the food. The partition factor is taken as L and thus the contaminant concentration is the same on both faces of the bottle-food interface, v) - The contaminant does not evaporate through the external surface of the bottle. 2.2. Mathematical treatment The equation of radial diffusion either in the package and the food is : 3Crt i-Siii + l ^^^t (1) dt dv^ r* ar with the constant diffusivity : D^ in the bottle and D^ in the food, and Q ^ is the concentration of contaminant at the radial abscissa r and time t. The boundary conditions are : for the extemal surface of the bottle :

f =0

r = R,

,2)

at the middle of the food : ^ =0 r=0 dr at the bottle-food interface :

(3)

D p - — l = Df.| — I

W

2.3. Numerical analysis The problem of diffusion of the contaminant through the bottle and the food with the initial and boundary conditions is resolved by using the Crank-Nicolson method.

737 3. RESULTS For this study, the food is assumed to be in solid state, or in gel state and thixotropic, so that the transfer of the contaminant into the food is controlled by radial diffusion through either the package or the food. Three kinds of results are of interest, e.g.: 1) the profiles of concentration of the contaminant developed either through the package and Sie food ; 2) the kinetics of contaminant transfer into the food ; 3) the concentration-time histories in various places of the food. Dimensionless numbers are used, leading to master curves which can be of value to those wishing to resolve a particular case : thus Dpt/Lp is used instead of time ; the relative abscissa for position ; the concentration C at time t and position r/R as a fraction of the initial concentration in the recycled polymer ; the amount of contaminant transferred at time t as a fraction of the corresponding amount after infinite time. 3.1. Profiles of concentration of contaminant developed through the package and food The profiles of concentration of the contaminant which migrates through the package and into the food are drawn in Fig. 1 and 2 for the package when the volume of food is 2 and 1 L, and in Fig. 3-6 for the food with various volumes ranging from 2 L to 0.125 L. 1 I

1

1

r;—y^

::3=-

n

1

o o

1

1.002

1.004

1

1.006

Fig. 1. Profiles of concentradon through the bi-layer package with 2 L food.

1.002

1.004

1.006

1.008

Fig. 2. Profiles of concentration through the bi-layer package with 1 L food. 0.016

0.016 H

0.012 h

H

0.008 h

0.004

Fig. 3. Profiles of concentration through the food of diameter 9.5 cm (2 L).

Fig. 4. Profiles of concentration through the food of diameter 7.5 cm (1 L).

738 Studies of these curves can lead to some conclusions : 1 - The profiles of concentration are expressed at various values of times (or rather Dpt/Lp), using the dimensionless numbers. The profiles are about the same in the package whatever the volume of the food. 2 - The profiles of concentration developed through the food using the same dimensionless numbers differ notably with the volume of the food. The larger the volume of the food, the longer the time of diffusion of the contaminant. 3 - The process of contamination is controlled by diffusion either in the package or through the food. It takes time for the contaminant to reach the food, with Dpt/Lp around 0.03. Thus the virgin layer acts upon the contamination as a functional barrier. _-

0.016

,

J

,

r

^

1

1

0.012

o

i

^ 4.0-

^

/

1.0

y .

1

_.

0.2

H

1 ,.^,

h

0.4

0.6

0.012

-i

0.008 h

n

0.004 h

H /H 0.1 J

0.4

0.004

n

0.016 h

y^ / J

2____^2.0-"

0.008

'

1 0.2

0.8

Fig. 5. Profiles of concentration through the food of diameter 6 cm (0.5 L).

0.4

0.6

0.8

Fig. 6. Profiles of concentration through the food of diameter 4.7 cm (0.25 L).

3.2. Kinetics of contaminant transfer Kinetics of contaminant transfer are drawn in Fig. 7 for all volumes of food, by plotting the ratio M/M^ vs. time or rather Dpt/Lp. Some facts of interest are obtained from these curves : 0.12

L

0.1

1

T "-^

1

J

"T

1

T

1

1

0.016

/A A

r -

0.08

M.

T

0.06

A

0.04

A

1

-

0.012

/ 0.008 /

0

0.02

1

0.04

1

1

0.06

1

i

0.08

Dp t/L?,

Fig. 7. Kinetics of transfer into the solid food (M/MJ for various volumes of food.

0

0.1

1

1

^^„-—

A J

/ /

/

-j

°-^

/ /

/ ^A-"^1

1

0.25

/ /

0.02 1

-"T

^ 0.125 /

0.004 n '

•

1

^''•0 ^y"^""^

^ x ^

^^^2.0"^

\

3

4

1

0

1

2 Dpt/L?,

Fig. 8. Concentration-time histories at the centre, for various volumes of food.

739

0.016

Fig. 9. Concentration-time histories at r=R/2, for various volumes of food.

Fig. 10. Concentration-time histories next to the package,for various volumes of food.

1 - The kinetics of transfer, expressed by plotting M/M„ vs. Dpt/Lp are the same for all the volumes of food. Thus they do not depend on the radius of the food, at least over a large period of the process. 2 - It clearly appears that no transfer of contaminant takes place for Dpt/Lp lower than 0.01. 3 - After this time, the rate of transfer increases slowly with time, passes through a maximum, and thus decreases up to equilibrium. This fact is of interest, because with a package made of a recycled polymer alone, the kinetics starts at time 0 with a vertical tangent meaning that the rate is very high. 4 - As a conclusion, not only is the functional barrier responsible for a period of protection of the food but also the rate of transfer of contaminant is very low at the beginning of the process. 5 - Of course, after infinite time, the contaminant is distributed in the package and food depending on the partition factor K. Min = M^ -h C^.2 7IR.AR.K (5) When the partition factor K is 1, eqn. 5 gives : M. 2R.AR M, (6) (R-hAR)^ M;, 3.3. Concentration-time histories of contaminant in various places through the food When the food is not in Hquid state, the transfer of contaminant is controlled by diffusion through the solid food. The concentration in the food thus varies not only with time but also with position. The concentration-time histories of contaminant are drawn in various places through the food : at the middle of the food for r=0 (Fig. 8), at position with r=R/2 (Fig. 9), at the food in contact with the package r=R (Fig. 10). Calculation is made by using the values in Table 1. Some results are worth noting from these profiles : 1 - The concentration-time histories vary with the position in the food. 2 - The concentration-time histories largely vary with the volume of the food, or with the radius of the bottle, at various positions in the food. 3 - A typical pattern is obtained in the food next to the package, as shown in Fig. 10. After a time of protection which depends only on the thickness of the functional barrier, the

740

contaminant concentration increases rather quickly up to a maximum, and thus decreases slowly with time to the equilibrium value. 4 - The equiUbrium value of the contaminant in the food is given by : Min = Cin.27cR.AR = C^ (27cR.AR.K+7cR^) (7) When the partition factor K of the contaminant between the package and food is 1, meaning that the concentration of contaminant is the same on both sides of the package-food interface, this equation reduces to eqn. 6 . The concentration at equilibrium decreases inversely with the radius of the food, while the thickness of the package is kept constant. 5 - The contamination of the product starts at different times depending on the position of the food, beginning next to the package. 4. CONCLUSIONS Old polymer containing a potential contaminant can be reused in new food packages if bylayer packages are made of a virgin polymer layer playing the role of a functional barrier. The case of a bottle is of interest, especially when the food is in solid or gel state and the process of contaminant transfer is controlled by radial diffusion either through the bi-layer package or the food. The functional barrier acts upon the transfer in two ways : firstly, a period of time over which the food is protected exists ; secondly, when the contaminant reaches the food, the kinetics of contamination is typical with a low rate which increases with time. The volume of food is a parameter of interest, while the thickness of the package is kept constant. The period of time over which the food is protected depends only on the characteristics of the package, namely, the diffusivity and thickness. The concentration of contaminant in the food varies not only with time but also with space ; this concentration-time history largely varies with the volume of the food. Table 1 Characteristics Thickness of the package : 0.03 cm virgin layer = 0.015 cm recycled layer = 0.015 cm Df= D = 10-^cmVs ^ - lO-'cmVs i^-5™2/. 6 4.7 3.75 2 R (cm) =9.5 7.5 0.5 0.25 0.125 V(l) = 2 1 Height of the bottle = 2Rx3 5. REFERENCES 1 Polymer Recycling, 1 (1996) 222. 2 T.H. Begley and H.C. Hollifield,. Food Technology, 47 (1993) 109. 3 A.E. Feigenbaum, V.J. Ducruet, S. Delpal, N. Wol, J.P. Gabel, and J.C Wittman, J. Agric. Food Chem, 39 (1991) 1927. 4 K. Figge, Prog. Polym. Sci., 6 (1980) 187. 5 H.L. Frisch, J. Polym. Sci., 16 (1978) 1651. 6 J.M. Vergnaud,. Liquid Transport Processes in Polymeric Materials. Prentice Hall, Englewood Cliffs, USA, (1992) pp 45-61. 7 Commission of the European Communities, 1994. Draft synoptic Document 7 on plastic materials and articles to come in contact with foodstuffs, CS/PM 2536 Bruxelles. 8 Council of Europe, 1994. Committee of Experts on Materials intended to come in contact with foodstuffs, 26^^ session, Strasbourg.

741 9 Food and Drug Administration, May 1992. Points to consider for the use of recycled plastics : food packaging, chemistry considerations. Division of Food Chemistry and Technology, HP 410, Washington. 10 Food and Drug Administration, 1993. Food additives : Threshold of regulation for substances used in food articles, 21 CFR. Federal Register, 58, 52719-52729 11 S. Laoubi, A. Feigenbaum and J.M. Vergnaud, Pack. Technol. Sci., 8 (1995) 249. 12 S. Laoubi and J.M. Vergnaud, Plastics, Rubber and Composites. Process and Applic, 25 (1996) 83. 13 I.D. Rosea and J.M. Vergnaud, Plastic, Rubbers and Composites. Process AppL, in press. 14 I.D. Rosea and J.M. Vergnaud, J. Appl. Polym. Sci., in press. 15 G. Haesen and A. Schwarze. Migration Phenomena in Food Packaging, Commission of the European Communities, 1978. 16 E.M. Kampouris, Europ. Polym. J. 11 (1975) 705. 17 J. Crank,. The Mathematics of Diffusion. Clarendon Press, Oxford (1975). 18 J.N. Etters, Ind. Eng. Chem. Res. 30 (1991) 589. 6. NOMENCLATURE C^ J: concentration of contaminant at radius r and time t Dp, Df: diffusivity of the contaminant in the bottle, the food, respectively K: partition factor M-„: amount of contaminant initially in the recycled polymer M^,M^ : amount of contaminant transferred in the food after time t, after infinite time, respectively Lp: thickness of the bottle r: radial abscissa R: thickness of the recycled polymer layer

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

743

Polypropylene as active packaging material for aroma sorption from model orange juice A. Feigcnbaum % R. Lebosse'', V. Ducruet^' - INRA SQuAlE, CPCB - Moulin de la Housse, 51687 REIMS - France i> INRA SQuAlE, Domaine de Vilvert, 78352 JOUY EN JOSAS - France

Abstract In fruit juices, aroma compounds usually undergo successive chemical reactions which may lead to undesirable compounds, responsible for off-odors and off-taste. We have investigated the possibility that such reactions are quenched by sorption of offflavor precursors in plastic packaging materials. Interactions of a model orange juice with three polypropylene based packaging films were tested: (i)- a standard polypropylene, (ii)- the same material coated with silica deposited by plasma and (iii)an uncoated microcrystalline polypropylene. The reactivity of pinenes strongly depends on the film. This is exi)laiiied by different sorption behaviors.

1. INTRODUCTION Plastic materials display many advantages for food packaging, justifying their very wide use. However, as far as food aromas are concerned, plastics are usually considered to have a negative impact on food quality. Sorption of aroma compounds by the lipophilic polymers may induce both weakening of flavor and taste intensities and changes in the organic profile of foods. This has been mainly reported for aqueous foodstufi's Hke fruit juices, where the partition coefficient of aromas between food and packaging strongly favors the plastic material [1,2]. Rcxently an idea emerged that these sorption effects might not be as detrimental for food quality as expected. For instance limonene, well known to be sorbed to a high extent, has only a reduced influence on aroma and taste [3,41. Since aromas are reactive compounds, their sorption could even have positive effects, by preventing the formation of undesirable reaction products, possibly responsible of off-flavors [5,6]. To evaluate such effects, one must know the partition coefficients between food and packaging, as well as the reactivity of aroma compounds. Unfortunately there are many discrepancies in reported partition coefficient values [5]. The reasons for this are numerous: when reactivity is not taken into account, the depletion of aroma compounds from the food may be wrongly attributed to sorption; when model solutions are used, excessive aroma concentrations may induce heterogeneous liquid media resulting in non realistic interactions; when kinetics are not carried out, it is not certain whether equilibrium conditions are reached. The objective of the present study is, therefore, to reinvestigate the behavior of the aroma compounds of orange juice in the presence of pol5^ropylene (PP), one of the most common packaging polymers, taking into account all these possible sources of error. We also wish to know how far changes in the structure of the polymer can influence aroma sorption and stability.

744 2. MATERIALS AND METHODS Materials. The polymer packaging materials used were a semi-crystalline ethylenepolypropylene copolymers (PP) (thickness: 40 pm, d = 1.018, Elf Atochem, Serquigny, France). Analytical grade aroma compounds were studied: a-pinene, p-pinene (Muka, St. Quentin Fallavier, France), myrcene (Interchim, Paris, France), d-Hmonene, octanal, decanal, ethyl 2-methylbutyrate (E2MB), nonan-2-one, (x-terpineol and citral (Aldrich, Strasbourg, France). Citric acid and sodium citrate were obtained from Prolabo (France). Solutions were made up with ultrapure water, obtained from a MiJH-Q system (Millipore, Bedford, MA, USA). Hexane (Prolabo, Fontenay s/Bois, France) and IIPLC grade pentane (l^'isons, Arcueil, France) were distilled to get the proper gas chromatography purity. ParafBn oil (Almo, St Genis Laval, France) was used as a solvent for standards in the headspace technique. Model solution. The aroma compounds (5 to 12 [iLflj) were dissolved at 4°C in a citrate buffer (0.976 gfl^ citric acid and 0.103 g/L sodium citrate in ultrapure water), at pH = 3.0. Sodium azide (Aldrich, 0.02% w/w) was added to prevent microbacterial changes during storage. The solutions were homogenized by sonication (TK52, Labo-Moderne, Paris, France) for 20 min. Contact conditions. Strips of PP (13 x 13 cm) were immersed in 266 mL glass flasks filled with the model solution and tightly stoppered with teflon caps. Experimental samples and model solution without film (control sample) were stored at 18 ± 1 °C in a dark room. After different contact times (1, 3, 6, 14 days), experimental samples (two samples for each time) and control samples were analyzed, using two different extraction methods: solvent extraction and headspace analysis. Solvent extraction. At each contact time, film and solution from experimental samples were separated. The solution (lOOmL) was extracted three times with pentane (20 mL) in a 150 mL flask in a room at 4°C. 1 mL of a solution of myrcene and nonan-2-one in pentane (800 pL/L) was added (internal standards). Extracts were dried with sodium sulphate and filtered through glass wool. The film was carefully rinsed and wiped, separated into two parts, then immediately plunged into a 100 ml^ flask containing pentane (80 mL). 1 mL of a solution of mjrrcene and nonan-2-one in pentane (800 jiL/L) was added. Extraction was carried out for 2 hours, alternating magnetic stirring and ultrasonic treatment (US) periods (15 min each). Multiple Headspace Extraction (MHE) using a P u r g e and Tr'^n Tn ice tor (PTI). An automatic PTI (Chrompack, Les Ulis, France) coupled ic a gao chromatograph (GC) was used for dynamic MHE of the volatile compounds from both aqueous solutions and from films [7,8]. A flow of purge gas (hefium, also the carrier gas) passed through the purge cell containing the sample (film or solution) and swept the volatiles into a cryogenic trap (0.53 mm x 15 cm; CP-Sil 5 DB) held at -120°C. After the purge time, trapped volatiles were thermally flash desorbed and directly injected onto the GC column. With aqueous samples, a water condenser at -10°C was page 2

745 used between the purge cell and the cryogenic trap to prevent blocking of the latter by ice crystals. The whole procedure was automatically controlled. The sample solution (} niL) and ultrapure water (ca 3 mL previously saturated with helium) [8] were introduced into the purge cell. A solution of myrcene in water (1 mL, 5 jiL/L) was added as internal standard. The first purge was started after thirty minutes equilibration at 40°C [10]. Repeated headspace extraction enabled a complete desorption of all volatiles after 3 purge cycles of 10 minutes. The film (5 cm2) was placed into the purge cell together with 10 fiL of a solution of myrcene and nonan-2-one (800 pL/l^) in parafiin oil (internal standards). Four purge cycles of 5 min at 60°C were used. Gas Chromatography. GC analytical conditions both for solvent extracts and for DHE have been reported elsewhere [5].

3. RESULTS The aroma compounds used in this study are typical of orange juice (table 1). Their structures range from apolar to polar, and from Hnear to branched and to cyclic compounds. In order to be certain of the homogeneity of the model solution, their individual solubihty in water was determined (table 1) [9]. Using the same method, we also checked that these compounds were soluble when used together at 10 mg/L.

Table 1 Aroma compounds studied. N° 1 2

3 4 5 6 7 8 9 10

Aroma Compounds

Kovats index^

Boihng Solubihty^' Concentration MW Point (°C) (mg.LQ (mg.LQ (g.mol ^)

(X -Hnene Ethyl 2-methyl butyrate (E2mb) P -Pinene

1023

156

1066

132

1122

165

Limonene Octanal Decanal Linalol Neral'^ (X -Terpineol

1204 1312 1515 1563 1692 1718

178 171 208 199 229 220

(jeranial^

1746

229

4.8

4.3

136

10.4

130

12.0

4.2

136

9.5 242 15.6

-

4.2 6.1 9.8 9.7 6.2 11.3

136 128 156 154 152 154

289

11.1

152

1990

241

«the respective proportion of geranial and neral in citral (measured by GC) were 35.5% and 64.5% ; ^see reference [9] ; ^measured with DB-Wax column ; FID relative responses to myrcene or to nonan-2-one standards are available from [5].

746 Three PP materials which had similar percentages of cristaJlinity (42±2%), were used in this study: * PP-Ref, a standard PP (40 fim), whose formulation is typical of those found in the form of a layer in contact with foods in multilayer structures. * PP-Nucl, a microcrystaUine PP (35 .um). It contained a nucleating agent, known to favor the formation of microcrystals [11]. * PP-Sil (40 M^m), a PP coated with a thin (50 nm) silica layer, deposited by a plasma created by an electric discharge in a [(Me.'jSi)20-02] atmosphere. This glass-like silica layer is likely to improve the inertness of the PP [12]. The solutions were analyzed after different times, using two comphmentary methods, relying on different principles: - extraction by pentane, followed by Kudema-Danish concentration and by GCanalysis. - dynamic headspace extraction (DHE), with trapping on a cryogenic trap [7,8]. During extraction from aqueous solutions, a condenser trapped the swept water, in order to prevent breakage of the cryogenic trap. This condenser was also likely to stop the less volatile compounds.

3.1. Reactivity of aroma compounds in the absence of PP films Aroma compounds have been reported to undergo reactions due to acid and to oxygen [6,13,14]. Since the aqueous solutions were sonicated, most oxygen was removed during work-up. Ijhe concentrations of aroma compounds in the model solution were monitored over two weeks, as shown in figure 1.

ln(aCo)

Time (days) Figure 1: concentrations of reactive aroma compounds 1 (•), 3 (O), 8 (A), and 10 (n).in the model solution (10 mg.L-^) at pH 3 over 14 days. (X-Pinene and p-pinene are highly reactive, and disappeared within a few days. Neral and geranial were degraded to a large extent after two weeks in the model solution at 20°C. The other compounds (hmonene, ethyl 2-methylbutyrate, linalol, octanal and decanal) were stable. GC-MS analysis after 14 days showed the presence of reaction products already described in the literature, as shown in figure 2 [5,13,14].

747 All the compounds shown in figure 2 were identified by GC-MS on the basis of both their retention indexes and their mass spectra, except the diols 20 and 21. The major compound issued from pinenes was terpineol 9 besides 11, 12 and 13. Based on FU) peak area, we could account for 95 % of the pinenes alter 14 days. The mass balance was not as good for citrals: compounds 14-19 were identified, and accounted only for 35 % of the initial aldehydes |5]. According to the hterature, the major compounds are the diols 20 and 21, which were not eluted in our GC conditions. These assignments were confirmed by studying the reactivity of 1, 3, 8 and 10 individually in the buffer solution.

Figure 2: reaction products of pinenes 1 and 3 and of citrals 8 and 10 [5,13,14]; 11 is borneol (Kovats index 1(>95); 12 is (x-terpinolene (1287); 13 is a-fenchol (1574); 14 is trans p-mentha-2,8~dien-l-ol; 15 is isopiperitenol; 16 is p-mentha-],5-dien-8-ol; 17 is p-mentha-l(7),2-dien-8-o]; 18 is 2,3-dehydrocineol; 19 is p-cymenol (2045); 20 is p-menth-2-en-l,8-dio]; 21 is p-menth-l-en-3,8-diol.

3.2. Behavior of aroma compounds in the presence of PP-Ref. To put PP-Ref in contact with the model solution, it was more convenient to immerse the film ia the solution than to make up a bag [15] or a package. After given times (0, 1, 3, 6, 10 and 14 days) film samples and their solutions were separated. Each was analyzed using the two methods described above, namely solvent extraction

748 and DHE. For analysis of the film by D]ffi, the water condenser was not used, so t h a t higher boiling compounds could also be transferred to the crj^ogenic trap. Analysis by solvent extraction and by DUE gave consistent results. DHE was much faster and more convenient, despite the fact that the higher molecular weight compounds were not swept. For each compound, at each time, analysis of the film (white bars) and of the solution (slashed bars) are shown in figure 3, in order to display the mass balance. The full Line corresponds to the analysis of aroma compounds in the absence of film (control sample) and the values are the same as in figure 1. Aroma compounds display three tjrpes of behavior: (a) - the overall recovery (film + solution) is higher in the presence of film for a-pinene 1 and p-pinene 3, (b) - the overall recovery of 2, 7, 8 and 10 is not afiected by the presence of PP-Ref, (c)- the overall recovery of octanal and decanal seems to be slightly lower in the presence of a film. Due to their long alky] chain, they were probably not extracted easily from the film under our conditions. Group (a) compounds are both apolar and reactive. Group (b) compounds are rather polar and may be reactive or not. Group (c) compounds are not reactive. GC-MS analysis after 14 days revealed the presence of the same products as in the absence of film including both the aroma compounds and their reaction products. Only their intensities at a given time were difi'erent. Sorption by PP-Ref thus had as consequence a slowing down of the degradation of pinenes 1 and 3. A slow equilibrium took place; the sorbed compounds reverted back to the solution, where they were rapidly hydra ted.

3.3. Behavior of aroma compounds in the presence of PP-Nucl and P P ^ i l . Since terpineol 9 is the major reaction product of pinenes, the compound was dropped from the model solution. PP-Nucl andPP-Sn were immersed in this model solution at 20°C, and the aroma compounds were monitored exactly as in the case of PP-Ref. Results are shown in figures 3 (middle) and 3 (lower) respectively. Replacement of PP-Ref by PP-Nucl resulted in a much better stabilization of (X pinene and of P-pinene (figure 4). Despite its lower thickness, this film seems to be much more absorbent than PP-Ref. This was not due to a lower percentage of crystallinity as both films had the same crystalhnity. In the presence of PP-Nucl, all reactions were considerably slowed down (figures 2 and 4). When PP-Sd was used, the decrease of a -pinene and P -pinene was faster than with PP-Ref. However no terpineol, theix major reaction product in solution, was detected. In contrast, Hmonene increased, indicating that a different pathway had been followed. It is possible to interpret this result by assuming that pinenes were adsorbed on the sihca surface, and reacted there, in a poorly hydrated medium. This could be explained by assuming that the intermediate cation 23 would undergo isomerization into Hmonene rather than hydration into 9 (figure 4).

749 Recovery (%)

1

2

3

Figure 3: changes in the concentration of aroma compounds 1 (cx-Pinene), 2 (E2mb), 3 (p-Pinene), 4 (Limonene), 5 (Octanal), 6 (Decanal), 7 a-inaJo]), 8 (Neral) and 10 (Geranial) in the solution (slashed bars) and in film (white bars) over 14 days. The full hne in each figure corresponds to the situation without film (cf figure 1).

750

Stable with PP-Nucl

PP-RelVN

PP-Sil

Figure 4: tailoring the reactivity of terpenic hydrocarbons by playing with the structure or surface state of PP-based packaging material. (PP-Ref is a usual food contact polypropylene; PP-Nucl is a microcrystalline PP; PP-SiJ is the l^P-Ref coated with with silica).

4. DISCUSSION AND CONCLUSION The results shown here suggest that polypropylene could play an active role in the evolution of aroma compounds in solution, a-terpineol 9, the normal reaction product of (x- and of p-pinene has been reported to be responsible for off-odors, contributing to musty aroma in orange juice [161. Also fenchol 13 has been reported to be responsible for a medicinal taste [13]. ITieir formation is observed in the test sample without film. The rate of their formation is considerably reduced in the presence of PP-Ref, and even more in the presence of PP-Nucl. a -pLnene was even -^^ al^ o m the r-rc:;ence of inis film. Of course since they are located in the film, pinenes are not available to the consumer. But without the film, they are not available either, since they are degraded. In real juices sorption may occur on pulp particles. The silica coated PP-Sil film does not play the role of an inert, barrier, since it is able to modify the reactivity of terpenic hydrocarbons. This can also be attributed to a specific sorption behavior on a porous sHica layer. Further studies are necessary to interpret the interaction with such films. With this objective, permeation experiments through all these films are currently in progress. This casts a new light on possible use of aroma and plastic packaging interactions. If the fruit juice contains large amounts of reactive hydrocarbons, packaging in polyolefins can have positive effects on quality. Obviously this statement does not take into account possible transfer of chemicals fi:om the material to the juice by migration [17]. However our study opens the way to possible improvements of quality by playing with the structure of the poljrmer.

751 5. REFERENCES 1 O.Y. Kwapong and J.li. Hotehkiss, J. Food Sci., 52 (1987) 761. 2 C.E. Sizer, P.L. Waugh, S. Edstam and P. Ackermfmn, Food TechnoL, 6 (1988) 152. 3 A.B. Marin, T.E. Acree, J.H. Ilotchkiss and S. Nagy, J. Agric. Food Chem, 40 (1992) 650. 4 J.H. Hotehkiss; P. Ackermann, M. Jagerstad and T. Ohlsson (eds), Food and Packaging Materials Chemieal Interaetions, UK, (Cambridge, 1995. 5 R. Lebosse, V. Dueruet and A. Feigenbaum, J. A^rie. Food Chem. (1997). 6 V. Kutty, R.J. Braddock and G.D. Sadler, J. Food Sci., 59 (1994) 402. 7 J. Ehret-Henry, V. Dueruet, A. Luciani and A. Feigenbaum, Analusis, 21 (1993) 277. 8 J. 1-ve Sech, V. Dueruet and A. Feigenbaum, J. Chromatogr. A, 667 (1994) 340. 9 R. Lebosse, V. Dueruet and A. Feigenbaum, J. High Resol. Chromatogr., 19 (1996) 413. 10 B. Kolb, L.S. Ettre and P. Hoqijat, Chromatographia, 32 (1991) 505. 11 J. Jansen; R. Gachter and H. MiiUer (eds). Plastics Additives Handbook, Hanser Pubhsher, USA, New York (1990) 863. 12 L. Agres, Y. Segui, R. Delsol and P. Raynaud, J. Appl. Polym. Sci., 61 (1996) 2015. 13 B.C. Clark and T. S. Chamblee; (}. (3haralambous (ed.), Off Flavors in Foods and Beverages, Netherland, Amsterdam, 1992. 14 J. Pokorny, F, Pudil, J. Volfova, H. Valentova, Ninth International Flavor (conference, G. Charalambous Memorial Symposium, Limnos July 1-4, 1997. 15 J. Koszinowski and O. Piringer, Verpack. Rund., 41 (1990) 15. 16 J.H. Tatum, S. Nagy and R.E. Berry, J. Food Sci., 40 (1975) 707. 17 A.E. Feigenbaum, M. Hamdani, V.J. Dueruet and A.M. Riquet, J. Pol. Engineering, 15 (1995/96) 47.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

753

Identification of the source of an off-odor in premiums intended for use with dry mix beverages Dimitrios Apostolopoulos Kraft Foods, Technical Center, Packaging Research and Technology, 801 Waukegan Road, Glenview, IL 60025, USA

Abstract Premiums intended to be inserted inside packaged dry mix beverages were tested for off-odor. Headspace gas chromatography/mass spectrometry was used in combination with an odor test to identify the volatiles in the premiums and also evaluate the odor barrier characteristics of the overwrap film. Data obtained from the headspace analysis of unwrapped premiums showed extraordinarily high amounts of chemicals with the major residual component being identified as cyclohexanone. Toluene, 2-methyl heptane, and 3-methyl hexane were the next sizable components. The source of those residual chemicals was considered to be solvents used with either the resin or the paint employed in the manufacturing and painting of the premiums. Odor testing of unwrapped premiums demonstrated a very strong, "solvent-like", objectionable odor. Residual cyclohexanone in overwrapped premiums was found to be about 16 times less than in the unwrapped premiums. Furthermore, the overwrapped premiums exhibited no off-odor. This clearly suggested that the solvent barrier provided by the plastic film used for overwrapping of the premiums was sufficient to prevent any residual solvent contamination of the dry mix beverages. It is apparent, however, that an unsealed premium overwrap or a premium with a punctured overwrap film would allow significant amounts of residual cyclohexanone and other residual solvents to transfer into the packaged contents and cause a severe off-odor problem. For that reason, it was recommended that those premiums should not be used with packaged dry mix beverages.

1. INTRODUCTION A common promotional tool is the insertion of a premium inside a food package. The volafiles in these inserts, if not controlled, can impart an undesirable flavor to packaged foods. Common practice is the use of an overwrap film to prevent direct contact and contamination of the product (1). The present study was undertaken with the objecfive to identify any product quality issues associated with the insertion of premiums inside packaged dry mix beverages. More specifically, i) identify any volatiles of the premiums that could potentially impart an off-odor

754

to the dry mix beverages, ii) quantify the odor impact of such volatiles upon their potential transfer into the product, and iii) determine whether the barrier provided by the premium overwrap film was sufficient to prevent transferring of the premium volatiles into the packaged contents. 2. MATERIALS AND METHODS 2.1 Premiums The premium samples under evaluation were molded polyethylene bodies shaped as potato men and women, which were painted and sealed in a nylon overwrap film. 2.2 Identification of Residual Species Present in Premiums by Using Headspace Gas Chromatography/Mass Spectrometry The identification of residual species present in the premiums under evaluation was performed in accordance with the ASTM F 151-86 method, modified as described below. Two over wrapped or two unwrapped premiums were placed into half-quart Mason jars. The jars were fitted and sealed with teflon-lined lids, equipped with sampling ports. Mason jars containing the premium specimens were placed inside a mechanically convected oven and heated at 110°C for 90 minutes, to ensure vaporization of the premium residuals into the headspace of the Mason jars. Using a preheated gas-tight syringe to avoid condensation of the volatiles, headspace aliquots of ImL were withdrawn from the Mason jars and injected into a gas chromatograph/mass spectrometer (GC/MS), equipped with a CP-Sil 8 CB chromatographic column operated at 20°C for 2 minutes and then increased at 10°C/min to 250°C. The compounds present in the injected aliquots were separated resulting in GC/MS scans with tentative mass spectral identification presented in Figures 1, 2, and 3.

I i ° r 99a i»

Figure 1: GC/MS profile of residual compounds for overwrapped premiums

755

I, 371

I Mrs

1244

1356

H5S

ISS-I

Figure 2: GC/MS profile of residual compounds for unwrapped premiums

Overwrapped Premiums

Figure 3: GC/MS profile of residual compounds for overwrapped versus unwrapped premiums

2.3 Odor Test The odor test was performed as described below. Three overwrapped or unwrapped premiums were placed in thoroughly cleaned one quart Mason jars, sealed with aluminum foil lined lids and heated at 49°C for 1 hour. Blanks (empty jars) were prepared and carried through the entire odor test to assure against any extraneous odors resulting from the jars or the screw caps. The Mason jars containing the premium specimens, as well as the blanks, were cooled to room temperature and then presented to an experienced panel for odor evaluation. The panel was composed of four people. All the panel menbers were familiar with the odor of solvents commonly used by the packaging industry. Odor evaluation entailed removing the screw caps of the Mason jars, opening a hole through the aluminum foil to allow sniffing of the headspace and determinating the odor givenoff by the premiums.

756 The odor evaluation panel was asked to briefly describe the type of odor present, rate the odor intensity on a 0-10 scale, and also indicate whether the odor was objectionable or not. 3. RESULTS AND DISCUSSION As indicated by the GC/MS profiles presented in Figures 1 and 2, both the overwrapped and unwrapped premiums exhibited a rather large number of residual compounds. The most abundant of all the residual compounds identified was cyclohexanone, followed in concentration by toluene, 2-methyl heptane, and 3-methyl hexane. The source of such residual chemicals was considered to be either the polyethylene resin or the paint used with the premiums. A comparison of the GC/MS profiles of the overwrapped and unwrapped premiums showed that the amount of cyclohexanone given-off by the unwrapped premiums was about 16 times more than the amount of cyclohexanone displayed by the overwrapped premiums (See Figure 3). This clearly suggested that the plastic film used for overwrapping of the premiums possessed very good solvent barrier characteristics and provided sufficient protection against any odor transfer under this test procedure. The conclusion above was also supported by the results of the odor test. Overwrapped premiums exhibited essentially no odor at all, unlike their unwrapped counterparts which exhibited a very strong solvent-like, objectionable odor, as shown by the table below. Table 1 Results of Odor Test for Premiums Type of Premium Overwrapped Unwrapped

Type of Odor

Odor Intensity on a 0-10 Scale*

None

0

Very strong, solvent-like, objectionable

* Where: 0 corresponds to essentially no odor characterized as objectionable

9

and

8-10 to excessive odor, usually

Based on the data generated, it can be stated that the overwrapped premiums inserted in the package were expected to contribute no odor to the dry mix beverage. However, the same data suggests that an occasionally unsealed or punctured overwrap could allow significant amounts of residual cyclohexanone, as well as other odorous residual compounds to transfer very easily into the packaged contents and cause a severe off-odor problem. For that reason, it was recommended that those premiums not be used in dry mix beverage packages.

757 4. REFERENCES 1. M.G. Heydanek, Jr., G. Woolford, and L.C. Baugh, J. Food Sci., 44 (1979) 850 2. ASTM Designation: F151-86. Standard test method for residual solvents in flexible barrier materials, (1986) 812-816.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

759

Effect of microwave heating on the migration of dioctyladipate and acetyltributylcitrate plasticizers from food-grade PVC and PVDC/PVC films into ground meat. A.B. Badeka and M.G. Kontominas

Laboratory of Food Chemistry and Technology, Department of Chemistry, University of loannina, GR-45110, Greece.

Abstract Migration of dioctyladipate (DOA) and acetyltributylcitrate (ATBC) plasticizers from plasticized polyvinyl chloride (PVC) and polyvinylidene chloride (PVDC/PVC) (Saran) films into ground meat of varying fat content (3%, 12%, 30% and 55%) has been studied. Meat samples were wrapped in PVC or Saran film and cooked for 0.5, 1, 1.5, 2, 2.5, 3, 4 min in a microwave oven on full power (~700W). The plasticizer migrating into meat was determined using an indirect GC method after saponification of the ester-type plasticizer (DOA or ATBC) and subsequent collection of the alcohol component of the ester, namely, 2-ethyl-lhexanol and 1-butanol, respectively. Migration was dependent on heating time, meat fat content and initial concentration of plasticizer in the film. The migration of plasticizer into meat did not reach equilibrium after heating for 4 min even in high fat content (55%) meat samples. Migration values of DOA and ATBC into 55% fat content meat samples after heating for 4 min was 846.0 mg/kg (14.7mg/dm2) and 95.1 mg/kg (2.5 mg/dm^) respectively.

1. INTRODUCTION

Microwaving is becoming an increasingly used process for the heating of foodstuffs in both the industrial and home sectors in Europe. The microwave oven is used for a variety of purposes such as cooking, baking, frying, defrosting, reheating, drying, enzyme inactivation, pasteurization, sterilization etc. [1-3]. Microwave processing offers several advantages over conventional heating methods. These advantages include speed of operation, energy savings, precise process control and faster start-up and shut-down times [1-3].

760 A variety of foods have been developed and modified over the past few years for the microwave market. Many of these food products are cooked with the packaging material (container or wrapping film) in the microwave oven. Such microwavable materials include plastics, paperboard and composites [4-8]. However, during cooking due to a significant increase in temperature, a variety of plastics additives contained in most packaging materials, i.e. plasticizers, antioxidants, stabilizers, residual monomers etc. may migrate from the packaging material into the food. This may result in the deterioration of food quality i.e. off-flavor and/or safety problems [9-12]. The migration of low molecular weight compounds from a polymeric material into a food-contacting phase is dependent on: the nature and thickness of the packaging material, the nature of the food in contact, initial concentration of the additive in the polymer, compatibility of the additive/polymer system, temperature, time of contact etc. [13-15] Polyvinylchloride (PVC) and polyvinyhdene chloride (PVCD)/PVC (Saran), films have found wide applications in the packaging of a large variety of foodstuffs. Also for covering foods during cooking in the microwave oven in order to prevent the drying out of the food surface and when baking dishes, reheating precooked meals and covering frozen dishes during reheating from the freezer. The most commonly used plasticizer for PVC is dioctyladipate (DOA) added at levels up to 40% (w/w), and for Saran it is acetyltributylcitrate (ATBC) added at levels up to 5% (w/w). There are several published studies dealing with the migration of plasticizers into different foods and/or food simulants during microwave heating [9, 11, 12, 15, 16-19]. All of them report equilibrium migration values. The present work involves a kinetic study of the migration of DOA and ATBC plasticizers from food grade PVC and PVDC/PVC (Saran) films respectively, into ground meat of varying fat content.

2. MATERIALS AND METHODS

2.1. Materials The PVC film used was MX-B LM, 15|im in thickness, containing 28.3% DOA (w/w) supplied by Borden, Chemical Division, N.Andover, Mass., USA. The Saran film used was 12|im in thickness containing 4.9% ATBC (w/w) supplied by Dow, Indianapolis, Ind., USA. Analytical grade DOA was purchased from Fluka (Buchs, Switzerland). Analytical grade ATBC was purchased from Unitex, Greensboro, N.C., USA. Analytical grade 2-ethyl-l-hexanol was purchased from Merck, Darmstadt, Germany. Analytical grade 1-butanol was purchased from Mallinckrodt, St. Louis, Mo., USA. Beef meat and fat were purchased locally.

761 2.2. Migration experiments Ground meat of 3% fat content was uniformly mixed with fat in a meat chopper so as to achieve meat samples of fat content 12%, 30% and 55%. Fat content was determined according to AOAC Soxhlet method [20]. Ground meat patties, approximately 40g in weight, were wrapped in PVC or Saran film and cooked for 0.5, 1, 1.5, 2, 2.5, 3 and 4 min in a microwave oven on full power (~700W). The total area of the film in contact with meat was 1.45dm2. All experiments were carried out in triplicate. For comparison purposes identical unwrapped samples (controls) were cooked in the microwave oven.

2.3. Analysis of DOA plasticizer The contaminated meat (~40g) was extracted in a Soxhlet apparatus with hexane for 6 h. After evaporation of hexane the residue was saponified with KOH 2N in methanol for 3 h. The volume of KOH 2N in methanol was dependent on meat fat content (8 mL/g fat). Thus DOA was decomposed to adipic acid and 2ethyl-1-hexanol. After saponification, methanol was evaporated in a rotoevaporator, the residue was acidified with HCl solution (1:1 v/v) and subjected to steam distillation until 200 mL of distillate had been collected. The distillate was extracted four times with 50 mL diethyl ether. The combined ether extracts were left overnight with 30 g anhydrous Na2S04. Diethyl ether was evaporated after separation from Na2S04. The residue was redissolved in CS2 and the CS2 solution was used to determine 2-ethyl-l-hexanol by GC using an appropriate standard curve. The recovery factor of the above method was obtained by addition of known amounts of DOA to ground meat samples and determination of the plasticizer content using the same procedure as for unknown samples.

2.4. Analysis of ATBC plasticizer The contaminated meat (~40g) was extracted in a Soxhlet apparatus with hexane for 6 h. After evaporation of hexane the residue was saponified with solid KOH for 7 min. The quantity of KOH was dependent on meat fat content (Ig/g fat). Thus ATBC was decomposed to citric acid and 1-butanol. After the saponification stage, the procedure followed was the same as that described above for DOA analysis. The recovery factor of the above method was obtained by addition of known amounts of ATBC to ground meat samples and determination of the plasticizer content using the same procedure as for unknown samples.

2.5. GC operational conditions The alcohol component of DOA, namely, 2-ethyl-l-hexanol, was determined under the following conditions: The GC unit was a Varian model 3700 GC equipped with a dual flame ionization detector. The column was of aluminum (1.90m long, 6.35mm o.d.) packed with 10% SE-30 stationary phase on Anachrom

762 ABS 60/80 Mesh. The temperatures used were: column, 165°C; injection port, 220°C; detector, 220°C. The alcohol component of ATBC, namely, 1-butanol, was determined under the following conditions: The GC unit was the same as that described above, the column was of stainless steel (Im long, 3.17mm o.d.) packed with 10% Carbowax 20 M on Supelcoport 80/100 Mesh. The temperatures used were: column, 65°C; injection port, 200°C; detector, 220°C.

3. RESULTS AND DISCUSSION

3.1. Migration of DOA Recovery factors of 74.5%, 74%, 84% and 82.3% for DOA into ground meat of 3%, 12%, 30% and 55% fat content were obtained respectively. The amounts of DOA migrated from PVC film into ground meat of varying fat content during microwave heating on full power (~700W) as a function of time are given in Figure 1. As shown in Figure 1 DOA migration into meat is time and fat content dependent. Migration of DOA into ground meat did not reach equilibrium after heating for 4 min even in high fat content (55%) meat samples (845.96 mg/kg or 23.5 mg/dm2). This value represents a 46.0% loss of the DOA plasticizer migrated from the PVC film into the ground meat sample.

^ 1000 (DO

>

800

<

600 i

o Q

400 4-

o '^o

200 -L

Time (min) Figure 1. Migration of DOA from PVC film into ground meat of fat content a) (A) 3%, b) (•) 12%, c) (•) 30% and d) (•)55% during microwave heating on full power.

763 DOA was not detected in unwrapped (control) meat samples. The migration amount of DOA into ground meat of fat content 3%, 12%, 30% and 55% after heating for 4 min was 195.2 mg/kg (5.42 mg/dm^), 565.1 mg/kg (15.70 mg/dm^), 771.8 mg/kg (21.44 mg/dm2) and 846.0 mg/kg (23.50 mg/dm2) respectively. Bishop and Dye [9] reported an average migration value of 21.2mg DOA/20 mL vegetable oil (1060 mg/1) after 10 min of cooking in the microwave oven. No further details were given on the dimensions and thickness of the film used, power setting of the oven, or the plasticizer content of the film used. Our DOA migration value into meat of 55% fat content after heating for 4 min is comparable to the above mentioned value (1060 mg/1), given the differences in nature of the food product (fat content), time of heating, area of contact between the foodstuff and PVC film, temperature etc. Startin et al [19] studied the migration of DOA into a variety of food products during microwave cooking and reported values of 435 mg/kg for peanut biscuits, 351 mg/kg for pork spare ribs, 191 mg/kg for cakes and 3 mg/kg for carrots. The film contained 17.2% DOA while its thickness, contact area between film and food and the cooking time were not specified. It is clear that as the fat content of the food increases the migration amount of the plasticizer will also increase. Our migration values are generally higher than the above, a fact which can be attributed to differences in experimental conditions, area of the film used, percentage of the plasticizer in PVC film, fat content etc. Harrison [12] reported DOA migration values ranging from 146 to 435 mg/kg for fatty foods such as chicken (152 mg/kg), pork (351 mg/kg), trout (146 mg/kg) and peanut biscuits (435 mg/kg). No further details were given in this work so it is difficult to attempt comparison of these values to ours. Finally, Castle et al [16] studied the migration of DOA into foods during the "reheating" process in a microwave oven and reported values ranging from 27 mg/kg for pizza to 2.6 mg/kg for potatoes. The film used contained 10% DOA along with polymeric plasticizer. It is obvious that the migration amounts under the above mentioned experimental conditions are much lower than our migration values.

3.2. Migration of ATBC Recovery factors of 65.3%, 64.7%, 66.4% and 65% for ATBC into ground meat of fat content 3%, 12%, 30% and 55% were obtained respectively. The amounts of ATBC migrated from Saran film into ground meat of varying fat content during microwave heating on full power (~700W) as a function of time are given in Figure 2. As shown in Figure 2 ATBC migration into meat is time and fat content dependent. Migration of ATBC into ground meat did not reach equilibrium after heating for 4 min even in high fat content (55%) meat samples (95.1 mg/kg or 2.50 mg/dm2). This value represents a 21.5% loss of the ATBC plasticizer which migrated from Saran film into the ground meat sample. ATBC was not detected in control meat samples. Furthermore ATBC was not detected in meat samples of fat content 3%. The migration amount of ATBC into

764 ground meat of fat content 12%, 30% and 55% after heating for 4 min was 40.2 mg/kg (1.06mg/dm2), 72.4 mg/kg (1.91 mg/dm2) and 95.1mg/kg (2.50 mg/dm2) respectively.

Time (min)

Figure 2. Migration of ATBC from Saran film into ground meat of fat content a) (A) 12%, b) (•)30% and c) (•)55% during microwave heating on full power.

Health and Reilly [18] reported that the migration of ATBC into poultry products during microwave cooking was time dependent, reaching equilibrium after 8 min of cooking (0.834 mg butanol/g). This value is much higher than our value 0.05 mg/g for meat of fat content 55% heated for 4 min at full power. This can be attributed to differences in time of heating and fat content of the foodstuffs used in the two studies. Health and Reilly also showed that an increased fat content of the food resulted in increased migration of the plasticizer. Castle et al [17] studied the migration of ATBC into various foods and reported values ranging from 79.8 mg/kg for peanut biscuits to 0.9 ^glhg for Brussels sprouts. The migration value of 79.8 mg/kg for peanut biscuits coirelates well with our value of approximately 95.1 mg/kg obtained for meat of fat content 55% after 4 min of microwave heating at full power. Finally, Castle et al [16] reported a migration value for ATBC of 79.9 mg/kg for high fat biscuits and 35.0 mg/kg for pizza under reheating conditions in a microwave oven. Both these values are of the same order of magnitude as our values, given the differences in experimental conditions between the two studies. What should be stressed is that under present experimental conditions the DOA migration is higher than the upper limit for global migration (60 mg/kg or

765 10 mg/dm^) set by the E.U. even for meat samples of low fat content. The ATBC migration is higher than the upper limit for global migration only for meat samples of high fat content. The present results indicate that PVC film should not be used in direct contact with foodstuffs in the microwave oven while Saran film may be used with appropriate caution in the microwave oven avoiding direct contact of high fat foodstuffs with the film. Further study on migration of the two plasticizers (DOA, ATBC) from cling films (PVC, Saran) into solid foodstuffs (pizza, sausage etc.) is in progress.

4. REFERENCES

1. J. Giese, Food Technol. 46 (1992) 118. 2. K. Knuntson, E.H. Marth and M.K. Wagner, Lebensm. Wiss. Technol. 20 (1987) 101. 3. N.N. Potter, "Food Science", 3^^ edn. AVI Publishing, Westport, Conn. (1986) 320. 4. P. Harrison, Packag. Technol. Sci. 2 (1989) 5. 5. M.R. Perry, J. Packag. Technol. 1 (3) (1987) 87. 6. M.R. Perry, J. Packag. Technol. 1 (4) (1987) 114. 7. H.A. Rubbright, Cereals Foods World 35 (1990) 927. 8. L.A. Sheridan, Microwave World 8 (4) (1987) 5. 9. C.S. Bishop and A. Dye, J. Environ. Health 44 (5) (1982) 231. 10.L. Dixon-Anderson, R.J. Hernandez, I. Gray and B. Harte, Packag. Techno. Sci. 1 (1988) 117. ll.S.M. Jickells, J.W. Gramshaw, L. Castle and J. Gilbert, Food Addit. Contam. 9 (1) (1992) 19. 12.N. Harrison, Food Addit. Contam. 5 (1) (1988) 493. 13.W.D. Bieber, K. Figge and J. Koch, Food Addit. Contam. 2 (2) (1985) 113. 14.K. Figge, Food Addit. Contam. 5 (1) (1988) 397. 15.J.M. Vergnaud, Polymer Plast. Technol. Eng. 20 (1) (1983) 1. 16.L. Castle, S.M. Jickells, J. Gilbert and N. Harrison, Food Addit. Contam. 7 (6) (1990) 779. 17.L. Castle, S.M. Jickells, M. Sharman, J.W. Gramshaw and J. Gilbert, J. Food Protect. 51 (12) (December 1988) 916. 18.J.L. Heath and M Reilly, Poultry Sci. 60 (10) (October 1981) 2258. 19.J.R. Startin, M. Sharman, M.D. Rose, I. Parker, A.J. Mercer, L. Castle and J. Gilbert, Food Addit. Contam. 4 (4) (1987) 385. 20.Official Methods of Analysis of AGAC methods, 16^^ edition, Vol II, (1995) 39(1) 08.

This Page Intentionally Left Blank

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

767

Effects of ionizing radiation on properties of monolayer and multilayer flexible food packaging materials K.A. Riganakos^. W.D. KoUer^, D.A.E. Ehlermann^, B. Bauer^ and M.G. Kontominas^ ^University of loannina, Department of Chemistry, Laboratory of Food Chemistry and Technology, GR-45110, loannina, Greece. ^Federal Research Centre for Nutrition, Institute of Process Engineering, Engesserstr. 20, D-76131, Karlsruhe, Germany.

Abstract Volatile compounds produced in flexible food packaging materials (LDPE, EVAc, PET/PE/EVOH/PE) during electron beam irradiation were isolated by purge and t r a p technique and identified by combined gas chromatographymass spectrometry (GC/MS), after thermal desorption and concentration. For comparison purposes non-irradiated films were also studied. Film samples were i r r a d i a t e d a t low (5 kGy, corresponding to cold pasteurization), intermediate (20 kGy, corresponding to cold sterilization) and high (100 kGy) doses. It was observed that a number of volatile compounds are produced after irradiation in all cases. Furthermore the amounts of all volatile compounds proportionally increase with increasing irradiation dose. Both primary (methyl- derivatives etc.) as well as secondary i.e. oxidation products (ketones, aldehydes, alcohols, carboxylic acids etc.) are produced upon irradiation. These products may affect organoleptic properties and thus shelf-life of prepackaged irradiated foods. No significant changes were observed in the structure of pol5mier matrices as exhibited by IR spectra after irradiation of the materials at doses tested. Likewise, no significant changes were observed in gas permeability of plastics packaging materials after irradiation.

1. EVTRODUCTION Irradiation of prepackaged foodstuffs using gamma and electron beam radiation is steadily gaining ground as a method of food preservation worldwide. While many workers have dealt with specific eff*ects of ionizing radiation on various food constituents, relatively few investigations have dealt with the effects of ionizing radiation on plastics packaging materials. [1-6].

768 Another use of ionizing radiation is in the improvement of polymer mechanical properties. [7-8]. Physical and chemical changes resulting in plastics are of prime importance in applications such as food packaging where such changes may directly affect the shelf-life of packaged foodstuffs. [9]. Radiation-induced changes in pol5m[iers are shown to depend on: a) chemical structure of the polymer, b) additives used to compound the plastic, c) processing history of the plastic, and d) specific irradiation conditions, namely, the absorbed dose, the irradiation atmosphere and, in certain cases, the dose rate. [10]. The major chemical changes that are reported to occur in polymers as a result of ionizing radiation are: a) simultaneous scission and cross-linking of the polymeric chains, their net effect determining the changes in polymer properties, b) formation of gases and low molecular weight radiolysis products and c) formation of unsaturated bonds. [10]. In the presence of oxygen, there is additional oxidative chain scission and oxidation of the polymer, leading to the formation of peroxide, alcohol and carbonyl fi:*actions as well as carbon monoxide and carbon dioxide and various oxygen-containing low molecular weight compounds. Large amounts of hydrogen, methane and hydrocarbons may also result. As a general rule, the amounts of gases produced increase with increasing radiation dose. Such products, upon migration into the packaged foods, may affect the sensory properties (off-odors and off-flavors) and/or safety of prepackaged foods. [11-12]. Radiolysis compounds produced upon irradiation may also be responsible for an unpleasant odor of the packaging materials after irradiation. [13-18]. The FDA h a s regulated the use of ionizing radiation in single layer plastic packaging m a t e r i a l s while today multilayer m a t e r i a l s (laminated or coextruded) are almost exclusively being used in advanced food packaging applications. [19]. There is practically no information in the literature on the effect of irradiation in substrates such as multilayer coextruded plastic packaging materials. [10]. The objective of this work was: i) to identify volatile compounds produced during electron beam irradiation (in the presence of air) at low, intermediate and high doses of two monolayer plastic food packaging films (LDPE, EVAc) and one multilayer film (PET/PE/EVOH/PE), ii) to investigate the possible structural changes in the above plastic films, as a result of irradiation and iii) to investigate possible changes in barrier properties of the above materials as a result of irradiation. Such composite flexible packaging materials are being experimentally produced in our laboratory and are being used in specific food packaging applications, (i.e. the extension of shelf-life of specific foodstuffs such as meat, sausages, poultry etc.).

769 2. EXPERIMENTAL Materials and Methods 1. Materials Three types of food packaging materials were used:l)LDPE (thickness 30 |im) typical representative of a homopolymer, 2)EVAc (thickness 20 [im) typical representative of a copolymer and 3)PET/PE/EV0H/PE (thickness 70 |im) one of many coextruded multilayer materials, all widely used in food packaging applications. [LDPE = low density polyethylene, EVAc = ethylene-vinyl acetate copol3m[ier, PET = poly(ethylene terephthalate), EVOH = ethylene-vinyl alcohol copolymer]. 2. Methods a) Irradiation Approximately 1.2-1.6 g of each packaging material were weighed into glass head space vials 22 mL in capacity. The vials were sealed using an aluminium covered silicon rubber disc and an aluminum crimp cap. The plastic films were then irradiated at doses of 5, 20 and 100 kGy in the presence of air. Irradiation doses were measured using a Far West dosimeter, at an energy of 10 MeV. b) Isolation of volatiles Helium gas, at a flow rate of 30 mL/min, was passed for 20 min through the head space vials which were maintained at SO^C. Volatile compounds were collected on a Tenax GC plug filled into the glass liner of an Optic PTVInjector. c) Identification of volatile compounds from non-irradiated and irradiated samples Volatile compounds were identified using a GC/MS system consisting of an HP (Hewlett Packard) 5890 II Gas Chromatograph, an HP 5985 B Mass Spectrometer and a Teknivent Data base System. Gas Chromatography (GC) conditions were: Carrier Gas: Helium (at 2 bar column pressure). PTV-Injector program: 50oC to 220oC, at a rate of le^C/sec, splitless injection mode, cryofocussing with liquid N2. Oven temperature program was 40oC/5min then 6^0 to 220^0. Separation was carried out on a 60 m x 0.25 mm i.d. CW 20m Quadrex fused silica capillary column. Mass Spectrometry (MS) conditions were: 70 eV electron energy and 200^0 ion source temperature. Volatile products were identified by comparing the mass spectra of the relevant chromatographic peaks with those from the GC/MS system library. d) IR analysis IR spectra of irradiated and non-irradiated films fi:-om all the samples were recorded on a Perkin Elmer 783 IR spectrophotometer. Cleaned plastic films,

770

cut into appropriate pieces, were used for the spectrophotometric measurements of irradiated and non-irradiated films. IR spectra were recorded in the wavelength region 4000-200 cm"^ e) Permeability measurements Permeability coefficients were estimated for oxygen with the Mocon Oxtran MH 2/20 instrument. All tests were performed at RH=80% and at a temperature of 20^0. 3. RESULTS AND DISCUSSION a) Determination of volatile compounds Gas chromatograms for the three plastic food packaging films nonirradiated and irradiated at doses 5, 20 and 100 kGy, are given in Figs. 1-3. Comparison of chromatogram Fig. la to lb, Ic and Id shows that a nimiber of volatile compounds are formed during irradiation. These compounds are absent in the chromatograms of the non-irradiated samples. Furthermore, the amounts of all volatile compounds proportionally increase with increasing absorbed radiation dose. The same pattern is shown in Figs. 2 and 3.

3.5e+007

3.0e*007

2.5e*007

2.0e^007

1.5e+007 L-Awu JUi(A_*-

(d)

l.Oe+007

5e+006

AJLJU

12.8 Oe+000-

.

I 10

1

1

»_JU_^..J1

i JL

1

(b)

16 15

20 Time ( m i n u t e s )

Figure 1. Chromatograms for non-irradiated (a) and irradiated samples of LDPE at 5 kGy (b), 20 kGy (c) and 100 kGy (d).

771 2.5e+007

2.06+007-^

1.5e+007

wyuUduLjiLL^

l.Oe+007

•-*AJ>AJUUL_A_A.

(d) (c)

«._

5e+006 (b)

Oe+00010

IMt-

15

20,18

25.79 ,AA^ i

20 Time (minutes)

(a)

, -

25

30

32

Figure 2. Chromatograms for non-irradiated (a) and irradiated samples of EVAc at 5 kGy (b), 20 kGy (c) and 100 kGy (d). 3.0e+007

2.5e+007

2.0e+007

1.5e+007 Uius

.JLLJU^LliJ^

IAA-AJIJ.

(d)

l.J3e+007

uyjiUiiLj

(c)

^4

5e+006

Oe+OOO-l

ii.

(b)

IJL^^LJUJLOJL

JsLLo^iiiii. 10

15

18.9 20 Time (minutes)

25^63 25

(a) 30

32

Figure 3. Chromatograms for non-irradiated (a) and irradiated samples of PET/PE/EVOH/PE at 5 kGy (b), 20 kGy (c) and 100 kGy (d).

772 Volatile compounds identified in LDPE, EVAc and PET/PE/EVOH/PE using GC/MS are given in Tables 1-3. These include several aldehydes, ketones, alcohols, hydrocarbons, carboxylic acids etc. Table 1 gives volatile compounds recorded in non-irradiated and irradiated at 100 kGy LDPE. Of 52 volatile compounds recorded in non-irradiated LDPE, 35 were identified by GC/MS. Of 74 volatile compounds recorded in irradiated LDPE, 49 were identified by GC/MS. Main classes of compounds identified in non-irradiated LDPE include s a t u r a t e d hydrocarbons (from hexane to hexadecane), aldehydes (such as hexanal, octanal), carboxylic acids (from acetic acid to octanoic acid), ketones (such as acetone etc.), phenols (such as dimethylphenol, BHT), etc. In addition to the above, unsaturated hydrocarbons (hexene, heptene), other ketones (such as hexanone, heptanone, octanone), esters (hexyl formate, octyl formate), aromatic compounds (such as benzene, ethylbenzene), as well as several methyl derivatives were identified in the irradiated LDPE film samples. It has been reported in the literature t h a t unsaturated carboxylic acids are produced upon thermal oxidative degradation of PE [20]. However, no production of u n s a t u r a t e d carboxylic acids was observed in the irradiated PE film samples. Table 2 gives volatile compounds found in non-irradiated and irradiated at 100 kGy EVAc. Of 58 volatile compounds recorded in non-irradiated EVAc, 45 were identified by GC/MS. Of 102 volatile compounds recorded in irradiated EVAc, 63 were identified by GC/MS. Main classes of compounds identified in noni r r a d i a t e d EVAc include s a t u r a t e d hydrocarbons (from h e x a n e to heptadecane), aldehydes (such as hexanal, heptanal, octanal, nonanal), alcohols (butanol, heptanol), carboxylic acids (from acetic acid to octanoic acid), several aromatic compounds (such as toluene, ethylbenzene, xylene, limonene, naphthalene, dimethyl alcohol, BHT), esters (propylene carbonate, dimethyl phthalate), etc. In addition to the above, unsaturated hydrocarbons (hexene, heptene), ketones (heptanone, octanone), as well as several methyl derivatives were identified in the irradiated EVAc film samples. Table 3 gives volatile compounds identified in non-irradiated and irradiated PET/PE/EVOH/PE. Of 70 volatile compounds recorded in non-irradiated PET/PE/EVOH/PE, 54 were identified by GC/MS. Of 102 volatile compounds recorded in irradiated PET/PE/EVOH/PE, 79 were identified by GC/MS. Main classes of compounds identified in non-irradiated PET/PE/EVOH/PE include saturated hydrocarbons (from hexane to hexadecane), carboxylic acids (fi:'om acetic acid to nonanoic acid), several aromatic compounds (such as derivatives of benzene, menthol, phenol, naphthalene, BHT), etc. In addition to the above, u n s a t u r a t e d hydrocarbons (nonene), aldehydes (hexanal, octanal), ketones (hexanone, h e p t a n o n e ) , alcohols (methyl-butanol, h e p t a n o l ) , methyl derivatives, other aromatic compounds (toluene), etc were identified in the irradiated PET/PE/EVOH/PE film samples. Based on the above information the following observations can be made: a) All three plastic materials produce similar volatile compounds (such as s a t u r a t e d hydrocarbons, carboxylic acids, aldehydes, ketones, aromatic compounds, esters etc.). b) Irradiation produces methyl-derivatives, unsaturated hydrocarbons, esters, other aldehydes, ketones, aromatic compounds, etc. c) EVAc (non-irradiated and irradiated) produces more odor or flavor active substances t h a n the other two polymer materials, such as limonene, xylene.

773

naphthalene etc. which may affect the odor/flavor of foodstuffs packaged in this plastic material. The results for LDPE are in general agreement with those of Azuma at al. [14]. These authors irradiated LDPE films in air at a dose of 20 kGy and recorded more than 100 volatile compounds in the headspace of film samples. 50 of the compounds were identified by GC/MS. Rojas de Gante and Pascat (6) irradiated LDPE and OPP films in air at doses of 10 and 25 kGy and obtained similar results. Irradiation in air produced 100 volatiles in LDPE and 58 in OPP films.

Table 1 Volatile compounds identified by GC/MS of irradiated (100 kGy) and nonirradiated LDPE films. Irradiated at 100 kGy No

T"

2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2A 25 26

Name Hexane 1-hexene ?

3-methyhexane Heptane 2-heptene Octane Acetone 3-ethyl-4-methyl- 1-pentene T r a n s - l-butyl-2-methylcyclopropane 3-ethylheptane Nonane Benzol ?

Decane ? 3-hexanone ?

Hexanal ?

Undecane Ethylbenzol 1- [ 1-cy clohexen- 1-yl] ethanone 4-hydroxy-4-methylpentanone

? 3-heptanone

Non-irradiated Retention

Name

time (min ) 7.76 Hexane 7.81 Heptane 7.87 Octane 7.92 Acetone 7.96 Trans-l-butyl-2-methylcyclopropane 8.02 Nonane 8.28 Decane 8.40 Hexanal 8.49 Undecane 8.53 1- [ 1-cyclohexen- 1-yl] ethanone 8.67 4-hydroxy-4-methylpentanone ? 8.91 ? 9.28 9.76 Dodecane 9.93 2,3-dehydro-4-methylfuran 10.26 Dimethyphenol 10.77 1,3,5,7-cyclooctatetraene 11.08 Octanal 11.26 Tridecane 9 11.30 11.50 6-methyl-5-hepten-2-one 9 12.03 12.05 Tetradecane 12.26 12.45 12.69

Retention time (min) ^778 7.96 8.23 8.38 8.46 8.84 9.88 11.26 11.35 12.01 12.26 12.80 13.24 13.42 13.73 14.55 14.83 15.51 15.55 16.02 16.69 17.41 17.91

2,3,6-trimethyl-l,5heptadiene

18.09

9

18.81 19.43

Acetic acid

774 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ^ 49 50 51 52 53 54 55 56 57 58 59

eo

61 62 63 64 65 66 67 68 69 70 71 72 73 74

? ?

Dodecane 2,3-dehydro-4-methylfuran Dimethylphenol 3-methyl- 1-butanol 5-methyl-3-heptanone 1,3,5,7-cyclooctatetraene 2-octanone Octanal Tridecane ?

2-methyl-4-octanone 6-methyl-5-hepten-2-one 5-methyl-l-heptene Hexyl formate ?

Tetradecane 2,3,6-trimethyl-l,5heptadiene 9

Acetic acid 9

12.80 13.26 13.48 13.73

? Pentadecane ? Propanoic acid

20.00 20.15 20.78 21.30

14.42 14.70 14.77 14.83 15.48

9 9

21.62 21.88 22.04 22.29 22.56

15.51 15.64 16.09 16.34 16.69 16.90 16.99 17.37 17.89 18.09 18.81 19.43

20.13 Pentadecane 20.20 ? 20.62 Propanoic acid 21.30 Octyl formate 21.50 ? 21.83 2,2-dimethylpropanoic acid 22.00 ? 22.28 Hexadecane 22.29 3,5,5-trimethyl-222.73 cyclopenten- 1-one ? 23.04 Butanoic acid 23.25 ? 23.63 Acetophenone 23.81 ? 24.00 ? 24.47 9 24.65 9 25.36 Pentanoic acid 25.76 3-methyl-2-butanoic acid 26.63 9 27.11 ? 27.40 Hexanoic acid 27.47 ? 29.10 Heptanoic acid 29.44 9 29.86 Octanoic acid 31.33

2,2-dimethylpropanoic acid Hexadecane 3,5,5-trimethyl-2cyclopenten-1-one Butanoic acid ? Acetophenone 9 9 9 9

Pentanoic acid 3-methyl-2-butanoic acid 9

Hexanoic acid 6,10-dimethyl-5,9undecadien-2-one Butylated hydroxytoluene 9 9

Heptanoic acid ? Octanoic acid

23.19 23.64 23.84 24.04 24.47 24.65 25.43 25.64 26.67 27.35 27.50 27.62 28.65 29.10 29.26 29.48 31.05 31.35

775 Table 2 Volatile compounds identified by GC/MS of irradiated (100 kGy) and nonirradiated EVAc films. Non-irradiated I

Irradiated at 100 kGy No

i"

2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

35 36 37

Name Hexane 1-hexene 3-methylhexane Heptane Trans-1,2-dimethylcyclopentane 3-heptene Octane ?

1-octene 3-ethyl-4-methyl- 1-pentene Nonane 3-methylbutanaI Methyl isobutyrate 7 7

Decane 7 7

Toluene 7 7

Hexanal Undecane 7

l-methyl-2-cyclohexene Ethylbenzene p-xylene 1,3-dimethylbenzene 7 7

3-heptanone 7

heptanal d,l-limonene

7

2,3-dimethyl-2-hexanol 3-octanol

Retention

Name

time (mir0 7.84 Hexane 7.89 Heptane 7.95 Octane 7.99 1-octene 8.11 Nonane 8.16 8.32 8.43 8.48 8.55 8.98 9.08 9.21 9.33 9.85 9.97 10.23 10.33 10.60 10.80 11.13 11.30 11.35 11.52 11.68 12.00 12.16 12.28 12.48 12.63 12.75 13.08 13.22 13.30

13.35 13.51 13.62

3-metliylbutanal Methyl isobutyrate 7

Decane 7

Toluene 7

Hexanal Undecane 7

l-methyl-2-cyclohexene-1ol Ethylbenzene p-xylene 1,3-dimethylbenzene 1-butanol heptanal d,l-limonene Trifluoroacetyl-alphaterpineol 7

1,3,5-trimethylbenzene Octanal Tridecane 7 7

6-methyl-5-hepten-2-one Nonanal Tetradecane 7

l-(l,2-dimethylpropyl)-lmethyl-2-nonylcyclopropane 9-methylnonadecane Acetic acid Cis-5-methyl-2-(lmethylethyl)cyclohexanone

Retention time (min) 7.77 7.92 8.23 8.44 8.84 9.08 9.21 9.74 9.89 9.93 10.57 10.77 11.26 11.36 11.49 11.65 11.96 12.12 12.24 12.50 13.20 13.26 13.62 15.13 15.30 15.49 15.60 16.22 16.41 16.63 17.86 17.91 18.20 18.85

19.20 19.43 19.55

776 38

Trifluoroacetyl-alphaterpineol

13.65

2-propyl- 1-pentanol

20.00

39 40

?

13.86 14.12

20.18 21.28

41 42 43 44

?

45 46 47 4S 49

?

Pentadecane 3,7-dimethyl-1,6-octadien3-ol Propanoic acid Hexadecane ? 5-methyl-2-(l-methylethyl)-cyclohexanol Heptadecane Naphthalene 1,3-butanediol ? 6,10-dimethyl-5,9undecadien-2-one Propylene carbonate Benzyl alcohol Butylated hydroxytoluene 1-phenylethanol ? Octanoic acid

50 51 52 53 5t 55 56 57 58 59

eo 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

? 3-methyl- 1-butanol ? ?

1,3,5-trimethylbenzene Octanal 2-octanone Tridecane 3-heptanol ? 9 ? ?

6-methyl-5-hepten-2-one ? Hexyl formate 4-hydroxy-4-methylpentanone Nonanal Tetradecane ?

l-(l,2-dimethyl)cyclopropane 9-methylnonadecane Acetic acid 9

Cis-5-methyl-2-(lmethylethyl)cyclohexanone 9

2-propyl- 1-pentanol 2-ethyl- 1-hexanol Pentadecane ? Benzaldehyde 3,7-dimethyl- 1,6-octadien3-ol Propanoic acid ? Octyl formate Dimethylpropanedioic acid Hexadecane 9

Butanoic acid 5-methyl-2-( 1-methylethyD-cyclohexanol

14.30 14.79 14.85 15.17 15.20 15.34 15.50 15.53 15.64 15.70 15.78 16.18 16.25 16.46 16.72 16.88 17.07 17.58 17.81 17.85 18.20 18.85 19.10 19.20 19.28 19.55 19.88 20.00 20.05 20.18 20.50 21.05 21.08 21.14 21.41 21.50 21.82 22.28 22.65 23.08 23.34

9

? Dimethyl phthalate

21.34 22.31 22.65 23.34 24.37 25.65 25.74 27.49 27.56 28.12 28.30 28.59 28.99 30.68 31.37 32.00 33.15 35.75

777 82 83 84 85 86 87 88 89 90 91 92 98 9i 95 96 97 98 99 100 101 102

Nonanol 2-methoxy- 1-phenylethanone ? Heptadecane 9 9

Pentanoic acid Naphthalene 1,3-butanediol 9 9

6,10-dimethyl-5,9undecadien-2-one Propylene carbonate Benzyl alcohol ? Heptanoic acid

? ? Octanoic acid 9

?

23.61 23.80 24.00 24.37 25.05 25.14 25.37 25.62 25.74 27.10 27.46 27.54 28.12 28.30 29.05 29.43 30.68 31.02 31.34 32.60 33.15

Table 3 Volatile compounds identified by GC/MS of irradiated (100 kGy) and irradiated PET/PE/EVOH/PE films. Non-irradiated

Irradiated at 100 kGy No

Name

exane T" HHeptane

2 3 4

5 6 7 8 9 10 11 12 13

3,4,5-trimethylheptane Octane Trans-1,2-dimethylcyclopentane Cis-l-butyl-2methylcyclopropane 4-methyloctane Trans-l-butyl-2methylcyclopropane 3-ethylheptane 4-methyl-3-heptene

? Nonane 3,5-dimethyloctane

Retention time (min) 7.74 7.91 8.11 8.22 8.35 8.43 8.56 8.59 8.62 8.67 8.80 8.83 9.06

Name Hexane Heptane Octane Trans-l,2-dimethylcyclohexane Cis-l-butyl-2methylcyclopropane Trans-l-butyl-2methylcyclopropane 4-methyl-3-heptene

? Nonane 3,5-dimethyloctane 9 9 9

Retention time (min) 7.77 7.90 8.30 8.35 8.50 8.59 8.67 8.83 8.92 9.09 9.17 9.40 9.55

non-

778 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

?

2-nonene ? 4-methylnonane 2,6,7-trimethyldecane ?

Tetramethyloctane ?

Pentanal Decane ?

? 2,6-dimethylundecane Toluene ?

3-hexanone ? 5-( l-methylpropyl)-nonane ? ?

Hexanal Undecane ?

Ethylbenzene 1,3-dimethylbenzene ?

3-heptanone ?

9.14 9.20 9.36 9.40 9.50 9.55 9.58 9.71 9.73 9.89 10.12 10.24 10.47 10.53 10.58 10.75 10.80 10.97 11.02 11.08 11.23 11.60 11.89 12.18 12.31 12.48 12.60 12.83

Dodecane Propylbenzene ?

12.94 13.17 13.41 13.50 13.68 13.94

^

l-ethyl-3-methylbenzene

14.06

49 50 51 52 53 54 55

14.27 14.39 14.52 14.64 14.83 14.89 15.04

56 57 58 59 60

Tert-butylbenzene Cyclodecane 1,3,5-trimethylbenzene 3-methyl- 1-butanol 1,3,5,7-cyclooctatetraene l-ethyl-4-methylbenzene l-methyl-2-(lmethylethyD-benzene 1,2,4-trimethylbenzene 2-octanone Octanal Tridecane l-methyl-3-propylbenzene

15.38 15.44 15.48 15.70 15.78

61

1,4-diethylbenzene

15.84

42 43 44 45 46 47

1-methylethylbenzene 2-heptanone ?

Tetramethyloctane Decane ? 9

? 5-( l-methylpropyl)-nonane 7 Undecane Ethylbenzene 1,3-dimethylbenzene 9

? 1-methylethylbenzene 1,3-dimethylbenzene 9

Dodecane Propylbenzene l-ethyl-3-methylbenzene Cyclododecane 1,3,5-trimethylbenzene 1,3,5,7-cyclooctatetraene l-ethyl-4-methylbenzene l-methyl-2-(lmethylethyl)-benzene 1,2,4-trimethylbenzene Tridecane l-methyl-3-propylbenzene 1,4-diethylbenzene l-ethyl-3,5-dimethylbenzene 1,2-diethylbenzene a-methylstyrene 2,5-dimethylbenzaldehyde 1,3,5-trimethylbenzene ? 2-ethyl-l,4-dimethylbenzene l-methyl-2-(lmethylethyD-benzene Tetradecane ? Cyclotetradecane Acetic acid 9

Pentadecan 1,7,7-trimethylbicyclo[2,2,l]heptan-2-one ? Hexadecane Cyclohexadecane Menthol 2-methoxy- 1-phenylethanone Trichloromethylbenzene

9.58 10.05 10.17 10.58 10.80 10.97 11.08 11.60 12.18 12.31 12.48 12.83 12.94 13.23 13.44 13.50 13.70 14.06 14.39 14.52 14.83 14.89 14.99 15.38 15.70 15.78 15.94 16.29 16.43 16.49 16.56 16.63 16.95 17.10 17.28 17.95 18.85 18.88 19.46 20.00 20.10 20.83 21.50 22.28 23.23 23.32 23.86 24.69

779 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 98 94 95 96 97 98 99 100 101 102

l-ethyl-3,5dimethylbenzene 1,2-diethylbenzene a-methyls t y r e ne

16.24

Naphthalene

25.63

16.40 16.44

27.51 27.61

1,3,5-trimethyl-benzene 6-methyl-5-hepten-2-one

16.60 16.63 16.87 16.92 17.10

Hexanoic acid 6,10-dimethyl-5,9undecadien-2-one Butylated hydroxytoluene

?

Hexyl formate 2-ethyl-l,4-dimethylbenzene l-methyl-2-(lmethylethyl)-benzene Tetradecane l,3-bis-(l,l-dimethyl)benzene ?

Hexanol Cyclotetradecane Acetic acid ?

Pentadecane 9

1,7,7-trimethylbicyclo[2,2,l]heptane-2-one Propanoic acid Octyl formate 3-methyl- Ih-pyrrole 2,2-dimethylpropanoic acid Hexadecane Butanoic acid Cyclohexadecane Menthol ?

2-methoxy- 1-phenylethanone Trichloromethylbenzene ?

Pentanoic acid Naphthalene Dimethylbenzenemethanol Hexanoic acid Heptanoic acid ?

Phenol Octanoic acid Nonanoic acid Bis-(l,l-dimethylethyl)phenol

17.28 18.20 18.68 18.85 19.24 19.28 19.70 19.98 20.12 20.70 20.83 21.21 21.44 21.57 21.97 22.28 23.08 23.23 23.32 23.62 23.86 24.69 25.05 25.36 25.69 25.84 27.45 29.43 30.28 30.68 31.31 33.09 35.36

? Phenol Octanoic acid Nonanoic acid Bis-( 1, l-dimethylethyDphenol

28.61 30.28 30.68 31.33 33.15 35.42

780 b) IR analysis No significant changes were observed in IR spectra of irradiated film samples at all doses tested, suggesting that irradiation does not affect the molecular structure of the films under study. Present results are in agreement with those of Rojas de Gante and Pascat (6) who have observed no significant changes in the IR spectra of LDPE and OPP with absorbed doses of 0-50 kGy and Bersch et al. (18) who found no "definite and assignable changes" with HDPE, LDPE, PET, PS, PVC, PMTE [poly(monochloro-trifluoro-ethylene)] and r u b b e r hydrochloride films irradiated with 56 kGy either under vacuum or in air. c) Gas permeability measurements Table 4 gives oxygen permeability values of the three polymeric films. As shown in Table 4 there are no significant changes observed in gas permeability of irradiated film samples at all doses tested. Table 4 Oxygen permeability values of non-irradiated and irradiated at 100 kGy polymeric films. No

Material

O2 Permeability

(cm^/m^.day)

Non-irradiated

Irradiated at 100 kGy

1

LDPE (30 ixm)

6,268

6,520

2

EVAc(20pim)

11,250

11,865

3

PET/PE/EVOH/PE (70 [xm)

0.68

0.70

Present work is being extended to a series of 3-,4- and 5- layer coextruded flexible food packaging films experimentally produced in our laboratory to be irradiated u n d e r various atmospheric conditions (modified atmosphere packaging) in contact with selected food products. PREFERENCES 1 H.G. Le Clair and W.H. Cobbs, Ind. Eng. Chem., 50(3) (1958) 323. 2 J.J. Killoran, Modem Packaging, 40 (1967) 179. 3 I. Varsanyi, I. Kiss and J. Farkas, Acta Aliment. 1(1) (1972) 5. 4 J.J. Killoran, Act. Rep., 20(2) (1977) 104. 5 P.S. Elias, Chem. Ind., (1979) 336. 6 C. Rojas De Gante and B. Pascat, Packag. Technol. Sci., 3(2) (1990) 97. 7 A. Charipao, Radiat. Phys. Chem., 22 (1983) 10

781 8 9 10 11 12 13 14 15 16 17 18 19 20

H. Kim-Kang and S.G. Gilbert, Applied Spectroscopy, 45 (4) (1991) 572. J.J. Killoran, in: "Preservation of food by Ionizing radiation", E. Josephson and M. Peterson (eds), CRC, Florida, Vol II (1983) 317. R. Buchalla, C. Schliittler and K.W. Bogl, J. Food Prot., 56(11) (1993) 991. J.J. Killoran, Radiation Res. Rev., 3 (1972) 369. K. Ishitani, Y. Yamazaki, T. Hipora and S, Kimura, Nippon Shokuhin Kogyo Gakkaishi, 23 (1976) 474. C.E. Feazel, R.E. Burks, B.C. Moses and G.E. Tripp, Packag. Eng., 5(4) (1960)43. K. Azuma, T. Hirata, H. Tsunoda, T. Ishitani and Y. Tanaka, Agric. Biol. Chem., 47(4) (1983) 855. K. Azuma, Y. Tanaka, H. Tsunoda, T. Hirata and T. Ishitani, Agric. Biol. Chem., 48(4) (1984) 2003. K. Azuma, H. Tsunoda, T. Hirata, T. Ishitani and Y. Tanaka, Agric. Biol. Chem., 48(8) (1984) 2009. R. Buchalla, C. Schliittler and K.W. Bogl, J. Food Prot., 56(11) (1993) 998. C.F.Berch, R.R. Stromberg and B.G. Achhammer, Modern Packag., 32 (8) (1959) 117. R.Reinke, in: "Plastic film technology: High barrier plastic films for packaging", K.M. Finlayson (ed.), Technomic Publ. Co., Lancaster PA(1979)70. M. Yamamura, in "Industrial Products Research Institute Annual Meeting" ,(1981) 19.

This Page Intentionally Left Blank

783

AUTHOR INDEX Acree, T.E., 1, 27, 69 Ahatnad, N., 345 Akrida-Demertizi, K., 125 Amarowicz, R, 597 Apostolopoulos, D., 753 Apriyantono, A., 279 Aristoy, M.C., 547 Am, H., 27 Amoldi, A., 529 Badeka, A.B., 759 Back, H.H., 271 Barta, J., 659 Bauer, B., 767 Begliomini, A.L., 315 Beirao Da Costa, MX., 133, 369 Bernardo Gil, M.G., 133 Bierenbaum, M.L., 695 Billi, M., 627 Bononi, M., 43, 143 Bunke,PR., 535 Cadwallader, K.R., 271 ChambersIV,E., 173, 187 Chambers, D.H., 187 Chang, C.Y., 353 Chaveron, H., 393 Chen, B.R., 493 Chen, M., 573 Chen, Y.S., 431 Chevance, F.F.V., 255 Choo, S.Y., 345 Conner, J.M., 111,385,615 D'Agostina, A., 529 Deibler, K.D., 69 Delahunty, CM., 117 Demertzis, P.O., 125, 719 Dirinck, P.J., 233 Ducruet, V., 743 Ebeling, S., 1 Ehlermann, D.A.E., 767 Escalona-Buendia, H., 615

Farmer, L.J., 255 Feigenbaum, A., 743 Flores,M.,331,547 Fouad, F.M., 647 Fox, P.F., 559 Franz, R., 719 Fu, H.-Y., 509 Fujisawa, K., 227 Garem, A., 207 Gaset, A., 79 Geronti,A., 219 Gimelfarb, L., 295 Gogoris, A.C., 15 Golding, J.B., 375 Goubet, L, 245 Guillard, A.S., 195, 245 Hashim, L., 393 Ho,C.-T,493, 509, 519 Huang, L.-Z., 519 Huang, T.C., 509, 519 Indrawaty, 279 James, C , 331 Janda, V., 679, 707 Jiang, J., 345 Kato, M., 423 Katsaboxakis, 689 Koller, W.D., 767 Kontominas, M.G., 759, 767 Kooyenga, D.K., 695 Kuramitsu, R., 181 Kurata, T., 639 Kwok, K.C., 621 Lasater, J., 331 Lavin, E.H., 69 Le Quere, J.L., 195, 207, 245 Lebosse, R, 743 Liadakis, G.N., 219 Liang, H.H., 621 Lin, L.Y., 353, 493 Lloyd, S.W., 331

784 Lubian,E..43, 143 Ludwig, S.P., 15 Maciarello, M.J., 401 Mamer, O.A., 647 Margomenou, A., 385 Marsili,R.T., 159 Martello, S., 43, 143 Maurel, S., 79 McGlasson, W.B., 375 McGorrin, R., 295 Melanitou, M., 689 Miller, J.A., 331 Mirrissey, P.A., 117 Moldao-Martins, M., 133, 369 MoUe, D., 207 Monspart-Selnyi, J., 659 Montedoro, G.F., 315 Moreira, N., 369 Morrello,M.J.,415 Mottram,D.S.,483 Naczk, M., 597 Negroni, M., 529 Nobrega, I.C, 483 Nolasco, M.A, 133 Nomura, F., 639 O'Riordan, P.J., 117 Obretenov, T., 455 Omar, N., 345 Omori, M., 423 Papanicolaou, D., 689 Parliment, T.H., 99 Paterson, A, 111,615 Patkai, G., 659 Piggott,J.R., 111,385,615 Pokomy, J., 667, 679, 707 Preininger, M., 87 Pudil, F., 667, 679, 707 Reineccius, G.A., 573 Revilla, E., 583 Riganakos, K.A., 767 Rizzi, GP., 535 Robinson, K., 187

Rosea, ID., 735 Ryan, J.M., 583 Salles, C, 195, 207 Sanceda, N.G., 639 Sanz, Y., 547 Sauer, D.B., 173, 187 Sauriol, F., 647 Seitz,L.M., 173,187 Selvaggini, R., 315 Septier, C, 195 Servili,M.,315 Shaath, N.A., 443 Shahidi, F., 55, 597, 647 Shallenberger,R.S., 1 Sheehan, E.M., 117 Smith, E.G., 173 Sommerer, N., 207 Sousa, I , 369 Spanier, AM., 331, 547 Spiliotis, C, 219 Su,Y.M., 519 Suzuki, E., 639 Talou, T., 79 Tan, C.T., 29 Taoukis, P.S., 627 Tateo, F., 43, 143 Toldra, F., 547 Trigo, R., 133 Tsai,H.J.,431 Tucker, AO., 393 Tzia, C, 219 Valentin, J., 195 Valentova, H., 667, 679, 707 Van Opstaele, F., 233 Vareli, G., 125 Vendeuvre, J.L., 245 Vergnaud, J.M., 735 Vemin, G., 455 Volfova, J., 667, 679, 707 Wallace, J.M., 559 Watkins, T.R., 695 Wijaya, H., 707

785 Williams, M., 375 Withers, SJ., 111,385 Wyllie, S.G., 375 Xi, J., 509 Yang, R.D., 621 Yen, Y.H., 353 Yoshino, M., 227 Yu,T.H., 353,431,493

This Page Intentionally Left Blank

787

SUBJECT INDEX Acetaldehyde, 276,277 Acetic acid, 673, 682 Acetone, 424,670,671,673,682,713,714 Acetonitrile,145 Actinomycetes, 174 Activity coefficients ethanol concentration,115 temperature effect, 114 Adhyperforin, 144 Adsorption, 542 ethanol on wheat straw, 125 water on wheat straw, 125 AH-B theory, 1,4-11 Alcohol, 615,616 analytical determination of, 221 Aldehydes, 173,353,365,616 branched, 235 Allicin, 660,662,664 Allium sativum ,659 Allyl isothiocyanate, 663 Amine oxidase, 639 Amino acids, 286, 424,639 addition to Cheddar cheese, 561,566 alanine, 360,361 analysis in cheese, 563 analysis of, 550,552 arginine, 427 aspartame, 7,8 aspartic acid, 360,361,426,427 chocolate precursors, 544,545 cocoa bean, 539 concentration in meat products, 553 contribution to flavor, 547 cured cooked ham, 201 free, 72,359,564,565 glutamic acid, 360,361,426,427 glycine, 360,361 goat cheese, 216 histidine, 427 isoleucine, 360,361,427 leucine, 360,361,427 lysine, 360,361,427,530

phenyl alanine, 427 pochung tea, 435 proline, 360,361 serine,360,361,427 threonine, 427 tyrosine, 360,361,427 valine, 360,361 Aminopeptidases, 547,548,551 Ammonia, 509 Ammonium ion, 231 Anethole, 220,222 Anise, 80 Anise seeds, 220 p-Anisidine value, 56 Anthocyanins, wine, 585, 590 Antimicrobial, 659 Antioxidants, 55,529 effect on microwave cooking,760 endogenous, 695 mammalian, 698 oil preservation, 689 risk of cardiovascular disease, 695,

698-703 urate's importance as, 695 use of vitamins as, 695 Antrachinons, 144 Arachidonic acid, 55 Armoracia lapathifolia, 664 Aroma, 173,271,276,573 coffee flavor, 44,50 contribution of amino acids to, 547 oatmeal, 415 potent food components, 87 stability, 576 Aroma analysis, 69 acidic, 671,672,676,684,715,716 anise, 671,676,684,715,716 burnt, 274 buttery, 274 citral, 670 crabby, 274 dark chocolate, 274

788 Aroma analysis cont. earthy, 274,275 fatty, 274 fecal, 275 fishy, 274 floral, 671,676,684,715,716 fresh, 671,672.676,684,715,716 fruity, 671,676,684,715,716 heavy, 671,672,676,684,715,716 lemon, 672 lemon juice, 671,676,684,715,716 lemon peel, 671,676,684,715,716 malty, 274 meat, 272,274,276 melon, 274 menthol, 684,715,716 musty, 274 nutty, 274 orange juice, 671,676,684,715,716 orange peel, 671,676,684,715,716 plastic, 274 popcorn, 274 pork, 256,264,265 pototato, 274 pungent, 275,671,676,684,715,716 rancid, 274 rubbery, 262 sniffing port, 272,273 spicy, 671,676,684,715,716 stable, 275 stale, 274 sticky skin, 262 terpenic, 684 woody, 671,672,676,684,715,716 Aroma compounds, 573,746 database of, 27 milk, 393 reactivity of, 743,747-749 sorption of, 743 Aroma extract dilution analysis (AEDA), 272 Aromatic note, 83 Aromatic samples, 80 Arrhenius parameter, 541 Artificial, 388

Aspartame, 632 Aspergillus niger, 659,662,664 Aspirin, 699 Autooxidation, 667,707,711,714 B Bacillus cereus, 659,664 Bacterial flora, 642 Bales rose de bourbon, 407 Baker's yeast, 231 Balsamic, 80 Bananas, 377 delayed ripening, 383 total volatiles in, 380 Benzemethanol, 365 Benzoic acid, 599 Benzyl disulfide, 187 Bergamot oil, 679-681, 683, 685 Beverage, 29 packaging material for, 719 sorption measurements on bottles, 724-728, 729-732 Bilayer bottles, 735 Bioflavonoids, 144 Bitter, 388 Bitterness, cooked cured ham, 204 Black tea, 423-425,427 Bologna, 188 Borneol, 173 Brine cured, 233 Broiler, 279 Buccal headspace analysis, 111, 112-113, 117 Butanol, 424 Butter oil, 647

Caffeine, 45 California bay oil 405 Camphor, 82 Canola meal, 599 Caramelization, 354,359,365

789 Carbon dioxide, 134 production in bananas, 379 Carvacrol, 187 Carvone, 671 Catechins, 424,433 Catfish, 274 Cellulose, 573 Charm analysis, 69 Cheese, 385 Cheddar cheese, 117,559,560 addition of amino acids, 561 goat, 207 mold-ripened, 174 Chenopodium, 408,409 Chicken aroma, 285 breasts, 280 domestic, 279 Chios, chewing gum, 689 Chios mastic resin, 689 color influence of vitamin E, 693 hardness, 693 harvesting, 690 packaging, 689 storage studies, 689 Chloroanisoles, 173 Chloroform, 713 Chloromethanes, role in sweetness, 3 Chocolate, 535 Cinnamic acid, 599 Cinnamon, 188 Citral, 682 Citronellal, 707,713,714 Citronellol, 707,713,717 Citronellyl acetate, 713 Citronellyl formate, 713 Citrus hystrix, 679,683,707-711.714, 715-717 Citrus oils, 679 Clevenger distillation Chios mastic resin, 689,691 Cloves, 188 Cluster population, 82 Coalescence, 31

Cocoa bean, 69 Coffee, 43 aromatization solvents, 43,45,47,48 beans, 69 cherry, 70 conventional brew, 72 flavor, 69 ion chromatogram, 50 propylene glycol solvent, 45,47 quick brew, 72 separation, 102 Color banana, 378 measurement, Chios mastic resin,

689, 691 wine, 587,592 Composition banana, 381 durian, 345 oatmeal, 419 wines, 588,589 Condensed tannins, 603 Conjugated double bonds, 58 Consumer preference, 281 Contaminants, plastics, 720,721 Copper, effect on milk flavor, 167 Corn, 187 Crayfish, 271,272,276 Creaming, 30 Cresol, 188 Crustaceans, 275 Cyclohexanone, off odor, 756 Cyclopseudohypericin, 148 Cysteamine, 519 Cysteine reactions, 488,489

Database, Flavornet, 27 Date of invention, 21 Deaminase activity, 227 Decanal, 618 n-Decane, 713 Description, 386

790 Description, analysis, 281 Descriptive panel, 179,193 Desirability, 315 Dessert, dehydrated fruity, 627,632 Deuterated compounds, synthesis, 89 Dicarbonyls, 511 Dichloromethane, 355 Diethyldisulfide, 580 Dihydropyridine, 685 Diludine, 679,708 2,4-Dinitrophenyl hydrazine, 63,102 Di-n-propyl disulfide, 445 Diode array detector, 145,147 Distillates aroma compounds in, 219,222 aroma compounds in durian, 346 aroma compounds in, Mentha pulegiumL,^ze, 137 aroma compounds in. Thymus zygisL, 136, 137 Disulfides, 488 Diterpenoids, 144 Docosahexaenoic acid, 55 Dodecanal, 618,619 Durian, 345,347,351

Eicosapentaenoic acid, 55 Electromyography, 119 Electron paramagnetic resonance, 64 Electronic nose, 79 Emulsion, flavor, 29 Encapsulation, liquid membrane, 41 Enzymes catalase, 696 glutathione peroxide, 696 in fruit flavor, 369 measurement of activity, 549 superoxide dismutase, 696 Enzymology, dry-cured meat products, 550 Epazote, 408 Escherichia coli, 659,664 Essential oil, 79

Chios mastic resin, 689 Mentha pulegium L, 136,137 stability of, 689 Thymus zygis L , 136,137 Esters, 353,365 apple, 369,371 ethyl, 615,617 Ethanol, 146,615-617,619,620,682,713 as fuel, 125 distillation of seeds, 225 2-Ethyl-3,5-dimethylpyrazine, 581 Ethyl hexadecanoate, 365 Ethyl-2-mercaptopropionate, 573 Ethylene production, in bananas, 379 Eucalyptol, 82 Eugenol, 188 Euphoria longana, Lamarck, 353

Fast atom bombardment, 654 Fat content, 245 Fatty, 388 Fatty acids pouchung tea, 434 short chain, 386 Fenchyl alcohol, 176 Fermentation, 227,639 Fish sauce, 639 Flavor, 173, 188,279,597 aged, 233 agents, 447, 448 analysis, Cheddar cheese, 567 apple jams and jellies, 369,371 banana, 378 characteristics in \A^iskey, 111 chocolate, 535 coffee and tea, 43,44,45,431 development of, 547 durian, 345 effect, addition of amino acids to cheese, 559 milk, 393 oatmeal compounds, 419,420

791 Flavor cont. onion oil, 446 perception, 111,121,369 pineapple, 331 profile, tomatillos, 312 release, 114,117 role of sodium nitrite, 245 sulfur compounds, 483 synthetic, 262 tomato juice, 315 Flocculation Mentha pulegium L., 133 Thymus zygis L., 133 Food canolaoil, 695,701-703 contamination, 739 dehydrated, 627 melanoidins in, 455,459-461 milled flax seed, 695,701-703 packaging, 737,743 preservation, irradiation in,767,768 quality, 628 sensory attributes, 627 shelf life, 627 Fourier transform infrared, 64 Free radicals, 696,697-703 formation from fatty acid, 697 initiator, 534 Fruit, 331,353, 357 Furans, 353,365 2-Furfurylthiol, 75 Furylmercaptan, 573,579

milk, 161,393,395 model system, 104 oatmeal, 418 packaging materials, 722,767,770-771 pineapple, 331-332,337-338,340 tomatillos, 302,303 tomato juice, 320 tomatoes, 302,304 Gas chromatography - mass spectrometry 76, 235, 273, 335, 530, 667, 668 coffee, 45,46-48 jellies and jams, 369,371 packaging material, 767,773-779 pineapple, 331-332,337,340 Gas chromatography - olfactometry, 27, 69,-331-332,337,340 Gas sensors, 79 GATT-TRIP, 15 provisional application, 20,21 provisions of, 17,18-19 Gel filtration, 199,210 Geosmin, 179 Geraniol, 365,A761428 Geranyl acetate, 685 Glucosinolates, 597 Glutamate, 227 Glutamate, dehydroginase, 229 Glycine ethyl ester hydrochloride, 182 Glycolysis, 231 Guaiacol, 188 Gum arabic, 34 H

Garlic, 659,661 Gas chromatography, 425, 443,449, 450, 744 banana, 378,382 coffee, 44,46-48,49,106 ICG of wheat, 125,126-128 indirect method for plasticizers, 759, 761-762 jams and jellies, 369,371 maillard reactions, 485

Halloumi, 385 Ham cooked cured, 195 dry cured, 233 fatty acid analysis, 245,246,247-250 phospholipid analysis, 245-248 role of sodium nitrate, 245 taste improvement, 547,548 Headspace, 80,187,234,271,385,575

792 headspace^Qont. analysis, 754 analysis, dynamic, 315 analysis, static, 315 Hepatopancreas, 271,272 Herbs, Mentha puJegium L, 133 2,4-Hexadienal, 573,578 H^canat, 55;578,620 Hexanol, 428 Histamine, 639 Histidine, 641 decarboxylase, 639 Hoja santa, 402 Horse-radish, 659,662,774 High performance liquid chromatography, 424 cooked cured ham, 200 goat cheese, 213 Hunter L, B, a, 353,356,358 Hydrogen sulfide, 509 Hydroperoxides, 55,667,670 Hypericin, 143-M5,147 Hypericum perforatum L, 146. Hyperlipemic subjects, 695,699-703

K Ketchup, 659,661 Ketones, 240,353,365 Kinetics, 544,545 of transfer, 736

Lactobacillus brevis, 660,662,664 Lactobacillus plantarum, 659 Laurel, 82 Lavender, 82 Lavandin, 82 Lignin, 188 Limonene, 681,682,684,713 Linalool, 82,428,681,682,684,713,716 Linalool oxide, 365,682,685,713,714 Lipid oxidation, 55 Lipolysis, 233 Lysine hydrochloride, 182 M

I IMP reactions, 484 Infrafred analysis, 767,769,780 spectrum of onion oil, 447 Inosic acid, 227 Intellectual property, 15,16,24,25 Interfacial film, 33 Iodine value, 56 Ionizing radiation, 767,768-769 Isoamyl alcohol, 365,615

Jams and jellies, 369,370,371,372

Maillard reaction, 354,358-360,483, 529-530, 534, 547 amino acids, 497-506 glucose based, 499,501,504,505 Hunter "L", 495,503 in glycerol, 498,501,502 in propylene glycol, 497 lambda max, 495,497,498 melanoidins in, 455,459 steps in, 457 odor descriptions, 504,505,506,507 solubilities, 496,497 xylose based, 500,502,506,507 Malonaldehyde, 59 Marjoram, 188 Mass spectra, 513,514 ofonion oil, 450,451-452 Mastication, 119

793 Meat, 233 cooked cured, 245 curing, 195,547-549 Medicinal, 177 Melanoidins biological activity of, 468 chemical properties of, 466,467 in vivo, 473 mass spectra of, 455,469-472 methods of analysis, 455,463-465 study of, 455 synthesis of, 462 Memory, attention and behavior, 27 Mentha pulegium L., 133 p-Mentha-1,4(8)-diene, 711,713,714 p-Menth-8-en-3-ol, 711,713-714 Methanol, 145,147,713 1 -Methylcyclopropene, 376-377 2-Methylisoborneol, 173 Methyt-n-propyl disulfide, 445 1-Methylpyrrole, 573 Methyl salicylate, 428 2-Methylthiophene, 579 Microflora, 174,385 Microwave, 493 processing, 759,760 Milk, 385 analysis of, 393,395-399 effect of light on flavor, 167 off-flavors in, 159 sensory analysis, 393-394,396-399 UHT processed, 393,394 Mint terpene, 389 Minty, 80,179,388 Mixxor separatory device, 102,103 Model systems, 102,455,458 cysteine/IMP based, 488 cysteine/ribose based, 488,490 cysteine/ribose phosphate based, 488-490 study of whiskey, 112 thiazolidine formation, 520 thiazoline formation, 509

Monosaccharides, predictor of sweetness, 3 Monosodium glutamate, 183 Multicapillary gas chromatography, 79 Multivariate classification methods, 162 Musa sp, 376 N NAFTA, 22 Nanofiltration 197,207 Natural products, odor potency of, 27 Nerol, 707 Nerolidol, 428 Nitrites, 60 effect on curing, 554 Non-volatiles, tomatillos, 299 Non-volatiles, tomatoes, 299 Nuclear magnetic resonance, 64 Nutmeg, 188

Oak lactones, 672 Oatmeal, 415,421 Ocimene, cis, 681,682,685,713 Ocimene, trans, 681-683,685,713 Octahydro-3A-methyl-cis-2H-inden2-one, 713 Octanal, 615,618 Odor descriptor, 176 evaluation, 755 profile, 179 odor unit, 310 Odorants, 75,245 identification of, 27 Off-flavors, 55, 573,743,768 Off-odors, 174,753,756 Oleoresin, 79 Onion oil, 443,445,446,449 types, 443,444-445

794 Orange juice, 32,743,744 Ornithyltaurine hydrochloride, 182 OSI 7284, 667,684,708,710 Ouzo, 225 preparation of, 220,221 profile diagram of, 220,224 soaking of seeds, 220,224 Oxazolines, 509 Oxidation, 233, 682 in apple, 369,371 Oxidative stability instrument, 64 Oxidative state, 56 Oxido-redox reaction, 523 2-Oxoglutarate, 231 Oxygen elevated levels, 695,699 free radicals, 695,699 reduction of, 696 sensors, 639

Packaging, Chios mastic resin, 574,689 Packaging materials, 173,768 analysis of, 720,721,722,723 effect on microwave heating, 759 polycarbonate bottles, 719 polypropylene, 743,744 recycled beverage materials, 719 sorption measurements, 724-732 types of pofymers, 768,769 Palm oil, 280 Panelists, 172,281 Parma, 234 Partial least squares regression, 386 Particle size distribution, 37,38 Patent, 15 proof of \A/ritten record, 22,23-24 provisional application, 17,18-19 termofGATT-TRIP, 16 Pectin, as gelling agent, 370 Penicillum oxalicum, 660,662

Pepper, 188 Peppercorns, 407 Peppery, 255,261-264,268 Peptides, goat cheese, 207 Per/7/a frutescens, 406 Perilla oil, 406 Peroxide value, 56 Phenolic acids, 597,599,601 Phenols, 255,267,269.353,365 Phosphate buffer, 520 Phosphoric acid, 145 Phytotherapy, 143 Pineapple, 332,339,340 Piper auritum, 403,404 Plasticizers, 759,760,761,762-765 Polycarbonate, use as beverage containers, 719 Polycyclic aromatic hydrocarbons, 189 Polymers, plastic, 735 Polyols, taste of, 5 Polyphenols, 423,424,426,587,589 Polypropylene, juice packaging material, 743, 746-750 Potassium chloride, 185 Preclimacteric application, of 1-MCP, 379-380 Precursors, chocolate flavor, 538 Precursors, tea flavor, 436,440 Principal component analysis, 118.284,317,386 Proanthocyanidins, 606 Procambarus clarkii, 271,272 Profiling, 256 Pro-oxidant, 529,530 Propanal, 55 Proteolysis, 233 Protohypericin, 148 Pseudohypericin, 148 Pseudomicelles. 616 Pseudomonas aeruginosa, 659,662,664 Purine nucleoside phosphorylase, 229 Pyrazines, 173,292,419 Pyrolysis, 188

795

Quantitation, food aromas, 87,88

Rancid flavor, 234 Rancimat, 64 Rapeseed protein. 597 Rate constants, 541 Recycling polymers, 735 Reductive aminatlon, 231 Rennet, 385 Residual flavor, 263 Resinous, 80 Response surface modeling, 137, 139-140,318 Rice, 228 Roasted note, 292 Rosemary, 529,674,675,679, 683-684,708,710

Saccharomyces cerevisiae, 660,662,664 Saint John's Wort, 143 Sake, 227 Saliva, 121,267,268 Salt, 181,183,197,216,265 Sample preparation techniques, 100 Sample simplification, 101 Sanitizer, effect on milk flavor, 167 Saporlfic group, sweetness functional group 1,4-11 Sassafras albidum, 402 Sassafras oil, 402 Sausages, 256,265 Schiff base, 519 Scotch malt whiskey, 111 Seal blubber oil, 57 Sensory, 255,385 analysis, 281,627-628, 632-635, 668, 680 characteristics, 173

description, 282 evaluation, 756 evaluation, ouzo, 221,223-225 evaluation, cheddar cheese, 562, 568, 569, 570 evaluation, coffee, 103,108 evaluation, cooked cured ham, 203 evaluation of goat cheese, 215 evaluation, tomatlllo, 298 evaluation, tomato, 298 evaluation, wines, 593 flavor research of whiskey, 111 panelists, 187 proflle, 710 quality, 187 Seranno, 234 Serum cholesterol, 700 Sesquiterpenes, 713 Shelf Jife, 627,628.629-631,633 Simulated mouth, 111,112-113 Simultaneous steam distillation extraction, 235 SInapic acid, 599 Sinaplnes, 597 SInlgrIn, 663 Smoke, smokiness, 187,255,256, 261,264,268 Solid phase microextraction, 315, 668, 680, 709 Sorghum, 187 Sorption, measurements on packaging materials, 719,723,724-728,729-732 Sotolon, 75 Soy bean trypsin Inhibitors, 621 beans, 187 milk, 622,623,624 sauce, 181 Spices, 188 Spicy, 80,255.263,264,268 Stable Isotope Dilution Assay, 88 Staphylococcus aureus, 660 Starch, modified, 34 Steam distillation, 271

796 Stokes', 31 Storage studies Chios mastic resin, 689,690 pineapple, 332,333 Strecker aldehydes, 277 Structure-active research, 1 Sugars, 359 content, jams and jellies, 370 fructose, 359,360 glucose, 359,360 maltose, 359.360 pouchung tea, 434 ribose reactions, 483 sucrose, 359,360 xylose, 369,360 Sulfanilamide, 60 Sulfur volatiles, 483 Sulfur-containing compounds, 240 Supercritical fluids extraction of oatmeal, 417,421 Mentha pulegium L , 133 Thymus zygis L., 133 Sweet and sour, 388 Sweet flavor, 262 Sweeteners, 1,2 Syringol, 188

Tannin-protein interactions, 607 Taste compound, 227 description, 284 transduction, 8,9-11 cooked, cured ham, 203 Tea, 431,432 Temperature affect on adsorption in wheat 125 affect on milk, 393 Temperature controlled partial crystallization, 547 Terpenes, 255,265,267,269,427 terpenic, 656,671,715,716 in apple, 369,371

Terpinen-4-ol, 713

Texture, 386 Theaflavin, 423,424,426 theaflavin-3-3'-digallate, 426 theaflavin-3'-monogallate, 426 Thearubigin, 423 Theoretical profile equations, 739,740 Theoretical profiles transfer, 737,738,739 Thiazines, 520 Thiazolines, 509,519 2-Thiobarbituric acid, 56 2-Thiobarbituric reactive substances, 61 Thiols, 488 Thymus zygis L., 133 Tomatillo, 295 Tomato, 295 Tomato juice, 321 TOTOX value, 56 Toxic herbs, 365 Triacylglycerol, 647

Triglyceride, 255 1,2,4-Trithiolane, 240 Trypsin inhibitors, assay, 623 U Umami, cooked cured ham, 203 Umami, goat cheese, 216 Umbellularia californica, 405 Uric acid, 228 UV absorption spectrophotometry, 458

Vicinyl hydroxyl groups, 2 Vitamins beta-carotene, 695,697

C, 695,697 E, 695,697,698 E, effect on Chios mastic resin, 689, 693 Volatiles, 79,117,280,385 affect on ham flavor, 245 alcoholic beverages, 114

797 volatiies cont. aldehydes, 234 analysis in ham, 247,251,252 carbonyl compounds, 56 determination of in packaging, 770 extract from coffee, 44 jellies and jam, 369,372,374 milk, 393,394 \A/hiskey, model systems, 112 packaging material, 767,768 pineapples, 331-332,335,336 tomatillos, 306 tomatoes, 306 W Water activity, 353,356,358 Weibull hazard analysis, 627,630-631 Weighting agents, 32 Wheat adsorption in of ethanol, 125,129 adsorption of water, 125,129 enthalpy of adsorption, 125,131 Gibb's free energy, 125,130 use as fuel, 125 Whey, 385 Whiskey, 615 Wine, 615,586 addition of seeds and skin, 583 Frankinja, 587 Merlot, 587 red, 583 Wormseed, 408,409 WWW site, 27

Xanthine oxidase, 229

Yeast, 331 Verba santa acuyo, 403,404

This Page Intentionally Left Blank