DEVELOPMENTS IN FOOD SCIENCE 40
FOOD FLAVORS: F O R M A T I O N , ANALYSIS A N D PACKAGING INFLUENCES
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DEVELOPMENTS IN FOOD SCIENCE 40
FOOD FLAVORS: F O R M A T I O N , ANALYSIS A N D PACKAGING INFLUENCES
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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
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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
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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
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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
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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
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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.
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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 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
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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
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(11)
DsCMgl
O^A^
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n ^
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^
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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
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[Abundance 250000 BASES REMOVED
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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
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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
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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) 0.01 Highly significant; *** p.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.
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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
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Emodinanthranol Figure 1. Structures of hypericin and hypericin-like compounds present in Hypericum perforatum L.
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Figure 2. Structures of hyperforin, adhyperforin and of some flavonoids present in Hypericum perforatum L.
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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
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IS1
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3. 2-butanone 4. chloroform 5. dichloroethane
6. 3-methyl-2-butanone
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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 r^||f
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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
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IBERICO
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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.
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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
o o 00
o o (O
^
i
^
)^ X 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.
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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
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Loadings: p[ll/p[2|
•3ll^ • 104
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•3-
r^Jil.. •IS'i itdji •116
•100 • U
• Ift53 •141
,,
• Btf*'-'* P[2],
^ii64
•Tl^ •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
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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)
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.
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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?
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
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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 :?
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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).
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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
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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
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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.
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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.
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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
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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
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/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-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: - .J5 .J2 O )
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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 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.
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E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved
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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-
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Structures and formation of oxazolines and thiazolines in the reaction of 3-hydroxy-2-butanone Figure 6. and ammonium sulfide.
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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
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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.
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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].
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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
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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 —^ • •
'E
1 *^v5
I 60 0)
—•—0.11 ^ -•-0.33 -A-0.53 ->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.
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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^
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.
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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.
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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.
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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.
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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.
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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 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
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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
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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
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20.00
39 40
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13.86 14.12
20.18 21.28
41 42 43 44
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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.
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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
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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
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